Volume 52 Number 2S August 2018 ISSN 0023-6772 Laboratory ... · aboratory limited l a nimals Committees Committees Organizing Committee Magda Castelhano-Carlos1,2,3 (Chair) Alice

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SAGE Publications Press Ltd

Volume 52 Number 2S August 2018 ISSN 0023-6772

Offi cial Journal of AFSTAL, ECLAM, ESLAV, FELASA,

GV-SOLAS, ILAF, LASA, NVP, SECAL, SGV, SPCAL

THE INTERNATIONAL JOURNAL OF LABORATORY ANIMAL SCIENCE, MEDICINE, TECHNOLOGY AND WELFARE

Laboratory Animals

SPCAL Congress 2018, abstract book

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Volume 52 Number 2S August 2018

Contents

Acknowledgments 3

Organizing Committee 4

Scientific Committee 4

Transparency Agreement 6

Scientific Programme 6

Oral Communications 6

Opening Lecture 6

Session 1: Transparency & Communication in Laboratory Animal Sciences 7

Session 2: Quality and Animal Welfare in Laboratory Animals 8

Session 3: Managing Resources and Facilities 10

Laboratory Animals Associations - Past, Present and Future 11

Session 4: Legislation: New Perspectives and Updates 13

Session 5: Education and Training 14

Session 6: Animals and Models: Biological Perspectives 16

Closing Lecture 19

Poster Session 19

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Acknowledgments

We would like to acknowledge the invited speakers,chairpersons, the laboratory animal scientists, man-agers, technicians and caretakers working with animalsthat submitted abstracts to the IV SPCAL Congress.The scientific success of this congress depends largelyon their contribution.

We also want to express our appreciation to theSchool of Medicine and the Life and Health ScienceResearch Institute (EM/ICVS) of the University ofMinho, that contributed and supported from the begin-ning to the organization of this event with fully opendoors. We are very grateful for their kind hospitality.

We must also acknowledge Braga Municipal Councilfor their support from the beginning of the organizationof the event, helping us to organize the social eventswelcoming all participant to the city of Braga.

We are grateful for the generous support of our cor-porate partners that sponsored and/or are present inthe exhibition area.

We also wish to thank:

– All SPCAL members the trust deposited in theOrganizing Committee.

– All the Scientists involved in the Scientific com-mittee, for their contributions to the program.

– The staff of the Planning Unit of the EM /ICVS,namely Madalena Branco and Daniel Monteiro,for their contribution, helping us beyond theirobligations.

– The Honour Committee for welcoming this eventand the public announcement of the TransparencyAgreement in Portugal.

Magda Castelhano CarlosChair of Organizing Commit

Laboratory Animals

2018, Vol. 52(2S) 3

! The Author(s) 2018

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Committees

Committees

Organizing Committee

Magda Castelhano-Carlos1,2,3 (Chair)Alice Miranda2,3

Dolores Bonaparte4

Lucılia Diogo1,5

Luıs Antunes1,6,7

Nuno Henrique Franco1,8,9

Ricardo A. Afonso1,5,10

Sandra Pereira2,3

Tırcia Santos1,3,11

1Sociedade Portuguesa de Ciencias em Animais de Laboratorio (SPCAL) j Portuguese Society for LaboratoryAnimal Sciences (SPCAL)2Instituto de Investigacao em Ciencias da Vida e Saude (ICVS) da Escola de Medicina, Universidade do Minho,Braga, Portugal j Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho,Braga Portugal3Laboratorio Associado ICVS/3B’s, Braga/Guimaraes, Portugalj ICVS/3B’s – PT Government AssociateLaboratory, Braga/Guimaraes, Portugal4Fundacao Champalimaud, Lisboa, Portugal j Champalimaud Foundation, Lisbon, Portugal5Faculdade de Ciencias Medicas, Universidade NOVA de Lisboa, Lisboa, Portugal j Nova Medical School, NewUniversity of Lisbon, Lisbon, Portugal6DCV/ECAV – Departamento de Ciencia Veterinarias, Escola de Ciencia Agrarias e Veterinarias, Universidade deTras-os-Montes e Alto DouroVila Real, Portugal Vila Real, Portugal j DCV / ECAV – Department of VeterinaryScience, School of Agrarian and Veterinary Science, University of Tras-os-Montes and Alto Douro, Vila Real,Portugal7CITAB – Centro de Investigacao e Tecnologias Agroambientais e Biologicas, Universidade de Tras-os-Montes eAlto Douro, Vila Real, Portugal j CITAB – Centre for the Research and Technology of Agro-Environmental andBiological Sciences, University of Tras-os-Montes and Alto Douro, Vila Real, Portugal8i3S – Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto, Portugal j i3S – Institute forResearch and Innovation in Health,University of Porto, Porto, Portugal9IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal j IBMC – Institute for Molecular and CellBiology, Porto, Portugal10Departamento de Fısica, Faculdade de Ciencias e Tecnologia, Universidade NOVA de Lisboa, Lisboa, Portugalj Department of Physics, Faculty of Sciences and Technology, Nova University of Lisbon, Lisbon, Portugal11Grupo de Investigacao 3B’s – Biomateriais, Materiais Biodegradaveis e Biomimeticos, Universidade do Minho,Guimaraes, Portugalj 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho,Guimaraes, Portugal.

Scientific Committee

Ricardo A. Afonso1,2,3(Chair)Ana Isabel Moura Santos1,2,4

Anna Olsson5,6

Antonio Sykes7

Catarina Pinto-Reis8

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Dolores Bonaparte9

Isabel Campos9

Isabel Vitoria Figueiredo1,10

Luıs Antunes1,11,12

Magda Castelhano-Carlos1,13,14

Manuel Rebelo15

Margarida Correia Neves13

Maria Teresa Rodrigo Calduch16,17

Nuno Henrique Franco1,18,19

1Sociedade Portuguesa de Ciencias em Animais de Laboratorio (SPCAL) j Portuguese Society for LaboratoryAnimal Sciences (SPCAL)2NOVA Medical School j Faculdade de Ciencias Medicas, Universidade NOVA de Lisboa, Lisboa, Portugal jNOVA Medical School, New University of Lisbon, Lisbon, Portugal3Departamento de Fısica, Faculdade de Ciencias e Tecnologia, Universidade NOVA de Lisboa, Lisboa, Portugal jDepartment of Physics, Faculty of Sciences and Technology, Nova University of Lisbon, Lisbon, Portugal4Federacao Europeia das Associacoes de Ciencias de Animais de Laboratorio (FELASA) j Federation of EuropeanLaboratory Animal Science Associations (FELASA)5i3S – Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto, Portugal j i3S – Institute forResearch and Innovation in Health,University of Porto, Porto, Portugal6IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal j IBMC – Institute for Molecular and CellBiology, Porto, Portugal7Centro de Ciencias do Mar, Universidade do Algarve, Faro, Portugal j Centre of Marine Sciences, University ofAlgarve, Faro, Portugal8Faculdade de Farmacia da Universidade de Lisboa, Lisboa, Portugal j Faculty of Pharmacy, University of Lisbon,Lisbon, Portugal9Fundacao Champalimaud, Lisboa, Portugal j Champalimaud Foundation, Lisbon, Portugal10Faculdade de Farmacia da Universidade de Coimbra, Coimbra, Portugal j Faculty of Pharmacy, University ofCoimbra, Coimbra, Portugal11DCV / ECAV – Departamento de Ciencia Veterinarias, Escola de Ciencia Agrarias e Veterinarias, Universidadede Tras-os-Montes e Alto DouroVila Real, Portugal Vila Real, Portugal j DCV / ECAV – Department ofVeterinary Science, School of Agrarian and Veterinary Science, University of Tras-os-Montes and Alto Douro,Vila Real, Portugal12CITAB – Centro de Investigacao e Tecnologias Agroambientais e Biologicas, Universidade de Tras-os-Montes eAlto Douro, Vila Real, Portugal j CITAB – Centre for the Research and Technology of Agro-Environmental andBiological Sciences, University of Tras-os-Montes and Alto Douro, Vila Real, Portugal13Instituto de Investigacao em Ciencias da Vida e Saude (ICVS) da Escola de Medicina, Universidade do Minho,Braga, Portugal j Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho,Braga Portugal14Laboratorio Associado ICVS/3B’s, Braga/Guimaraes, Portugalj ICVS/3B’s – PT Government AssociateLaboratory, Braga/Guimaraes, Portugal15Instituto Gulbenkian de Ciencia (IGC), Lisboa, Portugalj Institute Gulbenkian of Science, Lisbon, Portugal16Sociedade Espanhola de Ciencias em Animais de Laboratorio (SECAL), Espanhaj Spanish Association forLaboratory Animal Sciences, Spain17Centros Cientıfico e Tecnologico, Universidade de Barcelona, Barcelona, Espanhaj Scientific and TechnologicalCenters, University of Barcelona, Barcelona, Spain18i3S – Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto, Portugal j i3S – Institute forResearch and Innovation in Health,University of Porto, Porto, Portugal19IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal j IBMC – Institute for Molecular and CellBiology, Porto, Portugal

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Abstracts

SPCAL 2018 abstract book

Transparency Agreement

Thursday, June 21

14:30–16:00

Public announcement of the Transparency Agreement in

Portugal

Round Table and public discussion

This public event counts with the presence of:A Scientist, Professor and Medical Doctor, discussingthe value of animals in science and medicine:Nuno Sousa

1,2

1Life and Health Science Research Institute (ICVS),School of Medicine, University of Minho, Braga,Portugal and2ICVS/I3Bs – PT Associate Laboratory, Braga/

Guimaraes, PortugalExecutive Director of EARA, the association propos-ing the establishment of the agreement:Kirk Leech

European Animal Research Association (EARA)President of SPCAL, the association facilitating theagreement in Portugal:Ricardo A. Afonso1,2,3

1Portuguese Society of Sciences in LaboratoryAnimals (SPCAL);2Nova Medical SchooljFaculdade de Ciencias

Medicas, Universidade Nova de Lisboa, Lisboa,Portugal; and

3Department of Physics, Faculty of Sciences andTechnology, Universidade Nova de Lisboa, Monteda Caparica, Portugal

Scientific ProgrammeOral Communications

Thursday, June 21

16:30–17:30

Opening Lecture

New perspectives in stress research

Nuno Sousa1,2

1Life and Health Science Research Institute (ICVS),School of Medicine, University of Minho, Braga,Portugal2ICVS/I3Bs – PT Associate Laboratory, Braga/Guimaraes, Portugal

AbstractStressful stimuli in healthy subjects trigger activa-tion of a consistent and reproducible set of brainregions; yet, the notion that there is a single andconstant stress neuromatrix is not sustainable.Indeed, after chronic stress exposure there is activa-tion of many brain regions outside that network. Inthe last decades the field has been mapping theeffects of chronic maladaptive stress on the finestructure of the brain and, in parallel, determiningits behavioral and functional correlates. This sug-gests that there is a distinction between the acute-and the chronic-stress neuromatrix. The emergingview is that several factors modulate a dynamicinterplay in brain connectivity. Its comprehensionwill allow for a more holistic perspective of how thebrain shifts ‘‘back and forth’’ from a healthy to astressed pattern and, ultimately, how the latter canbe a trigger for several neurological and psychiatricconditions.

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Friday, June 22

09:00–10:15

Session 1: Transparency & Communication inLaboratory Animal Sciences

Chairpersons:

Ana Isabel M. Santos

Margarida Correia-Neves

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Transparency & Communication inLaboratory Animal Sciences

Kirk LeechEuropean Animal Research Association (EARA)

AbstractIn a number of European countries, public and privateresearch institutions have made the decision to adoptnew persuasive practices and policies to engage withthe public on the benefits of using animals in scientificand biomedical research. These institutions believethat they can play a positive role in public engagementand education. The belief is that being more open andtransparent about their use of animals in researchcould help improve public understanding and accept-ance. The need for a collective commitment is alsoimportant. These commitments are that institutions;will be proactive in seeking opportunities to explainwhen, how and why they use animals in research;will provide information to the media and the generalpublic about the conditions under which research usinganimals is carried out and will explain the benefitsobtained from using them compared to other methodsof research; will develop initiatives that generategreater public knowledge and understanding aboutthe use of animals in scientific research; will place ananimal welfare statement on their institution’s website.In the UK through the Concordat on Openness onAnimal Research, and in Spain through theTransparency Agreement, this approach has been codi-fied into national agreements. The presentation willevaluate the experience in these countries, outlineplans for how this can work across the EU and makethe case why the scientific community needs to talkabout animal research.

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Signing the Basel Declaration forTransparency in animal research: apersonal responsibility and a profes-sional duty

Nuno H. Franco1,2,3

1IBMC – Instituto de Biologia Molecular e Celular;2i3S – Instituto de Investigacao e Inovacao em Saude;3SPCAL – Sociedade Portuguesa de Ciencias deAnimais de Laboratorio

AbstractIn November 2010, in response to acts of violence byextremist animal rights groups, more than 80 topscientists from across Europe joined in Basel todraft and sign the ‘‘Basel Declaration’’ on animalresearch, marking an unprecedented effort fromthe scientific community towards ‘‘more trust, trans-parency and communication on the sensitive topic ofanimals in research’’. In October 2011, the BaselDeclaration Society (BDS) was founded, to strengthenpublic awareness of the importance of animal modelsin experimental biomedical research, to foster com-munication between researchers and the public, andto further promote the Basel Declaration and itsgoals and values within the scientific community. Asof June 2018, more than 4.600 scientists (only 0.6% ofthem Portuguese), along with 58 scientific institutesand associations from across the world, havedeclared their support of the Declaration, and theirpersonal commitment to transparency and animalwelfare in biomedical research. Furthermore,almost 200 researchers are Basel Declarationambassadors in their countries and institutions.

