ABSTRACT CONGRESO FELASA 2019

OA2S4
SOMETHING OLD, SOMETHING NEW AND SOMETHING BLUE – FELASA
ACCREDITATION IN AQUATIC ORGANISMS
U. Strähle1, J. Weiss1, L. Kaufmann1, J. Maier2 and Almut Köhler3
1
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-
Leopoldshafen, Germany
2
European Zebrafish Resource Centre, Karlsruhe Institute of Technology, Eggenstein-
Leopoldshafen, Germany
3
Safety and Environment, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen,
Germany
Abstract
EU Directive 2010/63/EU demands professional and species-specific expertise of
experimenters working with laboratory animals. Therefore, the long-existing and well-
recognized FELASA courses were adapted according to the new regulatory requirements.
These courses mainly focussed on the use of mammals, especially rodents. The increasing
use of zebrafish in research required addition of zebrafish modules to the course portfolio to
offer appropriate training. Especially, there is still a very wide gap in laboratory animal work
between mammalian and aquatic species with only very few overlap in personnel. The
European Zebrafish Resource Centre (EZRC) built up a completely new training course
focusing mainly on zebrafish and Medaka to fulfil the needs of the experimenters and the
requirements of legislation. While parts of the core modules such as legislation and ethics
can be adopted from other courses, aquatic organisms have some peculiarities to consider:
(1) procedures to be applied are very different. Surgeries are much more uncommon while
in vivo imaging is much more prominent. In case of surgeries, these are very specific
treatments often only occurring in specialized labs. (2) Most research projects are performed
in larval or embryonic stages that do not require an authorization and are not regulated by
legislation. (3) Welfare assessment in fish is much behind that of rodents and diagnostic
options are limited. (4) Animal handling mostly bases on tank recognition and not on
individual animals. We are presenting our ideas in setting up and running a course
considering the differences and still fulfilling regulatory and scientific needs.
OA4S2

LABORATORY ANIMAL SCIENCE ONLINE TRAINING THROUGH THE EYES OF THE LEARNER:
PERCEPTIONS OF GAINS

Andreia Costa1,2, A. Costa3 and I.A. Olsson1

1Laboratory Animal Science group, i3S, Instituto de Investigação e Inovação em Saúde –

University of Porto / IBMC, Institute for Molecular and Cell Biology, University of Porto, Porto,
Portugal

2ICBAS, Abel Salazar Institute of Biomedical Sciences, University of Porto, Porto, Portugal

3FPCEUP, Faculty of Psychology and Education Sciences of the University of Porto, Porto,
Portugal

Abstract

The European directive 2010/63/EU issued clear recommendations concerning the training and
competence of researchers using animals in experimental procedures. Different courses and
training programs have emerged across Europe, and the Laboratory Animal Science
community is facing the challenge of offering qualified and flexible training. From similar
educational areas, E-learning appears to be a promising solution tool, fulfilling the flexible-time
training and quality criteria.

However, very few studies have addressed the participants’ perception of e-learning as a
training tool. For course organizers the e-learning approach is the key to the problem of
demanding programs and short training periods. However, participants’ perceptions, that is
known to be a relevant factor for learning have not been investigated.

Within an exploratory study framework, the e-learning approach perception of 229

researchers participating in LAS training programs was assessed. Participants answered the
Questionnaire of E-learning Acceptance (QELA), a Likert-type scale comprising 5 subscales that
explores how participant perceive the organization of the e-learning, its relevance for the time
management and for preparing for practical training. Results also explored the influence of
student’s sociodemographic and professional profile in the e-learning acceptance.

In general, e-learning was students’ well accepted by the majority of the participants as an
alternative to classroom lectures. We will further discuss how we have used the results to
improve our use of e-learning.
OA8S2

PROBLEMS AND SOLUTIONS IN THE PERFORMING WITH CO2 AS AN EUTHANASIA AGENT IN

MICE

Paul Friedemann Pohlig1, S. Wecker2 and B. Zevnik1

1in vivo Research Facility, CECAD- University of Cologne, Cologne, Germany

2medres – medical research GmbH, Cologne, Germany

Abstract

With the opening of our new large mouse facility within a research centre (CECAD- Cluster of
Excellence in Cellular Stress Responses in Aging-Associated Diseases) in 2013 we dealt
intensively with the subject of euthanasia, since killing of animals was not carried out solely by
the staff of the animal unit, but also by researchers.

Use of carbon dioxide (CO2) or cervical dislocation are the main methods employed for
euthanasia of mice.

Even if trained in mouse euthanasia, people often feel unsure how exactly to comply with CO2
euthanasia procedures. However, wrongdoing or misusage is likely to cause immediate stress
and pain for mice.

First we used a system to assure a constant flow rate and slow increase of CO2 in purposely
designed chambers. A remaining issue was, that we could not guarantee the required increase
of CO2 due to the design of the chamber.

Therefore, the procedure was purely visually monitored for signs of stress and pain in mice.

In order to prevent possible misuse, we developed in cooperation with medres a fully

automated fail-safe solution that guarantees a constant and measurable increase of CO2, in
which we use the air flow of the existing IVC cage that fully complies with our and the
requirements of the “AVMA Guidelines for the Euthanasia of Animals 2013“.
OA9S1

GOOD PRACTICE IN MOUSE HANDLING

Jane Hurst .Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom

Abstract

The traditional method of picking up mice by the tail induces aversion and high anxiety. Mice
do not readily habituate to tail handling, even if their weight is supported and the duration is
brief. This negatively impacts animal welfare, and can also interfere with reliable test
responses. We have shown that picking up mice in a tunnel or scooping them on the open
hand (cup handling) are refined methods that mice accept much more readily, particularly
tunnel handling. But are these refined methods practical for general use in the laboratory?
Here I will discuss good practice in using tunnel and cup handling for laboratory mice, the
training that is required to use these techniques efficiently and resources that are available to
help with this. I will discuss the costs and benefits of each method, barriers to uptake, and
recommend some practical approaches to aid the implementation of these refined methods in
busy animal facilities. Finally, I will report feedback from some of the facilities that have
already implemented refined mouse handling.

OB11S3

MICROBIOTA IMPLANTATION AND MONITORING: NEW CHALLENGES, ONGOING

INNOVATIONS AND EVOLUTIONS

Sylvie Rabot. Micalis Institute (Food and Digestive Microbiology to serve Health), INRA,
AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France

Abstract

Due to the increasing amount of research works on the effect of the commensal microbiota in
health and disease, the use of germ-free animal models is expanding. Germ-free animals are
animals devoid of living microorganisms. They are mostly compared with conventional
animals, in order to assess the importance of commensal microbiota in host physiology
regulation. Before undertaking any experiment with those models, it is mandatory to ascertain
their germ-free status. Microbial cultures, microbial cell viability tests, and molecular methods
based on microbial DNA extraction and amplification are currently in use. Procedures used in
the Anaxem facility of the Micalis Institute will be presented, and advantages and
disadvantages of the different methods will be discussed. Germ-free animals are also often
used as hosts of various kinds of microbiota, from a particular entity to a complex ecosystem.
Indeed, transplanting the commensal microbiota from a donor organism with a phenotype of
interest (such as a disease) to a naive recipient organism, allows demonstrating the causal role
of the microbiota in the phenotype. Yet, microbiota transplantations raise methodological
issues, including the preparation and conservation of the microbiota transplant, the use of a
microbiota transplant prepared from a single donor or from a pool of donors, the
transplantation method, the age of colonisation, the time required to reach an equilibrium
between the recipient organism and the microbiota transplant. So far, there is no consensus
on the best methodology. Several examples will be presented and the advantages and
disadvantages of the different strategies will be discussed.
OB15S5