Aside promoting transparency and campaigning infavour of – relevant, competent, and ethical – animalresearch, the BDS is involved in several other activ-ities, including media training events, grantingawards, carrying out surveys, among other. In add-ition, since the first 2010 meeting in Basel, four inter-national conferences have so far taken place – inBerlin, London, Rome, and latest in San Francisco,in February 2018 – where scientists and other spe-cialists gather to discuss progress in transparencyon animal research, as well as draft, discuss andissue policy documents. These documents provideguidance on various topics, including animal welfareand the 3Rs, ethics, legislation, synthesis of evidence,publication standards, risk assessment, crisis man-agement, use of primates, training, among others.

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While committed to bridge the information gapconcerning why and how animals are used inresearch, the BDS aims go beyond promoting betterpublic understanding of science, public relations orpolitical lobbying. It is essentially a movement of sci-entists for scientists, in which every signatory per-sonally commits to the highest standards ofintegrity, transparency, and the ethical treatment ofanimals. In this talk, I will review the main activitiesof this organization, and invite all to join us and signthe Basel Declaration.

10:45–12:15

Session 2: Quality and Animal Welfare in LaboratoryAnimals

Chairpersons:

Luıs Antunes

Nuno H. Franco

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Introducing Therioepistemology: Thestudy of how knowledge is gained fromanimal research

Joseph GarnerStanford University, Department of ComparativeMedicine, and by courtesy, Department of Psychiatryand Behavioral Sciences, Stanford, California, USA.

AbstractFor the first time, the scale of the reproducibility andtranslatability crisis is widely understood beyond thesmall number of researchers who have been study-ing it and the pharmaceutical and biotech companieswho have been living it. This talk will make the casethat this is not a time for despair, but a time of intel-lectual excitement and hope. The emerging literatureon these issues contains recurring themes whichrepresent a paradigm shift, and thus potentially thebirth of a new discipline (which we have proposed betermed ‘‘therioepistemology’’). At the micro level thisis a shift from asking ‘‘what have we controlled for inthis model?’’ to asking ‘‘what have we chosen toignore in this model, and at what cost?’’ At themacro level, it is a shift from viewing animals astools or reagents, to viewing them as patients in anequivalent human medical study. Thanks to thegroundwork laid over the last 15 years by a varietyof authors, we not only understand many of thecauses of poor reproducibility and translatability,but we also know the features of animal work that

lead to reproducible work with a good chance oftranslation. Accordingly, now is a time for actionwhere we manifest change in the status quo of howanimal research is done. This talk will outline sixquestions that serve as a heuristic for critically eval-uating animal-based biomedical research for oppor-tunities to make this shift in perspective.

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Industry Initiatives – Putting animalwelfare and 3Rs principles into action

Kirsty ReidEuropean Federation of Pharmaceutical Industriesand Associations (EFPIA)

AbstractAt EFPIA (European Federation of PharmaceuticalIndustries and Associations) there is a foreseenimportance of promoting good science and animalwelfare, as well as increasing understanding of howthe two are intertwined.

Medical research continues apace. It needs to, asmany health challenges still remain unmet. Animalstudies continue to play an invaluable role in meetingthese challenges – both in experimental research,and in ensuring maximum safety of treatmentsbefore their use in humans, which is legally required.However, research involving animals poses manydilemmas.

Although pharmaceutical companies cannot avoidthe use of laboratory animals to prove that medicineswork, it is our priority to meet high standards ofanimal welfare. To put animal welfare principlesinto action, we systematically replace animals withalternative methods where possible, reduce andrefine the use of laboratory animals (3Rs) andimprove standards of care. The presentation willgive an overview of the work we do to go beyondcompliance, lead by example and openlycommunicate.

Advances in science are leading to fewer tests andexperiments on animals, and to new ways to reducethe impact on animals. This is why dialogue andtransparency about the use of animals for medicalresearch and developments in science need to bedebated by everyone involved.

The pharmaceutical sector continues to beinvolved in a number of initiatives, which affirm thekey principles of the 3Rs or change the currentresearch paradigm (such as the InnovativeMedicines Initiative). Establishing, promoting and

8 Laboratory Animals 52(2S)

maintaining a good culture of care is also a funda-mental component if ethical, scientific and animalwelfare obligations. EFPIA members have produceda ‘‘checklist’’ to help engage in or enhance discus-sions on a Culture of Care within establishments.

EFPIA is part of the IMI which is the world’s largestpublic-private partnership in health with a totalbudget of E5 billion – half from the European Unionand half from the pharmaceutical industry, throughEFPIA. The public-private consortia develop newsolutions and validate them in R&D practice duringproject lifetime. This has led to the development ofin vitro and in silico methods; elimination of poorlypredictive models; development of new improvedmodels and development of alternative tools.

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Pinworms and mites in laboratory mice:deal with uninvited guests

Alice Miranda1,2, Daniela Goncalves1,2,Susana Santos1,2, Celina Barros1,2,Claudia Pereira1,2, Agostinha Costa1,2,Denise Silva1,2, Magda Castelhano-Carlos1,2 andMargarida Correia-Neves1,2

1Life and Health Science Research Institute (ICVS/3Bs), University of Minho, Braga Portugal;2ICVS/3B’s – PT Government Associate Laboratory,Braga/Guimaraes, PortugalE-mail: [email protected]

AbstractPinworm and fur mite infections can lead to signifi-cant negative effects on research and animal wel-fare. The most common found in laboratory rodentsare oxyurid nematodes of the genera Syphacia andAspiculuris, and mites of the species Myobia mus-culi, Myocoptes musculinus and Radfordia affinis.Despite the efficient treatment strategies describedin the literature, the persistence of pinworm andmite infections in rodent facilities indicates thatother factors are contributing to the failure of theeradication processes such as: environmental eggcontamination, longevity of egg viability and ineffect-iveness of traditional methods of sanitation. In thiswork we present a multidisciplinary approach forthe eradication of pinworm and mites in a rodentfacility, which includes (i) animal treatment, (ii)animal transfer and (iii) sanitation measures.

Pinworm and mite infection was detected routinelywithin our rodent facility with approximately 1100mouse 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 medi-cated diet with Fenbendazole (150 ppm) in anad libitum regime for 10 weeks. Two topicalselamectin administrations (10 mg/kg) wereapplied, 30 days apart from each other;

– Sanitation measures: Environmental decon-tamination was performed in facility B toreceive animals under treatment and includeddeep disinfection of surfaces with compoundscontaining 1% hydrogen-peroxide. Room disin-fection was completed after 3% hydrogen-per-oxide fumigation of the rooms, ventilationfilters and ducts. All the materials in contactwith animals were autoclaved before enteringthe facility;

– Animal transfer: Two weeks after fenbendazoletreatment, and after the first selamectinadministration, animals were transferred tofacility B where they were submitted to therest of fenbendazole treatment period and tothe second selamectin administration;

– In house and external testing: In house perianalcellophane tape testing and fecal flotationswere performed weekly from week 4 to 6.Fecal and body-swab pools were sent to twodifferent diagnostic companies for PCR analysison week 8 and 10 of treatment. The progeny ofthe animals submitted to the described treat-ment were tested quarterly for the followingtwo years;

In house testing were negative. The two externaltesting have shown similar negative results for bothindependent diagnostic companies. The quarterlyanalysis performed during the following two yearswas kept negative for pinworms and mites andremained negative until today.

The 10-week treatment with fenbendazole medi-cated diet and selamectin topic treatment, togetherwith animal transfer combined with environmentaldecontamination with hydrogen peroxide compoundsseems to be and efficient strategy for pinworm andmite eradication.

Acknowledgements

FEDER through the Operational ProgrammeCompetitiveness Factors – COMPETE and National Fundsthrough the Foundation for Science and Technology (FCT)

under the project POCI-01-0145-FEDER-007038; NORTE-01-0145-FEDER-000013, supported by Norte PortugalRegional Operational Programme (NORTE 2020), underthe PORTUGAL 2020 Partnership Agreement, through the

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European Regional Development Fund (ERDF); FCT doc-toral fellowship to A. Miranda (SFRH/BD/52059/2012).

14:00–15:30

Session 3: Managing Resources and Facilities

Chairpersons:

Isabel Campos

Magda Castelhano-Carlos

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How Lean Management can SupportEfficiency of Laboratory Animal FacilitiesOperations

Alberto Gobbi1,2

1COGENTECH S.c.a.r.l.,;2Fondazione Istituto FIRC di Oncologia Molecolare(IFOM)

AbstractModern laboratory animal facilities aim to providehigh standards of animal care and scientific supportwithin a framework of regulatory compliance andwith a strong focus on efficiency of operations.Meeting these objectives with limited resources is achallenge for facility managers and requires theapplication of a range of traditional and innovativemanagement techniques. Lean production is a man-agement philosophy derived from the ToyotaProduction System, which considers the expenditureof resources for any goal, other than the creation ofvalue for the end customer, to be wasteful, and thus atarget for elimination. Lean has been successfullyapplied to many activities of laboratory animal facil-ities from the design phase to the operation of animalcare programs. The authors manage a 600 m2 mousefacility housing around 6,000 ventilated cages. Overthe years, several upgrades (ranging from the intro-duction of dirty side automation systems, to optimiza-tion of the logistics) were implemented in order toincrease the efficiency of the operations. In order tofurther improve, the main animal care activities werere-evaluated and reorganized using a Lean approachduring the summer of 2011. The initial transform-ation was carried out with the support of a team ofconsultants and involved mainly cages and bottlesprocessing, both in the animal rooms and in thewashing area. Activities were balanced and newworkflows defined together with new process layoutsand time schedules. Lean was subsequently spreadalso to the areas not involved in the initial transform-ation, such as daily cages and health checks, breed-ing activities, etc. The results of the application of

Lean management were striking, both initially andover the seven years of continuous implementation,and will be discussed together with the challengesencountered during the process.

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Navigating in a facility with multipleaquatic species

Ana Cristina BorgesInstituto Gulbenkian de Ciencia (IGC)

AbstractFrom a historical perspective, aquatic animal modelshave greatly contributed to experimental biology,embryology and stem cells. Both invertebrate andvertebrate aquatic species have been used sincethe 18th century to uncover basic aspects of biology.From these, we highlight, the amphibian model, thefrog Xenopus laevis, as a classical model to studyembryology, cell biology and biochemistry. TheZebrafish (Danio rerio), which has emerged in the1990s, as a prominent fish model for studying embry-ology, organ regeneration, human disease, and toxi-cology amongst other areas of biology. Its popularityis rooted to well-described characteristics, such ashigh fertility, transparent embryos, short generationtime, small sized adult, evolutionary genetic conser-vation and genetic tractability. More recently, anotherfish species, the African turquoise killifish(Nothobranchius furzeri) has joined the group offish species used in biomedical research, due to itsshort lifespan, making it an excellent model to studyaging and associated diseases.

Research conducted at the Instituto Gulbenkian deCiencia (IGC), an institution with a long tradition ofanimal-based research, integrates not only rodentsand flies but also the above cited aquatic modelorganisms. Structured as a core facility to providehousing and services to the research community,the IGC Animal House Facility, has grown, adaptedand diversified strategies to progressively incorpor-ate zebrafish, killifish and the frog. However, the co-existence of multiple aquatic species in a vivariumposes challenges at many levels, namely biosafetyand health control, facility design, and specializedhuman resources. Moreover, the ever-increasingcomplexity of scientific projects prompts us to con-tinuously develop solutions to cope with researchneeds and animal welfare.


In this talk I will describe the strategies we havebeen developing over the years to overcome thesechallenges.

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CONGENTO: Supporting science througha synergy of wills and skills

Joao CruzConsortium for Genetically Tractable Organisms(CONGENTO)

AbstractCongento (Consortium for Genetically TractableOrganisms) was born as a response to the need ofan organized infrastructure to support research ingenetically modified animals in Portugal.Congregating the efforts of four Portuguese non-profit research institutions – ChampalimaudFoundation (CF), Gulbenkian Institute of Science(IGC), Molecular Medicine Institute (IMM), andChronic Diseases Research Center (CEDOC) –Congento gathers and integrates the expertise inDrosophila, zebrafish and mice in one research sup-porting facility. The aim is to provide state-of-the-artservices in the three models in three main domains:1) Maintenance and stocking of genetically modifiedlines (both live or cryopreserved), 2) Generation ofnew lines and development of new technologies, 3)Knowledge transfer and continuous education andcertification in animal research and technology. Bysynergizing skills and resources across institutionsand model organisms, Congento aspires to representan added value to scientific research. Such an endea-vor involves many stakeholders at very differentlevels and brings up new challenges frequently,requiring dynamic organization, effective communi-cation and constant adaptation.

15:30–16:30

Laboratory Animals Associations – Past, Presentand Future

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SPCAL: a leap into the future

Ricardo A. Afonso1,2,3

1Portuguese Society of Sciences in LaboratoryAnimals (SPCAL);

2Nova Medical School j Faculdade de CienciasMedicas, Universidade Nova de Lisboa;3Department of Physics, Faculty of Sciences andTechnology, Universidade Nova de Lisboa, Monte daCaparica, Portugal

AbstractThe first and main mission of the Portuguese Societyof Sciences in Laboratory Animals (SPCAL) is to pro-mote the implementation of the highest ethical andbehavioral standards in the use of laboratory animalsfor scientific purposes. This has been done throughinterdisciplinary approaches and cooperation amongprofessionals from the different Institutions, havingin mind the animal’s health and welfare, as well asthe best scientific results.

Since its first General Assembly, in 2004, SPCALhas evolved from a well-conceived idea into anactive, trusted and recognized partner ofResearchers and Scientific Institutions, helpingthem to achieve the best practices concerning theuse of laboratory animals. Indeed, over the years,SPCAL has established solid collaborations with anincreasingly higher number of Research andAcademic Institutions, to provide them the bestEducation and Training possible. From one singleInstitution in 2005, SPCAL is currently collaboratingin regularly basis with Scientific Institutions spreadthroughout 2/3 of the country, providing Theoreticaland/or Practical Courses to which ECTS credits havebeen attributed by several Academic Institutions.

Abroad, either as an Association or through itsAssociates individually, SPCAL has gained recogni-tion among peer European Associations. Investmentwas made in a pro-active participation in theFederation of European LAS Associations (FELASA),with several SPCAL affiliates nominated for FELASAWorking Groups.

More recently, SPCAL and its associates have beenfacing different challenges. The fact that we live in aglobal society, with easy access to information and anincreasingly higher conscience, makes it impossiblefor any Scientific Association to neglect the public.Such public awareness and demand has drivenSPCAL Board to take a different approach. Anapproach towards clarification and transparency, inclose cooperation with both the CompetentAuthorities and civil representatives, who acknowledgeSPCAL as a representative of Researchers inLaboratory Animals Sciences. Having such need forcommunication between Scientists and public inmind, we feel that the time has arrived for a higherdegree of transparency in the field of LaboratoryAnimals Sciences.

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SPCAL’s missions haven’t and will not change. Theonly thing that will change is the way those missionswill be pursued in the future. Which leads us to the IVSPCAL Congress – "Quality and Transparency inScience involving laboratory animals".

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FELASA today and tomorrow – how canyou contribute?

Ana Isabel and M. Santos1,2

1Federation of European Laboratory AnimalsAssociations (FELASA);2Nova Medical School j Faculdade de CienciasMedicas, Universidade Nova de Lisboa.

AbstractThe Federation of European Laboratory AnimalSciences Associations – FELASA – is for themoment composed of 21 constituent associationsrepresenting 27 countries. FELASA was establishedin 1978 to represent common interests in the further-ance of all aspects of laboratory Animal Sciences(LAS) in European and beyond. Membership is opento LAS Associations representing nations. FELASArepresents common interests of constituent associ-ations in the furtherance of all aspects of laboratoryanimal science in Europe. Laboratory animal sciencebeing that discipline whose objective is to ensureoptimal conditions for the humane and appropriateuse of animals for scientific purposes for the benefitof mankind and other animals, and to promote fur-ther development of those conditions. The boardshould work for:

– Advance and co-ordinate the development of allaspects of laboratory animal science and practicein Europe.

– Act as a focus for the exchange of informationon laboratory animal science amongstEuropean states.

– Establish and maintain appropriate links withnational, international or governmental bodiesas well as other organisations concerned withlaboratory animal science.

– Promote the recognition and consultation ofFELASA as the specialist European body onlaboratory animal science and welfare.Organise joint scientific meetings of the con-stituent associations.

– Training and Education accreditation.

Along the last 40th years there is a long list ofachievements to share and a door open for new activ-ities. FELASA establishes FELASA working groupsare established around current issues of lab animalscience with experts in the field nominated by thedifferent member Associations. The working groupsshould review available information and issue guide-lines or recommendations promoting animals’ healthand wellbeing.

OCi10

Keeping up with Portuguese AnimalWelfare Bodies and the RedeORBEAnetwork

Nuno H. Franco1,2,3

1IBMC – Instituto de Biologia Molecular e Celular;2i3S – Instituto de Investigacao e Inovacao em Saude;3SPCAL – Sociedade Portuguesa de Ciencias deAnimais de Laboratorio

AbstractAnimal Welfare Bodies (AWB) – ‘Orgao Responsavelpelo Bem-Estar dos Animais’ (ORBEA) – were firstset up in Portugal following the enactment of the2010/63/EU Directive, transposed by Decreto-Lei113/2013. The Directive sets as central obligationsfor AWBs: to advise the staff on animal welfare andthe 3Rs, to establish and review operational pro-cesses for monitoring and follow-up of animal wel-fare, to follow the development and outcome ofprojects taking into account the effect on the animalsused, and to identify opportunities to further the 3RS.In addition, Portuguese legislation confers to AWBsan additional responsibility: evaluating projects andissuing non-binding appraisals for project licencerequests to the Competent Authority (CA).

Establishing AWBs raised significant challengesfor ORBEAS at institutes and universities, not onlyin meeting the demanding regulatory requirementsset for them, but also in establishing their identity,place and responsibilities in institutions, as well asasserting their authority while establishing relation-ships of mutual trust and respect with researchers.However, it was also an opportunity for raising stand-ards of competence and functionality in planning,reviewing, and supervising animal research proced-ures, furthering a culture of care.

With little guidance from the CA – and without theinput from a National Committee for the Protection ofAnimals used for Scientific Purposes (established in


Portugal in 2016 but still not yet active) – it was up tolaboratory animal scientists, veterinarians, and tech-nicians to organize themselves, and find means ofacquiring and exchanging reliable information,asking questions, and sharing experiences. SPCALwas pivotal in this endeavour, by funding and sup-porting events organized by and for ORBEA mem-bers, as well as professionals wishing to set anORBEA. These included two workshops in 2014 –one at the IBMC (Porto), another at NOVA MedicalSchool (Lisbon) – and the I National Symposium forORBEAS in 2016 at the i3S (Porto), and a second edi-tion in 2017, at the Champalimaud Foundation(Lisbon). The success of these events has led threeORBEAs in Coimbra to join efforts and organize the IIINational Symposium in 2018, fulfilling the originalgoal of making it a regular, yearly event.

This talk will cover the progress in ORBEA imple-mentation in Portugal since the enactment of the2010/63/EU Directive, highlight main achievements,and report on the development of the RedeORBEA –a network of Portuguese ORBEAs – and its futurechallenges.

16:45–18:15

Session 4: Legislation: New Perspectives andUpdates

Chairpersons:

Ricardo A. Afonso

Margarida Caramona

OCi11

Shipping Live Animals – Best Practice

Antonio V. SykesCCMAR – Centro de Ciencias do Mar do Algarve,Universidade do Algarve

AbstractDespite the EU effort on funding infrastructures, withbioteriums that culture and maintain animals forresearch purposes, and also, to some extent, onfunding the dislocation of researchers to such cen-ters to perform their research in loco, there is stillthe need of shipping and transporting live regulatedanimals after collection from the wild or in betweeninstitutions.

According to the National Centre for theReplacement, Refinement and Reduction of Animalsin Research (NC3Rs, UK), ‘‘Transport can be a sig-nificant stressor that may have an impact on bothanimal welfare and research outcomes. The primary

objective for all those involved in animal transportshould be to move the animals in a manner thatdoes not jeopardise their well-being and ensurestheir safe arrival at their destination in good health,with minimal distress.’’ The Convention onInternational Trade in Endangered Species of WildFauna and Flora (CITES) have published Guidelinesfor the non-air transport of live animals and plants(which will take place up to 48 h), where they con-sider that International Air Transport Association(IATA) Live Animals Regulations (LAR) are appropri-ate for the non-air-transport of all species of ani-mals. However, for transports exceeding 48 hoursadditional requirements should be met. The trans-port of an animal constitutes an unnatural situationand is most likely to cause it some degree of stress,and eventually injuries if it is not well prepared andperformed. High levels of stress may increase meta-bolic rates, hazardous behaviour, chances of injuriesand susceptibility to diseases. Considering an animalwelfare perspective, animal transport should bequick, efficient and strive to avoid as much stressas possible to the animal. The transport of live ani-mals must be well planned, meticulously preparedand effectively performed.

In this sense, this presentation will highlight themost important and necessary steps that need tobe considered and taken when planning, preparingand shipping live animals. It will address: a)General Recommendations for Living AnimalTransportation; b) Living Organisms Classification,UN Identification number and Proper ShippingName; c) Packaging; d) Marking and Labelling; e)Documentation and Legislation; f) Arrival at newfacilities and Quarantine; and g) Traceability.

OCi12

Portuguese legislation on the use ofanimals for scientific purposes

Ana Paula MartinsDivisao de Bem-estar Animal, Direcao Geral deAlimentacao e Veterinaria

AbstractThe Portuguese legislation regarding the protectionof animals used for scientific purposes, the Decreto-Lei n� 113/2013, of 7th August, is derived fromDirective 2010/63/EU, of 22ndSeptember, and thus,this presentation aims to present what are the mainrequirements, innovations and the activities that havebeen held in order to implement it.

Abstracts 13

OCi13

Comissao Nacional para a Protecao dosAnimais Utilizados para Fins Cientıficos(CPAFC) – funcoes e objetivos

Yolanda Vaz1,2

1Comissao Nacional para a Protecao dos AnimaisUtilizados para Fins Cientıficos (CPAFC); and2Servicos de Protecao Animal, Direcao Geral deAlimentacao e Veterinaria (DGAV)

AbstractA Portaria n.� 260/2016, de 6 de outubro, fixou a com-posicao e o funcionamento da Comissao Nacionalpara a Protecao dos Animais Utilizados para FinsCientıficos (CPAFC), tal como preconizado no n.� 4do artigo 55.� do Decreto-Lei n.� 113/2013, de 7 deagosto, que transpos para a ordem jurıdica nacional aDiretiva n.� 2010/63/UE, do Parlamento Europeu e doConselho, de 22 de setembro de 2010, relativa a pro-tecao dos animais utilizados para fins cientıficos.

O Despacho n.� 673/2018, de 15 de janeiro, doGabinete do Secretario de Estado da Agricultura eAlimentacao, por seu lado, designou os elementosque integram a CPAFC, assim como o seu secretar-iado, a qual teve a sua primeira reuniao na Sede daDGAV, a 16 de fevereiro de 2018.

A CPAFC tem funcoes de aconselhamento daDirecao Geral de Alimentacao e Veterinaria e dosOrgaos Responsaveis pelo Bem-estar dos Animaisdas instituicoes que pratiquem experimentacaoanimal, de acordo com o constante no n.� 2, doartigo n.� 55, do Decreto-Lei n.� 113/2013, de 7 deagosto.

A CPAFC propoe-se desenvolver, a curto prazo,trabalho de orientacao sobre os seguintes assuntos:

– Reforco de competencias das pessoas queexecutam determinadas funcoes no ambito deaplicacao do Decreto-Lei n.� 113/2013, de 7 deagosto, nomeadamente, em alinhamento comum programa de supervisao ate demonstracaode competencia, de acordo com o disposto noartigo n.� 31, do Decreto-Lei;

– Clarificacao sobre as responsabilidadesatribuıdas a cada pessoa envolvida na criacao,fornecimento e/ou utilizacao de animais execu-tando qualquer uma das funcoes espelhadas noDecreto-Lei n.� 113/2013;

– Promocao de metodos alternativos a experi-mentacao animal;

– Integracao na Rede das Comissoes Nacionaisdos Estados Membros da Uniao Europeia para

troca de experiencias e entendimentos relati-vos a Protecao dos Animais Utilizados paraFins Cientıficos.

OCi14

Researchers and regulation incentive forimprovement or unreasonableinterference?

Anna S. Olsson1,2

1i3S – Instituto de Investigacao e Inovacao em Saude,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 andTraining Accreditation

Ana Isabel and M. Santos1,2

1Federation of European Laboratory AnimalsAssociations (FELASA);2Nova Medical School j Faculdade de CienciasMedicas, Universidade Nova de Lisboa

AbstractAn accreditation system for Education and Training(E&T) has become recognized as a robust way ofimproving training and at the same time that estab-lishes a golden standard for LAS education and train-ing. Quality assurance aims to assist in thedevelopment of high quality educational programmesin laboratory animal Science throughout Europe andinternationally. Another strong objective is to help onthe harmonization of the training programs to helpwith the individual mobility at the global level.

The Federation of Laboratory Animal ScienceAssociations (FELASA) accreditation aims to imple-ment best practices for both high quality science andimproved animal welfare by recognizing, supportingand enhancing the quality of training; establishes a


more uniform platform of competence of thosetrained, enabling greater mobility of researchersand animal care staff; enables the identification andsharing of good practice; and provides independentreassurance for National Authorities and the publicabout the competence of those working with labora-tory animals.

The process has evolved and adapted over theyears to provide an independent reassurance forNational Authorities and the public about the compe-tence of those working with laboratory animals. Anadded value of accreditation is that it facilitates themobility of scientists that are well educated andtrained in Laboratory Animal Science.

Although the FELAAS accreditation incurs in somefinancial and time commitment for course organisersthe impact of the FELASA Accreditation scheme inthe scientific community is a proof of success of thescheme.

OCi16

Continuing Professional Development inLaboratory Animal Science in Spain

Maria Jesus Molina CimadevilaInstituto de Neurociencias, Universitas MiguelHernandez

AbstractIn accordance with European regulations for labora-tory animals, Spain has developed its own normativein training topics with these species. The OrdenMinisterial ECC/566/2015 regulates both initial andcontinued education with laboratory animals in ourcountry. However, while initial education has beenconsolidated for several years, continued educationis brand new at the moment. We will go through sev-eral approaches adopted by different institutions andcompanies regarding this issue. However the dead-line for evaluations for the autorities has not arrivedyet, and many questions arrise in the comunity aboutits final application. Althougth the regulation isunique for Spain, the application complicates by theorganizational structure of Spain in autonomouscommunities, whose criteria in the interpretation ofthe normative is not always homogeneus. SECALswill is to serve as a forum to facilitate the comunica-tion between the scientific comunity and autoritiesfor a smoth and usefull application of continuouseducation in our country.

OCs2

The competence for trainingcompetencies

Andreia Costa1,2, Maria S. Gomes1,2,3, Manuel J.Costa4, I. Anna S. Olsson1,2

1i3S – Instituto de Investigacao e Inovacao em Saude,Universidade do Porto;2IBMC – Instituto de Biologia Molecular e Celular,Universidade do Porto;3ICBAS – Instituto de Ciencias Biomedicas AbelSalazar, Universidade do Porto;4Life and Health Sciences Research Institute (ICVS),School of Health Sciences, University of Minho

AbstractThe terms competence/competent are used in bothDirective 2010/63/EU and Portuguese national legis-lation (Decreto-Lei n.� 113/2013) in relation to per-sons carrying out procedures with animals. In theeducational sciences, the construct ‘‘competence’’is generically defined as the ability to perform some-thing successfully or efficiently, assuming the indi-vidual possesses the skills or abilities needed toperform certain task. In addition, a skill is definedas a person’s expertise or proficiency. In a reviewin clinical practical context, Epstein and Hundert(2002) stated that professional competence inte-grates several dimensions not only related with cog-nitive and technical domains but also affective and‘‘habits of mind’’. Unlike factual knowledge whichcan be acquired in a concentrated effort in a shorttime, long-term training seems to be the most effect-ive way to develop skills and competencies.