ZEBRAFISH PICORNAVIRUS: SURVEILLANCE FOR A NOVEL VIRAL PATHOGEN OF LABORATORY

ZEBRAFISH

M. Crim, Cindy Besch-Williford, M. Hart and R. Livingston

IDEXX BioAnalytics, Columbia, United States

Abstract

Subclinical infections of research animals can add confounding variability in animal studies and
have the potential to alter experimental outcomes. Zebrafish picornavirus 1 (ZfPV-1) is a
recently discovered novel picornavirus that subclinically infects laboratory zebrafish. ZfPV-1
replicates in the enteric mucosa and transmission is presumed to be fecal-oral, which is a
common mode of transmission among picornaviruses. While the virus can be detected in
clinically normal zebrafish, the impact of infection on the zebrafish immune system, gut
development, microbiome, and other research areas remain unknown. Health monitoring data
from zebrafish colonies indicate that the virus is prevalent, with 24% of samples testing
positive to date, and widely distributed among research institutions in Europe and North
America. ZfPV-1 can be detected by real-time PCR in a wide variety of sample types, including
environmental samples, feces, embryos (presumably reflecting fecal contamination), and
whole frozen zebrafish. Fecal samples, which are sensitive for viral detection, can be easily
collected from small groups of adult zebrafish and are useful as an antemortem test for
valuable zebrafish lines or fish held in quarantine. To prevent potential adverse impacts on
research from an unrecognized viral infection, inclusion of ZfPV-1 is recommended for colony
health monitoring and quarantine testing.

OC12S4

SEX SPECIFIC MODEL OF CHRONIC STRESS

Marta Balog1, Ž. Debeljak2, D. Mandić2, S. Blažetić3, I. Labak3, V. Ivić1, M. Zjalić1 and A.

Bardak1

1Department of Medical Biology and Genetics, Faculty of Medicine, Osijek, Croatia

2Clinical Chemistry Department, University Hospital, Osijek, Croatia

3Department of Biology, University of J. J. Strossmayer, Osijek, Croatia

Abstract

Introduction: Chronic stress (CS) research in rodents is often incomparable because of different
protocols used and is also biased because mostly males are used. We developed a protocol of
CS in male and female rats validated by serum hormones analysis.

Methods: Male and Female Sprague-Dawley-CR rats were divided in young and old groups.
Protocol was performed through 10 weeks. Chronic stress was induced by combining various
physical and psychological stressors: disturbance of the circadian rhythm by lights and noise
during the night phase, restraint in metal tubes, exposure to cold, forced swimming and
rotation of rats in the cages using a laboratory shaker. To validate CS protocol serum was
collected from all animal groups at the begining and the end of study. Corticosterone,
progesterone and testosterone were analysed by mass spectrometry. Study was approved by
Croatian Ministry of Agriculture (602-04/14-08/06).

Results: Corticosterone was increased in young males and old females (p = 0.036) upon CS
compared to control groups at the end of the study. Comparison of baseline and end
measurement showed increase in corticosterone concentration in young males (p = 0.008) and
old females (p = 0.009). Progesterone increased in old females upon CS at the end of the study
(p = 0.018) while in young CS females progesterone decreased at the end measurement in
comparison with control group (p = 0.03). Testosterone increased in old CS males compared to
control group at the end of the study (p = 0.012).

Discussion and conclusion: CS protocol is validated by increased corticosterone in young males

and old females. Progesterone could be important in neuroprotection upon CS in old females
and testosterone in old males. These results imply gender and age differences in mechanisms
of chronic stress response.

Acknowledgement: This study has been funded by Croatian Science Foundation and This study
has been funded by European Union through European Regional Development Fund,
Operational Programme Competitiveness and Cohesion, grant agreement No.
KK.01.1.1.01.0007, CoRE – Neuro.