Training for persons carrying out procedures withanimals is traditionally organized as courses.Whereas no format was prescribed in the FELASArecommendations, courses are time-concentratedinto one (Cat B) or two (Cat C) weeks. This formathas several advantages: it allows students toimmerse in the topic, it makes it easier for organ-isers to bring together a faculty of expert teachersand it is especially convenient when the course is nottaken in the students’ own institution. However, theneed to deliver all the theoretical content to accom-plish defined learning outcomes in a short time setsconstrains for the acquisition of competence.Practical training is usually limited to proceduredemonstration and replication by students.Competence evaluation is usually assessed, at thelast course day, through a set of theoretical ques-tions, less than two weeks after the start of training.

Abstracts 15

However, it is unlikely that students will be able togain necessary competence in such a short time andwith so exhaustive curricula. This imposes a substan-tial challenge to course organizers and instructors,who have the duty of attesting competence, in par-ticular as regards the need to practice and learn toexecute procedures in living animals. Individual trai-nee’s own progress time must be respected toensure safe and efficient training.

With this presentation, we aim to raise the discus-sion of the dilemma between course format and com-petence development in laboratory animal science.How can course organizers achieve a satisfying solu-tion for caring out demanding and time-concentratedcourses, while guaranteeing the acquisition of pro-fessional competencies, which take time to developand consolidate?

10:45–12:30

Session 6: Animals and Models: BiologicalPerspectives

Chairpersons:

Isabel Vitoria Figueiredo

Ricardo A. Afonso

OCi17

Cephalopods: Historical perspective andcurrent use

Antonio V. SykesCentro de Ciencias do Mar do Algarve (CCMAR),Universidade do Algarve

AbstractAre cephalopods not from Earth? A recent study pub-lished by Steele et al. (2018) connects octopuses andremaining cephalopods to the theory of panspermia.But, despite cephalopods might be seen as very‘‘weird’’ invertebrates this is not what makes themexcellent animal models for research and we willrevise why. Cephalopods are a class of invertebrateswith over 700 species. Cephalopods have been usedas experimental models since the beginning of the20th century in vast research areas, such as neuro-biology (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 andMedicine for their work on the behaviour of nerve

impulses, by using the giant axon of the Atlanticsquid Loligo pealei, the use of cephalopods asanimal models for research gained momentum.Some years later, two other researchers used squidas a model in their research and became NobelLaureates: the American G. Wald, who was honouredin 1967 for his research on chemical and physio-logical processes in the eye, and Bernard Katz ofGreat Britain, won the prize in 1970 for his discov-eries concerning the role played by chemicals innerve impulses. From that point on, cephalopodsbecame the most relevant neurological model, withno peer in the animal kingdom (Young, 1985).

A lot has happened since then, with cephalopodsbecoming animal models for emergent researchfields, such as mechatronics (Calisti & Laschi, 2017;Kim et al. 2013), evolution (Budelmann, 1995) and,more recently, climate change (Portner & Farrell,2008).

Despite the report of the first genome of a ceph-alopod only occurred in 2015 (Albertin et al., 2015), arecent paper in Cell (Liscovitch-Brauer et al., 2017) isshowing that cephalopods adapt to changing watertemperatures by altering their RNA more often thantheir DNA, which can be seen as proof that when theNobel Prize–winning biologist Sydney Brenner saidthat octopi were the first intelligent beings onEarth, he was right.

References

1. Albertin et al. (2015). doi: 10.1038/nature14668.

2. Breidbach O and Kutsuch W (eds) Budelmann. The ceph-

alopod nervous system: what evolutions has made of the

molluscan design. The Nervous System of Invertebrates.

An Evolutionary and Comparative Approach. Basel:

Birkhauser Verlag, 1995, pp.115–138.

3. Calisti & Laschi (2017). doi: 10.1142/

9789813149137_0088.

4. Gherardi et al. (2012). doi: 10.1007/s10071-012-0513-y.5. Hanlon and Messenger. Cephalopod Behaviour. New

York: Cambridge University Press, 1996.6. Kim et al. (2013). doi: 10.1016/j.tibtech.2013.03.002.7. Liscovitch-Brauer et al. (2017). doi: 10.1016/

j.cell.2017.03.025.8. Portner & Farrell (2008). doi: 10.1126/science.1163156.

9. Sio (2011). doi:10.1017/S0025727300005421.10. Steele et al. (2018). doi: 10.1016/

j.pbiomolbio.2018.03.004.11. Tricarico et al. (2011). doi: 10.1371/

journal.pone.0018710.12. Wells (1962). Brain and behaviour in cephalopods.

Heinemann, 171.13. Williamson and Chrachri. Cephalopod neural networks.

Neurosignals 2004; 13(1–2): 87–98.


14. Young. The functioning of the giant nerve fibres of thesquid. Journal of Experimental Biology 1938; 15(2):170–185.

15. Young. Cephalopods and Neuroscience. BiologicalBulletin 1985; 168(3): 153–158.

OCi18

Animal experimentation: examples ofrational approaches towards drugdevelopment

Catarina Pinto-ReisFaculty of Pharmacy, University of Lisboa,ImedULisboa/IBEB, Lisboa, Portugal

AbstractDespite large investments in drug development, theoverall success rate of drugs during clinical develop-ment remains relatively low. A possible explanationmay be related to the weakness of the preclinicalresearch. Indeed, animal experimentation is pivotalto bridge the translational gap to the clinic. Whencarefully selected, designed and conducted, animalmodels are an important part of any translationaldrug development strategy and intimately linked toadvances in scientific knowledge and technology. Theselection of a valid, adequate and predictive animalmodel is essential to address this question.

In this communication two different approacheswill be presented; one as therapeutic drug andanother as medical device.

Designing a new therapeutic drug is a lengthy andcomplex process. Those decisions on which drug orwhat respective formulation should proceed to thenext stage are taken using a wide range of data.Experiments using animal models are essential tounderstand how a drug will work. In general, a candi-date drug is initially tested in cell lines and only after-wards the studies move into animal subjects. If apotential drug appears to be effective in animals, andpasses initial safety screening, then it can be studied inhuman trials. In this communication, severalapproaches of an oral insulin formulation usinganimal models will be presented and discussed.

In the case of medical devices, the general focus ofanimal experimentation is on the device’s ability tofunction with living tissue without harming. Untilnow, most of the medical devices use biocompatiblematerials, such as gold nanoparticles. Again, animalmodels are critical to assess the safety of a newapproach in the treatment of skin cancer.

Both examples show that animal experimentationis crucial to pharmaceutical industry researchers todecide whether a potential therapeutic drug will beeffective and safe for use in humans. Furthermore,from the examples presented herein, we also con-cluded that the translational value of animals’models can be further enhanced when combinedwith other translational tools, which simultaneouslyallows us to decrease the number of animalsrequired. Nevertheless, all strategies will haveundeniably improved patient access to more effectiveand safer new drug therapies.

OCi19

Are we entering a new Era in genomeediting?

Ana NovoaInstituto Gulbenkian de Ciencia. Electronic address:[email protected].

AbstractThe CRISPR/Cas9 genome editing system is having atremendous impact on transgenic technology now-adays. This novel technology derives from the adap-tive immune system of prokaryotes, that was recentlyadapted to allow any kind of genetic modification,from basic gene inactivation to subtle edits, likepoint mutations, insertion of large cassettes oreven do gene replacements, via gene targeting, in awide variety of species and cell lines, with the onlyrestriction essentially being our imagination. Thisinnovative approach both democratized genomic edit-ing and, very rapidly, overcame the laborious, moreexpensive and time-consuming embryonic stem-cell-based recombineering systems. In my talk I will dis-cuss the use of CRISPR/Cas9 genome editing systemin the wider context of the in vivo transgenic technol-ogies used to modify the mouse genome: mouseoocyte pronuclear microinjection of transgenic con-structs, BACs, YACs, zinc-finger and transcriptionactivator-like effector nucleases, and also the micro-injection of targeted ES-cells on blastocysts. Finally,a brief discussion on the impact that this novelmouse genome-engineering tool has on the 3 R0s(Replacement, Reduction and Refinement) principle.

Abstracts 17

OCi20

Implementation of a mouse GnotobiologyFacility to host microbiota-relatedstudies

Ana Sofia LeocadioInstituto Gulbenkian de Ciencia (IGC)

AbstractThe gnotobiology research has exponentially grownin the last few years due to the important role ofhost–microbiota interactions in the study of severalmetabolic and autoimmune diseases. The use ofaxenic mice, which are rendered free of microorgan-isms, is a straightforward approach to investigate theoverall contribution of the microbiota in suchinteractions.

In 2005, the Instituto Gulbenkian de Ciencia (IGC)developed a dedicated platform for axenization of dif-ferent mouse strains reared, maintained andmanipulated in multi-cage isolators. This AxenicFacility is highly well established, with an annualcapacity of 400 axenic mice being produced for inter-nal requests and for the international community, inthe frame of the Infrafrontier-EMMA EU consortium.

Isolators represent a good strategy for axenic miceproduction at medium/large scale, however, subse-quent experimentation in this system is suboptimalfor gnotobiology research, often requiring diversifiedgenetically modified animals with multiple and par-allel experimental conditions. In 2013, the increaseddemand of gnotobiology experiments by theresearchers at IGC led us to develop a GnotobiologyFacility equipped with racks of Individually-VentilatedCages (IVC) under positive pressure, namely theISOcage P system. This system is designed to achievethe technical features of an isolator, allying the ergo-nomic, flexibility and density advantages of an IVC.Each cage acts as a micro-isolator, allowing multiplestudies in the same rack, excluding cage to cage con-tamination. This setting is completed with an ISOcageBiosafety Station (IBS), specifically conceived forsuitable manipulation of mice in a fully sterilizedenvironment.

Currently, the Gnotobiology Facility hosts allexperiments performed in axenic mice, accommo-dated in this ISOcage P IVC racks, set in a roomaccessible to trained researchers, while the AxenicFacility is exclusively dedicated to the production ofaxenic mice, and axenization of new strains, allocatedin multi-cage isolators in a room only accessible bythe facility personnel.

Since the implementation of the Gnotobiology/Axenic Facility, more than 250 different gnotobiologyexperiments were performed, with animals beingsubmitted to various procedures, from gavage andregular feces collection to sophisticated surgeries.To validate this working system, standard operatingprocedures (SOPs) and an axenic sentinel pro-gramme were applied and gradually optimized. Thedata relative to the microbiological status along timedemonstrates a significant decrease of contaminatedexperiments. Also, sentinels have been maintainingtheir axenic status for more than 18 months, indicat-ing this setup reveals to be efficient, optimizinghuman, financial and space resources.

Acknowledgements

We thank to the Animal Facility team and to all users of theGnotobiology/Axenic Facility, especially to JessicaThompson and Joana Dias for their contribution to theearly trials.

The content of this work also gave rise to aCommunication in the form of poster.

OCs3

Chitosan/b-glucan particles for hepatitisB surface antigen vaccination: prophy-lactic and therapeutic value

Edna Soares1,2, Sandra Jesus1,Rosemeyre Cordeiro1, Anthonie Groothuismink3,Andre Boonstra3, Henrique Faneca1 andOlga Borges2

1Center for Neuroscience and Cell Biology, Universityof Coimbra, Portugal;2Faculty of Pharmacy, University of Coimbra,Portugal;3Department of Gastroenterology and Hepatology,Erasmus MC Rotterdam, Netherlands

AbstractThe hepatitis B virus (HBV) killed 887 000 people in2015. The World Health Organization (WHO) set thegoal to eliminate HBV as a public health threat by2030. The major hurdles include high prevalence indeveloping countries and ineffective currently avail-able antivirals. Hence, new vaccine adjuvants can bedesigned to stimulate the chronic hepatitis Bexhausted immune system and to provide the antigenincreased stability to temperature variations, benefit-ing HBV vaccine coverage in developing countries.

Different adjuvants for the hepatitis B surface anti-gen (HBsAg) were developed and included b-glucan


particles, prepared from alkaline/acid treatment ofSaccharomyces cerevisiae, b-glucan/chitosan andchitosan particles prepared by a precipitation tech-nique. Vaccine formulations were tested in C57BL/6mice, following either three subcutaneous adminis-trations (n¼5), three oral administrations (n¼7) orone subcutaneous followed by two oral administra-tions (n¼5). At the end of the experiment each mousewas individually euthanized by cervical dislocation,away from the other animals to minimize stress,and death was confirmed by testing the absence ofboth respiratory rate and reflexes. Blood was col-lected to assess serum HBsAg-IgG; faeces and vagi-nal washings were collected to assess HBsAg-IgA;livers were collected for tissue interstitial fluid IFN-g, and spleens were collected for HBsAg restimula-tion ex vivo and HBsAg-specific cytokines detectionusing a multiplex assay. Animal studies wereapproved (ORBEA_50_2013/27092013) and carriedout in accordance with institutional ethical guidelinesand with National (Dec. No. 113/2013) andInternational (2010/63/EU Directive) legislation.

The oral vaccination approach resulted in 60 %mice seroconversion. The presence of HBsAg-speci-fic IgA on mucosal surfaces and IFN-g in the liverwere the major advantages found of this vaccinationroute. The fully subcutaneous vaccination resulted in100 % seroconversion with high serum anti-HBsAgIgG, mostly subtype IgG1 followed by IgG3. The b-glucan particles used as an adjuvant induced astrong and varied HBsAg-specific cell-mediatedimmunity observed by the secretion of cytokinesrelated with Th1, Th2, Th17, Th22 and Treg-biasedimmune responses.

The present work represents an important contri-bution to the knowledge of both b-glucan and chito-san/b-glucan particle adjuvant mechanisms, with agreat impact for antiviral immunotherapies.

12:30–13:30

Closing Lecture

Therioepistemology, a success story:mouse models of trichotillomania andcompulsive skin picking

Joseph GarnerStanford University, Department of ComparativeMedicine, and by courtesy, Department of Psychiatryand Behavioral Sciences, Stanford, California, USA.

AbstractIn this talk, I will illustrate the efficacy of therioepis-temology in the development, validation, and use ofan animal model. Trichotillomania (compulsive hairpulling) affects roughly 3.5% of women (making it the3rd most common disorder in women), yet this debil-itating disorder has been largely ignored byresearchers and clinicians alike. Hair and feather-pulling behaviors are widespread abnormal behav-iors in animals with many similarities to trichotillo-mania. This talk will survey how treating mice aspatients, allowed us to validate barbering and ulcera-tive dermatitis as models of trichotillomania andcompulsive skin-picking disorder, identify bio-markers predicting disease development and treat-ment response, elucidate disease mechanisms, andultimately identify a novel drug. Because the inter-ventions we have identified are tied to underlying bio-markers we have a rapid method for validating thesefindings in humans before investing in a drug trial –humans should show the same novel biomarkers asmice.

Finally, in parallel, by showing that ulcerativedermatitis is a behavioral condition, we have beenable to design behavioral interventions in mice thatresolve what would otherwise be life-ending lesionsin 95% of affected mice (Adams, Garner et al. 2016).As a result, we have been able to virtually eliminateulcerative dermatitis as cause of unplanned euthan-asia in our facility.

Poster Session

Friday, June 22

PS1

What is the most appropriate rat modelto study type 2 diabetes?

BF Melo1,*, JF Sacramento1,*, CS Prego1,MJ Ribeiro1, IB Martins1, MP Guarino1,2 andSV Conde1

1CEDOC, NOVA Medical School, Faculdade deCiencias Medicas, Lisboa, Portugal;2Escola Superior de Saude de Leiria – InstitutoPolitecnico de Leiria, Leiria, Portugal*both authors contributed equally for the work

AbstractObesity and Type 2 diabetes (T2D) have reached epi-demic proportions being clear that the existing thera-pies are clearly scarce to address this problem. Ratshave been widely used to mimic human diseases

Abstracts 19

aiming to understand the causes and progression ofdisease symptoms, as well as to develop new drugsand determine their mechanisms of action. With thiswork we aim to evaluate several experimental andgenetic rat models of T2D, in order to be able inthe future to select more accurately the most appro-priate T2D model given the specific researchrequirements.

Different experimental settings of Male Wistar rats(8–9 weeks old; n¼8–10 animals per experimentalgroup) were used: 1) 3 weeks of 60% lipid-rich diet(HF diet); 2) 19 weeks of HF diet; 3) 4 weeks of highsucrose diet (HSu, 35% of sucrose); 4) 16 weeks ofHSu diet; 5) 14 weeks of combined HF plus HSu diet(HFHSu); 6) 25 weeks of HFHSu diet; 7) 4 weeks HFdiet plus streptozotocin (HFSTZ, 25 mg/kg,i.p.).Additionally, a genetic model of T2D, the ZuckerDiabetic rat (ZDF), was tested at 17 and 23 weeksof age. Animals were compared with age-matchedcontrols. Caloric intake and weight were monitored.Insulin sensitivity and glucose tolerance were evalu-ated respectively through an insulin tolerance test(ITT) and oral glucose tolerance test (OGTT).Fasting glycemia, insulin and C-peptide were mea-sured. At a terminal experiment rats were anesthe-tized with pentobarbital (60 mg/kg i.p.), blood wascollected by heart puncture and tissues were col-lected, weighted and kept for further studies, as ana-lysis of the expression of proteins involved in glucosemetabolism. Laboratory care was in accordance withthe European Union Directive for Protection ofVertebrates Used for Experimental and OtherScientific Ends (2010/63/ EU). Experimental protocolswere approved by the Faculdade de Ciencias MedicasEthics Committee.

Rats fed with HSu and HFHSu diet gain less weightthan animals fed with HF diet and ZDF in comparisonwith respective controls. Insulin resistance wasachieved in all experimental settings, being higherin animals submitted to 25 weeks of HFHSu diet,except for HF animals with STZ. In contrast, glucoseintolerance was achieved in all animals, with ZDF andHFSTZ animals presenting higher levels of intoler-ance. Fasting glycemia values within the range forT2D diagnose were only obtained in ZDF animals.All animals were hyperinsulinemic in comparisonwith theirs controls, except HFSTZ animals thatwere hypoinsulinemic.

We conclude that the most appropriate rat modelto use for T2D studies depends on the pathologicalfeatures to be tested and that ideally, more than onerat model should be used to represent the diversityseen in human diabetic patients.

Acknowledgements

J.F. Sacramento and B.F. Melo were supported by the

Portuguese Foundation for Science and Technology grantsPD/BD/105890/2014 and PD/BD/128336/2017 respectively.

PS2

Parasympathetic tone Activity (PTA)evaluation to discriminate Ketorolac andKetorolac/Tramadol analgesia level inswine

C Leitao1,2, JR Lima-Rodrıguez3, F Ferreira4,5,C Avelino4,5,6, FM Sanchez-Margallo3 andL Antunes1,2,7,8

1Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB),University of Tras-os-Montes and Alto Douro (UTAD),Portugal;2Department of Veterinarian Sciences, School ofAgricultural and Veterinary Sciences School (ECAV),UTAD, Portugal;3Anesthesiology Department Co-ordinator, JesusUson Minimally Invasive Surgery Centre JUMISC,Spain;4Department of Mathematics, School of Science andTechnology (ECT), UTAD, Portugal;5Centre of Mathematics of the University of Minho –UTAD Pole (CMAT-UTAD), Portugal;6Center for Computational and StochasticMathematics (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 ofMolecular and Cellular Biology (IBMC), University ofPorto, Portugal.

AbstractEvaluation of nociceptive/anti-nociceptive balanceduring general anaesthesia is still challenging androutinely based on clinical criteria. Analgesic drugdelivered may be optimized with a parasympathetictone activity (PTA) monitor. This study comparesketorolac and ketorolac-tramadol balance analgesiausing a PTA monitor.

All procedures were approved by the MinimalInvasive Surgery Centre Jesus Uson ethical commit-tee and animal welfare body. Pain intensity responsewas assessed using a 0–100 numerical state scale(PTA) after nociceptive stimuli provide by a MayoHegar needle holder in nine Large White pigs, agedbetween 85–90 days and with 49 kg mean weight,


under stable sevoflurane twenty minutes anesthesia.Bispectral index, heart rate, non-invasive blood pres-sure and respiratory parameters were also mea-sured. Animals were divided into three groups ofthree: a) without analgesia b) ketorolac (1.5 mg/kg)and c) ketorolac/tramadol (1 mg/kg). Mean valuesor mean areas under the curve (AUC) in selectedtime periods were compared over time and betweengroups through a mixed-model repeated measuresANOVA and non-parametric Kruskal-Wallis tests, fol-lowed by Bonferroni or Dunn’s multiple comparisons.

We observed a significant decrease in the PTA AUCmean value after application of the stimulus in theanimals treated without analgesia and those withketerolac only. The PTA AUC mean value in the con-trol group was significantly lower than the corres-ponding mean in ketorolac group (p<0.5). Theketorolac/tramadol group showed the highest PTAAUC mean values, significantly different from thoseobtained for the other two groups, with no significantdifferences detected over time (p>0.5). Bispectralindex means showed no statistically significant dif-ferences neither over time nor between differenttreatment groups (p>0.5). Heart rate showed only astatistically significant higher AUC mean between thegroup without analgesia and the ketorolac/tramadolgroup (p<0.5), in the time period after the stimulusapplication, where without analgesia was higher.Non-invasive blood pressure means showed no stat-istically significant differences over time or betweentreatment groups (p>0.5).

This study shows that a low dose combination ofKetorolac and Tramadol is sufficient to block the painresponses induced with a needle holder in pigstwenty minutes after its administration. The PTAmonitor was able to clearly recognize and distinguishthe analgesic level between treatments and can thusbe 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

Costa E Duarte*1,2, R Faısca*1,2, P Reis3,CP Cabrita4,5 and S Figueiredo2,1,6

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 Farmacia,Universidade de Lisboa, Portugal;5IBEB, Faculdade de Ciencias, Universidade deLisboa, Portugal;6IBILI, Faculty of Medicine – University of Coimbra.e-mail: [email protected]

AbstractAccording to some studies, in 2015, there were 17.5million cancer cases worldwide and 8.7 milliondeaths. Between 2005 and 2015, cancer casesincreased by 33%, with population aging contributing16%, population growth 13%, and changes in age-specific rates contributing 4%. In women, the mostcommon cancer was the breast cancer. DMBA is con-sidered to be one of the etiologic factors of malignantneoplasms in humans. In research, DMBA has alsobeen used to chemically induce mammary carcino-genesis in experimental models with Sprague-Dawley rats. With this work, we pretend to charac-terize DMBA-induced breast cancer tumours.

Ten Sprague-Dawley female rats were orallyadministered with 65 mg/kg of DMBA diluted inolive oil, at 55–57 days of age. At 27 weeks after car-cinogenic induction, all animals were euthanized byoverdose of anesthetic. A full necropsy was per-formed and all tumours were excised, measuredand weighed. This work was approved by DGAV. Thelesions’ structural pattern were graduated as non-neoplastic, benign neoplastic, in situ malignant neo-plastic and invasive malignant neoplastic.Histological type was defined by the predominantpattern observed and classified accordingly to thedifferent histological types. In cases that presentedmalignant and benign lesions, the histological typegiven was the predominant on the malignant lesion.Benign lesions were considered for the total numberof patterns count. Grading of malignant lesions wasperformed applying the Nottingham Grading System.This system is based on evaluating and scoring threedistinct morphological features: degree of tubular/glandular formation, nuclear pleomorphism andmitotic index based. After scoring these features,the tumours were classified as grade 1, grade 2and grade 3.

Sixty fragments were collected and graduated in 15non-neoplastic (6 at right mammary chain and 9 atleft mammary chain), 11 benign neoplastic (5 at rightmammary chain and 6 at left mammary chain), 1 insitu malignant neoplastic (only at right mammarychain), and 33 invasive malignant neoplastic (19 atright mammary chain and 14 at left mammary

Abstracts 21

chain). All the benign neoplastic tumours were clas-sified as grade 1; all the in situ malignant neoplasticwere classified as grade 1; the invasive malignantneoplastic were distributed in 20 tumours of grade1, 6 tumours of grade 2 and 7 tumours of grade 3. 9 ofall animals presented invasive malignant neoplastic.

With these results, we conclude that this is a goodmodel to induce invasive malignant breast cancertumours with an incidence around 90%.

PS4

Which animal model should be applied inDMBA-induced breast cancer? A brief,non-systematic review

E Costa*1,2, CP Reis3,4, S Cabrita2 andIV Figueiredo1,5

1Pharmacology and Pharmaceutical Care Laboratory,Faculty of Pharmacy – University of Coimbra;2Experimental Pathology Service, Faculty of Medicine– University of Coimbra;3iMed.ULisboa – Faculdade de Farmacia,Universidade de Lisboa, Portugal;4IBEB, Faculdade de Ciencias, Universidade deLisboa, Portugal;5IBILI, Faculty of Medicine – University of Coimbra.e-mail: [email protected]

AbstractBreast cancer is a global public health issue as it isthe most frequently diagnosed malignancy in womenin the Western world and commonest cause ofcancer death in European and American women.DMBA is an HPA, known to be a ubiquitous environ-mental contaminant and is considered to be one ofthe etiologic factors of malignant neoplasms inhumans. It is present in cigarette smoke, coal,burned wood, and gasoline and diesel engines. Ithas been assigned various toxicological, immuno-toxic, mutagenic, teratogenic and carcinogeniceffects. It induces malignant transformation in celltransformation systems in vitro, being mutagenicfor bacteria and mammalian cells. With this work,we pretend to describe the main characteristics ofthis DMBA breast cancer induction model.

We used the PubMed database to search 11 art-icles based on keywords ‘‘Breast Cancer’’,‘‘Experimental model’’ and ‘‘DMBA’’.

There are different models of breast cancer withdifferent rat strains. During some studies, the out-breed strains of Sprague-Dawley and Wistar female

rats demonstrated to be more sensitive to chemicalcarcinogenesis when compared with others strains.More recent studies showed that the number ofmammary tumors developed in Wistar Rats waslower when compared with female Sprague-Dawleyrats(1–3). The breast tumors induction can be hor-mone-dependent or independent. DMBA induce hor-mone-dependent tumors, it expresses estrogen andprogesterone receptors, and it is very important toselect the time of DMBA administration(4–6). Thechemical carcinogenesis is maximal when DMBA isadministrated between 45 and 65 days of age, sexualmaturity age. When administered by gavage at 50–56days of age, in a single dose ranging from 10 to100 mg per Kg of body weight, it induces the devel-opment of a high number of mammary tumors (morethan 90% of incidence)(7–10). The carcinogenesis ini-tiates at 64–70 days of age, 14 days after administra-tion of DMBA. After initiation, the carcinogenesis cancause benign lesions such as cysts, adenomas,alveolar hyperplasias and fibroadenomas or malig-nant lesions with different histological patterns, forexample, papillary, cribiform and comedo(4). At 7th to10th weeks after administration of DMBA, mammarytumors were detected by palpation and the number oftumors increases over time(11). All references usedhad their work approved by the Institution EthicsCommittee.

We consider this a promising model because themammary tumors are easily induced, the experimen-tal protocols were defined, the animals developed ahigh number of tumors, and tumors were similar tothose found in humans.

References

1. E. Boyland, K. Sydnor, The induction of mammary

cancer in rats, Br. J. Cancer 16 (1962) 731–739.2. A. Gal, A. Baba, V.Miclaus, C. Bouari, M. Taulescu, P.

Bolfa, G. Borza, C. Catoi, Comparative aspects regarding

MNU-induced mammary carcinogenesis in immature

Sprague-Dawley and Wistar rats, Bull. UASVM Vet.