OD4S1

HOUSING AND HUSBANDRY OF ZEBRAFISH: THE CONTRIBUTION OF THE FELASA WORKING

GROUP

Livia D'Angelo

Dept Veterinary Medicine and Animal Productions, University of Naples Federico II, Napoli,
Italy

Abstract

Zebrafish (Danio rerio) are increasingly used as vertebrate organisms to model key questions
raised in basic and applied research including, but not limited to biomedicine, toxicology,
environmental science, biotechnology and aquaculture. In Europe, zebrafish research is
covered by the European Directive 2010/63. The Annex 3 of the Directive, on “Requirements
for establishments and for the care and accommodation of animals”, in the Part B does
provide only general requirements. Currently, numerous and varying husbandry procedures
are available from the different laboratories/facilities housing zebrafish. However,
standardisation protocols, providing ranges of husbandry parameters, are necessary to ensure
the animal welfare as well as reproducibility of experimental procedures. A joint working
group (WG) on zebrafish housing and husbandry recommendations, composed of members of
the European Society for Fish Models in Biology and Medicine (EUFishBioMed) and of the
Federation of Laboratory Animal Science Associations (FELASA) has been given a mandate to
address a FELASA list of parameters, "Terms of Reference", and give recommendations for
related guidelines. These recommendations for laboratory zebrafish care and use can help the
further implementation and the fulfilment of Annex 3, Part B of EU Directive 2010/63, both
concerning the housing and care of experimental animals.
OD4S2

FISHES ARE NOT WET MICE: HOW TO ESTABLISH A FISH FACILITY FULFILLING WELFARE
REQUIREMENTS

D. Soroldoni1, C. Jollivet1,2, F. Lang1, A.C. Oates2 and Xavier Warot1

1School of Life Sciences – Center of PhenoGenomics, Ecole Polytechnique Fédérale de

Lausanne – EPFL, Lausanne, Switzerland

2EPFL SV IBI UPOATES, Ecole Polytechnique Fédérale de Lausanne – EPFL, Lausanne,

Switzerland

Abstract

Working with the Zebrafish Danio rerio as an animal model was initiated at the Ecole
Polytechnique Fédérale de Lausanne in 2017, to support the development of new paradigms.
To this end, a new fishroom was built to meet the highest standards in cutting-edge research,
husbandry and animal welfare. Special attention was given to the design of the water supply
system, to ensure the highest quality of water delivered daily into the tanks. Similarly, the
facility was designed in such a way that sentinel animals tanks and quarantine tanks could be
integrated into the main water circuitry. The food delivery system was tailored to the
physiological needs of zebrafish, relying on recent developments of robotic feeding devices.
Those choices proved to be sound, as shown by the survival rate, the breeding efficiency and
growth rates of the fishes. Once established, our facility was accredited by the Cantonal
Veterinarian Authorities, according to the requirements of the Swiss Animal Welfare Act and
ordinances on Animal Protection and Experimentation. During this process we realized that
fishes were often considered as “wet mice” due to the lack of species-specific guidelines. The
Authorities paid close attention to the enrichment of the tanks, to the anesthesia and
euthanasia methods, and to the characterization of the transgenic lines exhibiting a
pathological phenotype. These regulatory requirements revealed the lack of deep scientific
knowledge in these fields, and the needs for more precise guidelines, which should be
addressed by the Zebrafishes research community in a coordinated way with the authorities.

OD4S4

CHALLENGES IN THE MAINTENANCE AND BREEDING OF WILD ZEBRAFISH IN A LABORATORY

ENVIRONMENT

Anita Rácz1,2, T. Dwyer2 and S.S. Killen2

1Department of Genetics, Eötvös Loránd University, Budapest, Hungary

2Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical,

Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom

Abstract

Zebrafish, Danio rerio, is a widely used model organism for a range of research topics. While
the majority of this work has been conducted using domesticated strains, there is an emerging
interest in the use of wild-origin zebrafish in research. The behaviour and physiology of wild
zebrafish is vastly different from domesticated strains and so traditional breeding and
maintenance practices are generally unsuitable. Here we outline three broad challenges and
solutions that have emerged during our recent work with a population of wild zebrafish. First,
the wild fish appeared agitated in the laboratory environment and so a step-by-step re-
housing protocol was required to acclimate wild fish to being kept in smaller tanks. The use of
enrichment (substrate and plants) aided in this process. Second, wild-origin zebrafish were not
amenable to breeding practices which are generally applied to domesticated strains. However,
placing domesticated fish in neighbouring spawning tanks triggered courting behaviour in wild
fish and resulted in a six-fold increase in the number of egg laying wild couples. The final
challenge was managing pathogens present among the wild fish. Histological analysis of wild
fish revealed the presence of mycobacteriosis. Here we present data on the efficacy (measured
by egg survival rate) of different methods for egg disinfection (NaClO; PVP-I; KMnO4) at a
range of concentrations and soak times, to prevent transmission of Mycobacterium spp. to the
next generation via waterborne exposure. These results will aid other facilities in overcoming
the challenges encountered while working with either wild zebrafish or domesticated lines.