Med. 68 (2011) 159–163.

3. Alvarado A, Faustino-Rocha AI, Colaco B, Oliveira PA.

Experimental mammary carcinogenesis – Rat models.

Life Sci.173 (2017) 116–34.4. J. Russo, I.H. Russo, Experimentally induced mammary

tumors in rats, Breast Cancer Res. Treat. 39 (1996) 7–20.5. S. Nandi, R.C. Guzman, J. Yang, Hormones and mam-

mary carcinogenesis in mice, rats, and humans – a unify-

ing hypothesis, Proc. Natl. Acad. Sci. U. S. A. 92 (1995)

3650–3657.6. G. Thordarson, A.V. Lee, M. McCarty, K. Van Horn, O.

Chu, Y.C. Chou, J. Yang, R.C. Guzman, S. Nandi, F.

Talamantes, Growth and characterization of N-methyl-

N- nitrosourea-inducedmammary tumors in intact and


ovariectomized rats, Carcino- genesis 22 (2001) 2039–2048.

7. J. Russo, I.H. Russo, Atlas and histologic classification

of tumors of the rat mammary gland, J. Mammary GlandBiol. Neoplasia 5 (2000) 187–200.

8. W.S. Al-Dhaheri, I. Hassouna, S. Al-Salam, S.M.Karam, Characterization of breast cancer progression

in the rat, Recent Adv. Clin. Oncol. 1138 (2008) 121–131.9. J. Cortes-Garcıa, A. Aguilera-Mendez, A. Higareda-

Mendoza, E. Beltran-Pena, M. Pardo-Galvan,

Induccion de cancer en rata wistar (Rattus norvegicus)mediante el uso de dimetil benzo(a)antraceno (DMBA),EDEMM. 1 (2009) 11–17.

10. N. Currier, S.E. Solomon, E.G. Demicco, D.L.F. Chang,M. Farago, H.Q. Ying, I. Dominguez, G.E. Sonenshein,R.D. Cardiff, Z.X.J. Xiao, D.H. Sherr, D.C. Seldin,

Onco- genic signaling pathways activated in DMBA-induced mouse mammary tumors, Toxicol. Pathol. 33(2005) 726–737.

11. A.I. Faustino-Rocha, R. Ferreira, P.A. Oliveira, A.

Gama, M. Ginja, N-Methyl-N-nitrosourea as a mam-mary carcinogenic agent, Tumour Biol. 36 (2015) 9095–9117.

PS5

Blood and Urine parameters in DMBAbreast cancer induction model

E Costa*1,2, R Duarte1,2, R Cabecas3,D Goncalves4, CP Reis5,6, S Cabrita2 andIV Figueiredo1,7

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 Farmacia,

Universidade de Lisboa, Portugal;6IBEB, Faculdade de Ciencias, Universidade deLisboa, Portugal;7IBILI, Faculty of Medicine – University of Coimbra.

e-mail: [email protected]

AbstractDMBA is considered to be one of the etiologic factorsof malignant neoplasms in humans. It is present incigarette smoke, coal, burned wood, coal tar andgasoline and diesel engines. It has been assignedvarious toxicological, immunotoxic, mutagenic, tera-togenic and carcinogenic effects. In the study ofmalignant human breast neoplasm, DMBA has also

been used to chemically induce mammary carcino-genesis in experimental models with Sprague-Dawley rats. With this work, we intend to describethe blood and urine parameters of DMBA breastcancer induction model.

Twenty Sprague-Dawley female rats were dividedin two groups, ten animals in control group with nomanipulation and ten animals in test group withDMBA administration. At 55–57 days of age, the ani-mals of test group were orally administered with65 mg/kg of DMBA diluted in olive oil. At 15 weeksafter carcinogenic induction, tumors started to bedetectable by mammary palpation. At 27 weeks, allanimals were euthanized under anaesthesia. A fullnecropsy was performed. At the same time bloodsamples was collected from each animal. Once amonth, during 6 months, each animal was placed ina metabolic cage, for 24h, in order to perform urinecollection. This work was approved by DGAV. In wholeblood count was made, and at serum we measuredglucose, urea, creatinine, alkaline phosphatase, TGO,TGP and total calcium. In urine, we performed phy-sicochemical determinations, such as quantificationof bilirubin, urobilinogen, ascorbic acid, ketones, red/white cells, nitrites, proteins, density, pH, urea andalso urinary ionogram (Na, CL, K) was made.Statistical analysis was made using R software.

In blood count, comparing both groups, there aresignificative differences in platelets with p-value¼0.037 and IC95% [11.541;304.409]. At serummeasurements, there were significative differencesin creatinine with p-value¼0.031 and IC95%[0.008;0.142]. Platelets and creatinine are decreasedin group with DMBA administration. In urinalysis,there were significative differences in urea, sodium,potassium and chlorine in different measurementsthroughout the experimental protocol.

With this work, we conclude that DMBA breasttumors model can cause some alterations in bloodand urine parameters which may have impact infuture protocols

PS6

Categorization of the TNBS-inducedmodel of colitis in rats

M Duarte1, S Goncalves-Monteiro2, S Capas-Peneda3, P Dias-Pereira4, M Morato1,2 andM Duarte-Araujo5

1Laboratory of Pharmacology, Department of DrugSciences, Faculty of Pharmacy of University of Porto,Portugal;

Abstracts 23

2LAQV@REQUIMTE, Faculty of Pharmacy of Universityof Porto, Portugal;3Laboratory Animal Science, IBMC – University ofPorto, Portugal;4Department of Pathology and MolecularImmunology, Institute of Biomedical Sciences AbelSalazar, University of Porto, Portugal;5Department of Immuno-Physiology andPharmacology, Institute of Biomedical Sciences AbelSalazar, University of Porto, Portugal

AbstractInflammatory Bowel Disease (IBD) is a highly preva-lent chronic relapsing immunologic disorder of thegastrointestinal tract. Among several animalmodels that are available to study IBD, the 2,4,6-tri-nitrobenzene sulfonic acid (TNBS) chemicallyinduced colitis has a pivotal role, especially in pre-clinical testing [1]. Despite its popularity, the varia-bility found in the induction and outcome of thisanimal model are confusing and often reported asinconsistent. The aim of this work was to character-ize and refine TNBS-induced colitis, in rats includedin a project that aims to investigate the role of renin-angiotensin system in the colon.

Protocols were approved by local animal welfarebody (179/2017 ORBEA ICBAS-UP), as well as thenational 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 rectallyinstilled with a 30% ethanolic solution of TNBS(20 mg/rat, n¼17). Analgesia was provided by theadministration of tramadol (20 mg/kg, SC) and acet-aminophen (5 mg/ml in drinking water). Eleven litter-mates were used as controls. The following week,animals were daily monitored to evaluate bodyweight, food and water intake, general welfare andgrimace scores, by cage side assessment. On the 7thday animals were euthanized by decapitation, thecolon was removed and its macroscopic diseaseactivity index (DAI) assessed.

TNBS induced different disease severity degrees,that were categorized in 3 groups: Mild, Moderateand Severe, according to macroscopic DAI scores(Mild: [0–4]; Moderate: [4–8]; Severe: [8–12]). Sevendays after induction Mild-TNBS animals recoveredtheir initial weight (0,58�0,04%, n¼5), butModerate-TNBS (�6,17�0,04%, n¼6) and Severe-TNBS (�4,54�0,02%, n¼6) lost weight. Food intakeaccompanied weight changes, so Mild-TNBS atemore (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 theopposite 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 gri-mace scores, it was clear that on the 7th day Mild-TNBS had a lower score (�0,5-1 points) thanModerate and Severe-TNBS (1,5-2 points).

It was possible to categorize the variabilityobserved with the TNBS-induced model of colitis inthree severity groups, with different characteristics.We hope that in the future these results can helpresearchers to anticipate the severity of TNBS-induced colitis, which is relevant for animal welfare,reproducibility of experimental protocols and alsodata analysis.

Acknowledgements

M. Morato thanks GEDII – Grupo de Estudo da DoencaInflamatoria Intestinal – for funding and Mrs. Ceu Pereirafor excellent technical assistance.

Reference

1. Antoniou et al. (2016). Annals of Medicine and Surgery11, 9–15.

PS7

Light cycle and cage change: behaviouraleffects over two different mice strains

RL Alves1,2,3, A Santos4, I Polonia4,T Summavielle1,2 and A Magalhaes1,2,4

1i3S – Instituto de Investigacao e Inovacao em Saude,Universidade do Porto, Portugal;2Addiction Biology, IBMC – Instituto de BiologiaCelular e Molecular, Portugal;3Lab of Neuropsychophysiology, FPCEUP –Faculdade de Psicologia e de Ciencias da Educacaoda Universidade do Porto, Portugal;4Instituto de Ciencias Biomedicas Abel Salazar,Universidade do Porto, Portugal

AbstractThe frequency at which mouse cages are changedhas a relevant impact in the welfare of animals, andconsequently on experimental results. The time ofday at which cage change takes place may have dis-tinctive effects on behaviour, since mice are noctur-nal animals with a well-defined circadian rhythm. Ifso, the effect of cage change should be minimized.The objective of this study was to determine theeffects of cage change in different periods of thelight/dark cycle, on the exploratory, social, and


anxiety-like behaviours of two mice strains (B6 andFVB-WT). A total of 12 cages (6 per strain), each witha pair of animals (N¼24) were initially subjected to acage change during the light phase of the cycle (2 hafter the lights on), and a week later during the darkphase (2 h after the lights off). Behavioural changeswere videotaped for 10 min in the new home cage,immediately after the change. After this period, onemouse per cage was tested in the Elevated Plus Maze(EPM) to evaluate anxiety. Results show that B6 miceincreased exploratory behaviour (mean diff: 178,8; CI95%: 70,39 to 287,1; p¼0,0027) and digging (meandiff: 22,56; CI 95%: 0,761 to 44,36; p¼0,0432) whenthe cage was changed during the dark phase. Whenthe change occurred during the light phase, B6 micespent more time hiding (mean diff: �19,7; CI 95%:�33,39 to �6,013,1; p¼0,0072) and peeking (meandiff: 42,96; CI 95%: �73,95 to �11,97,1; p¼0,0091)from the paper tube, which suggests that changesduring the dark phase lead to more proactive beha-viours when compared with changes during the lightphase. The FVB mice did not change behavior whenthe cage was changed during the dark or light phase.Furthermore, this strain was less anxious than B6mice in EPM (F(1)¼ 37,284; p¼ 0,001). This work sug-gests that the impact of the light/dark cycle on micebehaviour after the cage change is strain-dependent.These results suggest that changing cages duringthe dark phase increases exploratory behavioursand reduces passive-anxiety behaviours, leading toimproved adaptation to the new environment. Thisseems to be particularly relevant for more anxiousstrains.

This project was appraised by the Animal WelfareBody (ORBEA) at the i3S, and was not considered tosurpass the pain or distress threshold to be consid-ered 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 Orcamento

do Estado.

PS8

Estimating the Predictive Validity ofAnimal Models of Type 2 Diabetes inDPP-4 Inhibitor Studies – AssessingMethodological Quality and AnimalWelfare

SB Miranda1,2, O Varga3 and NH Franco2,4

1FCUP – Faculdade de Ciencias da Universidade doPorto;2I3S – Instituto de Investigacao e Inovacao em Saude;3University of Debrecen;4IBMC – Instituto de Biologia Molecular e Celular

AbstractType 2 diabetes (T2D) is a metabolic disorder respon-sible for millions of deaths annually. It is mainlycharacterized by hyperglycaemia, frequently accom-panied by obesity, arterial hypertension, andhypercholesterolemia as comorbidities, amongothers. Although several drugs are available to helpmitigate symptoms and alleviate the impact ofcomorbidities of T2D, more effective treatments arestill needed.

To both understand the underlying pathophysiolo-gical mechanisms of T2D and test the efficacy oftherapeutic drugs, animal studies are still of theutmost importance. Several animal models mimick-ing traits of human disease – with varying fidelity –are currently used. It is however central to establishtheir predictive validity – i.e. the extent to which thesecan predict human outcomes – as well as the impactof other variables, such as route of drug administra-tion or sex of the animals. To ensure that such stu-dies yield reliable – i.e. reproducible – and thusclinically relevant results, their design and executionmust uphold high methodological standards.

As part of an ongoing bilateral Portuguese-Hungarian bilateral project, we are currently carryingout a retrospective evaluation of the predictive valid-ity of animal models by a systematic review of pre-clinical and clinical data, to compare reported drugefficacy in both human patients and animal models ofT2D, focusing on dipeptidyl-peptidase-4 (DPP-4) inhi-bitors, namely linagliptin, saxagliptin, vildagliptin,and sitagliptin. We also aim to evaluate both metho-dological and animal welfare standards, as centralmeasures for ensuring that animal research is com-petent, meaningful, and responsible. Results fromthe latter assessment will be presented.

All articles so far analysed (N¼49) reportedcompliance with relevant animal welfare regulations,

Abstracts 25

18/49 did not have a conflict of interest statement,29/49 did not report randomisation of animals totreatment groups, and 40/49 did not perform blindoutcome assessment. Moreover, none reported allo-cation concealment, and only one justified samplesize. Clinical signs of disease were seldom reported,aside glycaemia levels, which minimum and maxi-mum values varied between 62 mg/dL and 603 mg/dL, respectively. As most animal models of T2D arenon-lethal, endpoints applied were mostly scientific(rather than being applied for animal welfare rea-sons), but in many cases this was impossible todetermine, due to insufficient information. Thesepreliminary results strengthens the available litera-ture suggesting that the design, execution, andreporting of animal studies often fails to meet basicstandards, with relevant implications for the reliabil-ity of available published data, as well as the social,ethical and legal acceptability of animal research.

PS9

Evaluation of animal well-being during anassay of parecoxib genotoxicity in FVB/nmice

T Ferreira1,5, S Campos1, S Santos1,5,JC Almeida1,5, MSS Moutinho2, RM Gil da Costa,I Gaivao3,4, M Ginja5,6, L Antunes1,2 andPA Oliveira1,2

1Departamento de Ciencias Veterinarias, UTAD,Portugal;2Centro de Investigacao e de TecnologiasAgroambientais e Biologicas (CITAB), UTAD,Portugal;3Grupo de Oncologia Molecular e Patologia Viral, CI-IPOP, Instituto Portugues de Oncologia do Porto,Portugal;4Faculdade de Engenharia, LEPABE, Universidade doPorto (FEUP), Portugal;5Departamento de Genetica e Biotecnologia, UTAD,Portugal;6Centro de Ciencia Animal e Veterinaria (CECAV),UTAD, Portugal

AbstractThe evaluation of animal well-being during experi-mental assays is crucial to improve and refinein vivo research. The comet assay is a simple tech-nique used to analyse DNA damages induced by sev-eral compounds. This technique determines simpleand double DNA strand breaks. The FVB/N mice are

widely used to evaluate drug efficacy and drug prop-erties in vivo. Parecoxib is a cyclooxygenase-2 (COX2)selective inhibitor that can be used to inhibit theinflammation. However, its effects in DNA damageare not known.

This research aimed to study animal well-beingduring the evaluation of genotoxicity effects of pare-coxib in female FVB/n mice.

This study was approved by the Universidade deTras-os-Montes e Alto Douro ethics committee(approval no. 10/2013) and the PortugueseVeterinary Directorate (approval no. 0421/000/ 000/2014). To achieve this goal we used 21 twentyweeks-old FVB/n mice, equally divided into twogroups: parecoxib group (n¼10) and control group(n¼11). Parecoxib was administered intraperitoneally(5 mg/Kg) daily for 22 consecutive days. During theexperiment, the following parameters were investi-gated: body weight, body condition, posture, groom-ing, mucosae, eyes, ears, whiskers, behaviour,answer to external stimuli (put a hand on themice’s cage), hydration, respiratory and cardiac fre-quency, faeces aspect, and convulsions. At the end ofparecoxib administration all animals were sacrificedby xilazyne-ketamine overdose, and blood was col-lected by intracardiac puncture. Five microliters ofblood were placed in PBS, then blood was embeddedin agarose and subjected to an electric field.

All animals survived the experiment and no animalshowed clinical signs of toxicity. All evaluated para-meters were normal. The mean frequency of arbitraryunities in 200 cells was 116.03 in the parecoxib groupand 51.77 in the control group, i.e. parecoxib groupshowed a significant increase of DNA damage in com-parison with the control group (p¼0.0003). Theseresults suggest that parecoxib induces DNA damagein FVB/n mice. More studies are however needed toconfirm the genotoxicity of parecoxib. However,according to our results parecoxib did not change phy-siological parameters and apparently did not impactanimal health and well-being.


PS10

Applying Lean Management to reducethe rodent cage changing time andanswer an increase in 100% in cagenumbers

A Costa1,2,*, C Rocha1,2*, C Barros1,2*,C Pereira1,2*, D Silva1,2*, , S Santos1,2*,D Goncalves1,2, S Pereira1,2 and MJ Castelhano-Carlos1,2

1Life and Health Sciences Research Institute (ICVS),School of Medicine, University of Minho, Braga,Portugal;2ICVS/3B’s – PT Government Associate Laboratory,Braga/Guimaraes, Portugal*These authors gave equal contributions to this work

AbstractIn order to increase the capacity for rodents housingin the clean zone of the Animal Facilities Unit (UBiot)of the Life and Health Sciences Research Institute ofthe School of Medicine (ICVS-EM), University ofMinho, a new building was built and we expected anincrease of the rodent cages capacity to double.Nevertheless, the number of hired animal caretakerswas the same, so we decided to face the challenge byapplying Lean management practices in order toimprove our cage changing time.

The work presented here was developed by thecaretakers’ team and manager of the UBiot, in colla-boration with the occupational health and safety offi-cer of ICVS-EM, being tested in practice by the 3animal caretakers working in the Clean Zone of theUBiot. The aim was to implement a cage changingprocedure that would reduce the time used for cagechanging by 50%.

The methodology applied to improve the cagechanging procedure was based on A3 problem sol-ving in 7 phases which include detailed analyses ofthe actual working conditions; identification of tar-gets of improvement; analysis of the root cause ofthe problem; definition of countermeasures; defini-tions of a plan of actions and follow up strategies.Two sessions of Lean practice training were givento the animal caretakers in order to prepare themfor the methodology.

The analysis of the cage changing procedurerevealed an actual takt time of 74 seconds per cagechange with several movements and actions of non-value-creating work. We developed working instruc-tions and tasks distribution including operators forcage changes and a logistic operator; reorganization

of the materials and animal facility rules forresearchers that contributed to reduce the timeused for cage changes that allowed us to accomplisha takt time of 37 seconds per cage. During the A3problem solving we have also identified ergonomicproblems in the repetitive work performed byanimal caretakers and we implemented a rotationsystem for tasks attributed to the caretakers eachweek.

The approach of a problem of work management inanimal facilities using the A3 problem solving meth-odology and Lean management practices was provenvery efficient and motivating.

The animal facility caretakers team achieved theestablishment of working instructions, improved theworking space organization and the communicationwith researchers, reduced the repetitive work, beingable to reduce the time spent changing cages by 50%as aimed.

We believe the application of Lean managementtools such as the example discussed here contributeto improve the working environment, not only witheffective practical results, but also by being a wayof stimulating team work and drive motivation of allthose involved.

Acknowledgements

The work presented in this poster was performed in theanimal facilities of the Life and Health Sciences Research

Institute (ICVS), School of Medicine, Minho University.Financial support was provided by FEDER funds throughthe Operational Programme Competitiveness Factors –

COMPETE and National Funds through FCT –Foundation for Science and Technology under the projectPOCI-01-0145-FEDER-007038; and by the project

NORTE-01-0145-FEDER-000013, supported by NortePortugal Regional Operational Programme (NORTE 2020),under the PORTUGAL 2020 Partnership Agreement,through the European Regional Development Fund (ERDF).

PS11

Report of an Animal Facility storehousereorganization based on LeanManagement principles

M Pereira1, A Ribeiro1 and M Rebelo1

1Instituto Gulbenkian de Ciencia (IGC), Lisboa,Portugal

AbstractFor the past few decades, much has been outlinedabout Lean Management and the application of its

Abstracts 27

concepts in the most diverse areas, from industriesto companies and institutes. Lean Management con-sists on a set of concepts, principles and tools usedto provide a better service while consuming fewerresources. In the laboratory animal sciences,resources refer mainly to work, but can also relateto space, material, equipment and anything else usedby animals to support the advancement of research.The Animal House Core Facility of the InstitutoGulbenkian de Ciencia (IGC) is an infrastructure thatallows the production, housing and experimentationof diverse model organisms like mouse, zebrafish,Xenopus and Drosophila for scientific studies. TheMouse Facility is regularly used by more than 100internal and external researchers. Currently itcovers a total area of 1100 m2 including storingspaces and 4500 animal cages, thus requiring a meti-culous organization at different levels: financial,human resources, product management and storage.An important area, but often overlooked, for theproper functioning of a unit is the warehouse. Thisplace allows having all the necessary material for thedaily operation, well-conditioned and ideally, in suffi-cient quantity for any emergency. The goal of thiswork is to show an example of an infrastructure(building) adaptation and application of LeanManagement principles, to improve the logistic andthe overall management of an Animal Facility. Sevenyears ago, the existing storage area of the IGC MouseFacility consisted in 4 rooms (5 to 30 m2), for anumber of 3500 animal cages in the Facility.Predicting an increase of 25% of mouse researchactivity in the following few years, it was decided toreorganize the warehouse to allow receiving highervolumes of consumables, namely bedding and diet.The first step was to create a unique and larger areaby removing the divisions between those rooms.Then, based on Lean Management principles createan area dedicated for each set of material. The out-come of this simple reshuffling became of crucialimportance for the routine of the Facility.Importantly, it allowed in the same area receiving aconsiderable larger quantity of diet (from 5 to 15 pal-lets), still increasing the space to store all the otherproducts. This had a crucial impact in the routine ofthe Facility, permitting stock management flexibility,and markedly decreased the risk of stock productoutbreak.

Acknowledgements

We thank to the Animal Facility team for their contribution

to reorganize the warehouse and daily support of itsmaintenance.

PS12

Male mice as bedding sentinels: a newstrategy to further apply the 3rs?

D Bonaparte1 and A Madalena1

1Fundacao Champalimaud, Lisboa, Portugal

AbstractTraditionally, mouse bedding sentinels are pairhoused females. Every quarter, one sentinel isshipped to the lab for terminal sampling and testing,and it is replaced by a new one. When sent to thelaboratory, sentinel mice are pooled together intoshipping crates. Due to conspecific aggression, bothcrate pooling and reintroduction of a new cage mateare not recommended in male mice. In our facility, wekeep a colony of SOPF NMRI mice which is used togenerate both surrogate mothers and sentinels. Sinceolder females have lower performances as surrogatemothers, when they surpass the optimal age or weightfor embryo implantation we redirect them for beddingsentinels. With this approach, we reduce the numberof NMRI females required in total. However, becausewe are a large facility, recycled females alone are notenough to populate all sentinel cages; we need toproduce additional females for this purpose.Moreover, we have no use for male NMRI mice,which are mostly euthanised before weaning. Sincewe have moved to health monitoring by survival sam-pling, we no longer need to ship our sentinels; nor toreintroduce new cage mates every quarter. For thisreason, we hypothesised whether NMRI males couldbe used as sentinels as well. If successful, this strat-egy would allow us to use both genders: we would notneed to eliminate the males, and we could reduce theglobal size of the colony. In December 2017, we haveset ten pairs of eight-week-old male NMRI sentinelsin one of our holding rooms, one cage per IVC rack. Allcages are enriched with nesting material and a redplastic shelter. Each cage receives soiled beddingfrom the respective rack every two weeks. We havemonitored these males regularly for aggression beha-viour and weekly for fight lesions or other signs ofpoor welfare. This new strategy has been in placefor six months and we have not observed any pro-blems, except for one sentinel cage. While we recog-nize that further studies need to be done as toincrease sample size and introduce further welfareassessment methods, we find these preliminaryresults quite promising. We have recently equippedanother holding room with male sentinels and, forthe time being, we are no longer euthanising our


NMRI males. Should this strategy continue to provesuccessful, we plan to definitely resize our NMRIbreeding colony.

Acknowledgements

We thank the Champalimaud Vivarium Team for helpingwith this study

PS13

Evaluation of histological postmortemchanges in mice liver, heart and lungs

P Dias-Pereira1, A Canadas1, S Goncalves-Monteiro2, S Capas-Peneda3 and M Duarte-Araujo4

1Department of Pathology and MolecularImmunology, Institute for the Biomedical Sciences ofAbel Salazar – University of Porto, Porto, Portugal;2LAQV@REQUIMTE, Faculty of Pharmacy of Universityof Porto, Portugal;3Laboratory Animal Science, IBMC – University ofPorto, Portugal;4Department of ImmunoPhysiology andPharmacology, Institute for the Biomedical Sciencesof Abel Salazar – University of Porto, Portugal

AbstractImmediately after death a series of irreversible bio-chemical and structural changes occur in the body, aprocess known as autolysis. Researchers often rejectmice tissues that have not been submitted to fixationimmediately after the animal’s death, fearing mas-sive autolysis, a practice contrary to NC3Rs princi-ples. Despite the limited information available, someresearchers demonstrated that the time course ofhistological and ultrastructural changes followingdeath is organ specific 1,2.

The objective of this study was to describe thesequence of autolytic changes observed in the first24 hours after death in mice liver, heart and lung.

Thirty adult male and female C57BL/6 mice with5,79 � 2,94 months of age and weighing approxi-mately 30 gr were used in this study. All animalswere maintained in accordance with Portuguese DLn� 113/2013 and kept under stable temperature (20–24�C) and relative humidity (40–70%) conditions, inIVC cages with ad libitum access to water and food,exposed to 12:12 hours light/dark cycle. The animalswere euthanized by a single intraperitoneal adminis-tration of pentobarbital sodium (300 mg/Kg) and keptunder stable temperature (20,96 � 1,05�C) and rela-tive humidity (39,55 � 8,71%) conditions, during a

period between 0 and 24 h after death. The materialwas collected, fixed in 10% formalin and routinelyprocessed for histological examination.

No significant histological changes were appre-ciated in any of the organs during the first 2 hoursafter death. Nevertheless, autolytic changes wereorgan-specific, since 80% of the mice dead for 2–4hours showed histological changes in the heart andliver, but only 26% had autolytic changes in the lungs(epithelial detachment in the airways). Furthermore,2–3 hours postmortem a separation of cardiacmuscle fibres (individualization) with a multifocalslight fine granular cytoplasm was observed.Hepatic autolytic changes were visible 3–4 hoursafter death and consisted on hepatocyte atrophyalong with sinusoidal ectasia.

Our data demonstrate that there is a time intervalafter the animals’ death in which histologicalchanges in heart, lung and mice liver are not signifi-cant, suggesting that the cadaveric material can stillbe used in some lines of research. Furthermore, incombination with a method to estimate the time ofdeath, previously described (unpublished data),researchers can estimate the time of death and,thus, use tissues of dead animals, contributing to areduction in the number of animals used.

References

1. El-noor MMA, Elhosary NM, Khedr NF, et al.Estimation of early post-mortem interval through bio-chemocal and pathological changes in rat heart and

kidney. American Journal of Forensic Medical Pathology2016; 37: 40–46.

2. Tomita Y, Nihira M, Ohno Y, et al. Ultrastructural

changes during in situ early postmortem autolysis inkidney, heart and skeletal muscle of rats. Leg Med2004; 6: 25–31.