OD6S1

ZEBRAFISH HEALTH MONITORING: RECOMMENDATIONS FROM THE FELASA-AALAS

WORKING GROUP

Jean-Philippe Mocho1, Chereen Collymore2, S. Farmer3, K. Murray4, E. Leguay5 and Nuno

Pereira6,7,8

1Joint Production System Ltd, Potters Bar, United Kingdom

2University of Ottawa, Ottawa, Canada

3University of Alabama, Birmingham (AL), United States

4Zebrafish International Resource Center, Eugene (OR), United States

5Vetofish, Châteauneuf-les-Martigues, France

6Instituto Gulbenkian de Ciência, Oeiras, Portugal

7Nova Medical School/CEDOC, Lisboa, Portugal

8ISPA, Instituto Universitário, Lisboa, Portugal

Abstract

When exchanging fish for research, facilities open their doors to a potential hazard as incoming
fish may carry relevant pathogens. To help mitigate that risk, FELASA and AALAS established a
joint working group, with three representatives of each association, to develop
recommendations on how to define the health status of a zebrafish colony, how to report the
screening data, and how to triage imports according to a facility’s specificity.

The working group started with a survey on current practices and some analysis of estimated
prevalence. Then it defined the list of relevant pathogens and the screening pattern
(frequency, type, and number of samples and assays) for routine health monitoring (based on
hypothesis of higher prevalence of pathogens). Lastly, a reporting template for historical data
and facility description allowing for biosecurity risk assessment was developed.
Some scenarios will be used to illustrate how various types of facilities can adopt and adapt
the recommendations. This leads to the question of reliable barriers for different aquatic
systems; specifically how to isolate a quarantine area – a requirement for Specific Pathogen
Free (SPF) status. Screening patterns for SPF status are then described, based on a hypothesis
of low prevalence of specific pathogens, requiring a higher number of samples. For all
screening patterns, environmental testing is considered as it can be a useful adjunct to fish
sampling.

OD6S3

OVERVIEW OF AKKAB AND NEXT TARGET: RECOMMENDATIONS AND GUIDELINES FOR

CLEANING AQUATIC HOUSING SYSTEMS

Florian Kellner-Fendt

AK KAB Arbeitskreis Käfigaufbereitung / Working Group for Cage Processing c/o TECNIPLAST
Deutschland GmbH, Hohenpeissenberg, Germany

Abstract

For more than 12 years the German Working Group for Cage Processing “AK KAB“ (Arbeitskreis
Käfigaufbereitung) has been working on summarizing and publishing state of the art
recommendations and guidelines for cleaning rodent cages in a proper way. AK KAB already
published 5 editions of their so called “orange booklet” in both German and English, either
available in printed or digital format (the latter available from the FELASA-website).

Besides rodents as main species in many laboratory animal facilities around the globe, more
and more fish or frogs are being used for research as well.

To address the specific needs of aquatic users, the AK KAB recently founded a new working
group, consisting of experts for cleaning aquatic equipment. This group inside the AK KAB has
now been working for one and a half years, putting together important specific aspects of
aquatics housing systems inside biomedical research laboratories. The ultimate goal is to
provide recommendations and guidelines for processing aquatics housing systems in a proper
way.

The group of experts will present a first structure and initial contents of this new planned “Fish
Booklet“. The idea of this presentation is not to present a final edition of the fish booklet, but
to get feedback from the interested audience regarding the planned contents in order to
understand what may still be missing or needs more details. The plan is to publish and present
the final new booklet in 2020, ready in time for the 15th International Zebrafish Conference.

OD7S2