PS14

Efficacy of different methods of eutha-nasia in adult zebrafish: anaesthesiaoverdose and rapid cooling

JM Ferreira1,2, IAS Olsson1,2 and AM Valentim1,2,3

1i3S – Instituto de Investigacao e Inovacao em Saude,UP, Portugal;2IBMC – Instituto de Biologia Molecular e Celular,University of Porto, Laboratory Animal Sciencegroup;

Abstracts 29

3CITAB, UTAD, Laboratory Animal Science[[email protected]]

AbstractWith the increasing use of zebrafish in research, ade-quate methods of euthanasia are required. The EUdirective allows euthanasia in fish by anaestheticoverdose, electrical stunning and concussion.However, more specific methods are required forthis small tropical fish and compatible with post-mortem tissue analyses. MS222 is the mostcommon agent used in fish, but it may induce aver-sion in zebrafish, contrary to clove oil and etomidate(1). Propofol/lidocaine is a new protocol proposed byour group with the potential to be used in zebrafish(2). Rapid cooling has shown to cause less distress tozebrafish euthanasia (3), obtaining clean tissues forpost-mortem analysis, but under present EU legisla-tion it cannot be used on conscious fish. Our aim is totest the efficacy of these euthanasia protocols inadult zebrafish.

All procedures were performed under DGAVlicenses. Sixty adult mixed-sex AB zebrafish wererandomly assigned to 5 groups of euthanasia:250 mg/L MS222; 20 mg/L propofol þ 100 mg/L lido-caine; 6 mg/L etomidate; 50 mg/L clove oil; rapidcooling (water at 2–4� C). Two minutes after opercu-lar movement ceasing, the animals were transferredto clean system water to ensure death.

Zebrafish euthanized with rapid cooling ceased theopercular movements significantly quicker (P<0.00001) compared with the other groups. Other dif-ferences were found in the latency to opercularmovement ceasing in the other groups: MS222 �clove oil �propofol/lidocaine� etomidate (P� 0.001for comparisons between etomidate and MS222 orclove oil). No zebrafish recovered after being placedin clean water.

All protocols used were efficacious to achieveeuthanasia, but rapid cooling was consistently thefastest and so more efficient. It has the advantageof being safe to the operator, easily available andcheap. Further studies are needed to refine this pro-tocol, evaluate animal welfare, and biological samplequality before legislation can be changed to includethis as a method for euthanasia for small tropicalfish.

Acknowledgements

This work was funded by the project Norte-01-0145-FEDER-

000008 at I3S, supported by NORTE 2020, under thePORTUGAL 2020 Partnership Agreement, through theFEDER, and by a postdoctoral fellowship from FCT.

References

1. Wong D, von Keyserlingk MA, Richards JG, et al.

Conditioned place avoidance of zebrafish (Danio rerio)to three chemicals used for euthanasia and anaesthesia.PLoS One 2014; 9: e88030.

2. Valentim AM, Felix LM, Carvalho L, et al. A new anaes-

thetic protocol for adult zebrafish (Danio rerio): propofolcombined with lidocaine. PloS one 2016; 11(1): e0147747.

3. Wilson JM, Bunte RM and Carty AJ. Evaluation of

Rapid Cooling and Tricaine Methanesulfonate(MS222)as Methods of Euthanasia in Zebrafish (Danio rerio).JAALAS 2009; 48: 785–789.

PS15

Optimization of husbandry protocols forZebrafish larvae experiments

LD Vale, I Santos, M Franco, AC Borges andM RebeloInstituto Gulbenkian de Ciencia, Animal HouseFacility

AbstractZebrafish has been widely used as a research modelin different fields of science such as embryology,regenerative biology, developmental genetics, toxi-cology, behavior, aquaculture, among other subject.This species is relatively easy to maintain in thelaboratory and due to technological advances andstate-of-the-art life support systems, water qualitycontrol is enhanced with minimal labor, hence sup-porting fast expansion of this model organism.Despite suitable for rearing embryos and adult spe-cimens, recirculating systems are not conceived forlarvae experimental manipulation and tracking. Asresearch based on larvae and juvenile zebrafish life-stages expands, it is crucial to develop approachesthat enable larvae rearing methods supporting closeobservation and manipulation of individuals. This ischallenging since larval survival and developmentrates are highly dependent on environmental condi-tions. To tackle this, we are developing an assaybased on a rotifer polyculture protocol1 that was fur-ther developed to support larval development untiljuvenile stage in static conditions.

We have tested several parameters to optimizesurvival and growth rates in static tanks. Conditionstested include: temperature (24�C and 28�C), larvaedensity (10, 20, 40 and 70 larvae/L), and water qualitymanagement by using different cleaning routines(daily, 2 and 3 times per week). This combinatorialstudy was done with wildtype (AB) and Nacre mutant


(mitfa-/-) strains and compared with a referencegroup that was held in a recirculating system at28�C at a density of 10 larvae/ L. Survival rateswere calculated by daily counts of living animalsthroughout the whole experiment. Larval meta-morphic development was characterized, since zeb-rafish age is not predictive of developmental stage2,therefore larvae growth was analyzed at specifictimepoints until the juvenile stage was reached. Forthis, larvae were sampled and photographed tomeasure standard length (SL) and to analyze externalanatomic characteristics.

The analysis of the results led us to set up a proto-col with optimal conditions to perform differentexperiments in static conditions, namely survivaltrials, drug treatment experiments and tracking ofchimeras.

References

1. Best J, Adatto I, Cockington J, et al. A Novel Method forRearing First-Feeding Larval Zebrafish: Polyculture with

Type L Saltwater Rotifers (Brachionus plicatilis).Zebrafish 2010; 7(3): 289–95.

2. Singleman C and Holtzman NG. Growth and

Maturation in the Zebrafish, Danio Rerio: A StagingTool for Teaching and Research. Zebrafish 2014; 11:396–405.

PS16

@EARA_PT – Public awareness of animalresearch on Twitter in Portugal

AR Barros1,2,3

1European Animal Research Association;2Faculdade de Ciencias Sociais e Humanas daUniversidade NOVA de Lisboa (UNL);3NOVA Medical School j Faculdade de CienciasMedicas, UNL

AbstractThe European Animal Research Association (EARA) isa communications and advocacy organisation whosemission is to support the interests of biomedicalresearch and healthcare development. As a pan-European advocacy organization, social media is an

essential way to inform, educate and unify audiencesin support of the biomedical field through providingaccurate and evidence-based information about theimportance of the humane use of animals in biomed-ical investigation. Twitter, a social network platformestablished in 2006, is one of the channels thatallows EARA to build a relationship with followersand respond immediately to news. This platformhas become a powerful tool to broadcast sciencemainly biomedical research and medical news butalso for fostering conversations. Twitter has evolvedinto a useful tool for researchers and other profes-sionals to rapidly communicate and debate reliableinformation. The activity on Twitter is initiated andextended by posting messages (i.e., tweets), repost-ing remarkable messages (i.e, retweeting), andattracting other individuals (i.e., followers) to theaccount. Tweets are limit to no more than 280 char-acters and can include links to webpages, picturesand hashtags. To increase public awareness ofanimal research, EARA has eight Twitter accountsacross Europe in the following languages: English,Belgian, Dutch, French, German, Italian, Spanishand Portuguese. The Portuguese EARA Twitter offi-cial channel (@EARA_PT) focus on post relevant con-tent about EARA activity, media, research and policyin Portuguese language to promote useful sources ofinformation on animal research and increase thepublic awareness on this topic.

PS17

Zebra fish facilities as a stage for ajournalistic report

MJ PerryFaculty of Pharmacy – University of Lisbon, MedicinalChemistry and Therapy

AbstractVisiting laboratories of animal experimentation sur-prises us by the different dynamics of the visit, com-pared with those found when visiting a museum or azoological park or an aquarium... Not only reveals therigor of the communication of the research labora-tories with the outside, as it constitutes a curiouschallenge to the reporter. This one will have toadapt what he sees and what he hears inside the

Abstracts 31

animal facilities, to the proper language of the jour-nalistic text that is, do it in a way comprehensible to anon-specialist public. It is intended to present to the4th SPCAL meeting the making-off of the report‘‘Onde esta o Danio? – nos bastidores doZebrafish’’, published in various media through theCiencia-Viva Regional Press platform (1), in whichthe stage of the journalistic work was the Zebrafishfacilities of the Instituto de Medicina Molecular (IMM)and of the Champalimaud Center for the Unknown(CF).

Acknowledgements

We thank to Ciencia-Viva Imprensa Regional, namely toProfessor Antonio Piedade, for the publication of the jour-nalistic report ‘‘Onde esta o Danio? – nos bastidores do

Zebrafish’’.

References

1. http://imprensaregional.cienciaviva.pt/home/.2. http://imprensaregional.cienciaviva.pt/conteudos/arti-

gos/?accao¼showartigo&id_artigocir¼1126.

PS18

Ethics and welfare in aquatic organismsused for research and education

J Carrola1, H Santos2 and AV Sykes3

1CITAB – Centre for the Research and Technology ofAgro-Environmental and Biological Sciences, UTAD,Portugal;2CIIMAR, Matosinhos, Portugal;3CCMAR – Centro de Ciencias do Mar do Algarve,Universidade do Algarve, Faro, Portugal.

AbstractThe issues related to the ethics and welfare of usingfish and, lately, cephalopods are relatively recent.However, there is an increasing amount of aquaticorganisms being used for research and educationthat lead to several questions related to ethics andanimal welfare. When using aquatic animals asmodels in research, similar to other protected verte-brates, one cannot overlook the 3Rs principles(Reduce, Replace, and Refine) and the ARRIVEGuidelines, which should be considered and followedto meet all regulatory requirements while guaran-teeing appropriate animal welfare, husbandry andresearch.

In order to promote the 3Rs and include cephalo-pods as protected animals, the EU published

Directive 2010/63/EU and Portugal has abide withspecific legislation (Decreto-lei n� 113/2013).Overall, this implies that, throughout the EU and inPortugal, students and researchers need to betrained to meet the existing law requirements,namely the accreditation from Direcao Regional deAlimentacao e Veterinaria (DGAV). In addition, theAnimal Welfare Committees (ORBEA) were legallyinstalled in institutions holding regulated animals,and have the responsibility for in loco supervision ofanimal welfare in both husbandry and experiments.The local ORBEAs tasks also include the increase ofawareness related to animal welfare, the 3Rs and theneed to comply with the existing legislation and gen-eral rules in teaching, extension activities andresearch performed in the lab or in the field.

Working with regulated animals (aquatic speciesincluded) requires appropriate training to obtainaccreditation through species-specific courses.However, there is a lack for a book (written inPortuguese) that compiles the necessary informationfor the aquatic organism course and that can be usedas a manual for education. The ‘‘Ethics and welfare inaquatic organisms used for research and education’’book will resume all the themes used in that type ofeducation and is an effort joining specialist research-ers and technicians from academia and public aqua-ria. It will focus on animals’ biology, capture,samples collection, transportation, water quality,life support systems, individual identification andmarking, biosafety, quarantine, ethology, nutrition,experimental design, main procedures, anaesthesia,analgesia, euthanasia, pathologies, etc.

The book aims at grouping the most relevant andupdated information in Laboratory Animal Sciencerelated to regulated aquatic animals. It will providea general approach to the different topics, which webelieve that will become an added value for theresearch and academia community, for educationand aquatic animals’ welfare.

PS19

Inside cage temperature and light var-iation for breeding mice within 4 dayspost-partum

GM Morello1, S Brajon, J Hultgren, C Gilbert,JM Ferreira1,2 and IAS Olsson1,2

1i3S – Instituto de Investigacao e Inovacao em Saude,Universidade do Porto, Portugal;


http://imprensaregional.cienciaviva.pt/home/

http://imprensaregional.cienciaviva.pt/conteudos/artigos/?accaoshowartigo&id_artigocir1126




2IBMC — Instituto de Biologia Molecular e Celular,Universidade do Porto, PortugalE-mail: [email protected]

AbstractMouse breeding rooms are often automatically con-trolled for temperature and light to keep thermal andlighting conditions standardized throughout the year.Temperature set-points in mouse breeding roomsare commonly between 20.0 and 26.0oC. Cage tem-perature is assumed to be consistent with room tem-perature, but very little has been done to confirmthis. Additionally, very little is known about theactual light intensity achieved inside the breedingcages when young pups are present.

The actual temperature and light intensitiesachieved in 50 mouse trio (two females and onemale) 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 ofan ongoing larger project investigating pup survivabil-ity. Cage temperatures and light intensities were auto-matically recorded once every 10 minutes inside thecages. Room temperature measurements were takenfrom the climate control system once a day. Lightintensity was measured once prior to the experimentnext to each cage, by using a luximeter, during thelight 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 of80 and 70% of the litters experienced cage tempera-tures of 21.0 to 21.9oC and 22.0 to 22.9oC, respect-ively, while 66% of the litters experiencedtemperatures either below 21.0oC or above 23.0oC.Room light intensity measurements revealed a vari-ation from 5Lx (cages in the bottom of a rack) to550Lx (at the top of the rack). Cage light intensitywas, however, less than 5 Lx for approximately 95%of the observation period for all the litters.Occasionally, mice were exposed to light levels ashigh as 120 to 980Lx, most likely during cagemanipulation on the work-table, by the care-takers.The behaviour and physiology of mice are known tobe affected by temperature and light but the conse-quences of the observed variations for the welfare ofthe mice and survivability of their pups are still poorlyunderstood. Meanwhile, temperature and light inbreeding facilities and in research are often managedat the room or at the rack level, rather than at theindividual cage level. Thus, there is an urgent needfor more research considering the micro- rather thanjust the macro- environment of breeding mice.

This work is a result of the project Norte-01-0145-FEDER-000008-Porto Neurosciences and NeurologicDisease Research Initiative at I3S, by Norte PortugalRegional Operational Programme (NORTE-2020),PORTUGAL 2020 Partnership Agreement throughFEDER.

Abstracts 33

Documents

Volume 52 Number 2S August 2018 ISSN 0023-6772 Laboratory ... · aboratory limited l a nimals Committees Committees Organizing Committee Magda Castelhano-Carlos1,2,3 (Chair) Alice