Volume 54 Number 2 April 2020 ISSN 0023-6772 Laboratory ......J-P Mocho El XV Congreso de la SECAL (Sociedad Espan˜ola para las Ciencias del Animal de Laboratorio) tuvo lugar en Sevilla

Volume 54 Number 2 April 2020 ISSN 0023-6772

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Laboratory AnimalsTHE INTERNATIONAL JOURNAL OF LABORATORY ANIMAL SCIENCE, MEDICINE, TECHNOLOGY AND WELFARE

Official Journal of AFSTAL, DALAS, ECLAM, ESLAV, FELASA, GV-SOLAS, ILAF, LASA, NVP, SECAL, SGV, SPCAL

Volume 54 Number 2 April 2020

Contents

Working Party Report

Genetic quality assurance and genetic monitoring of laboratory mice and rats: FELASA WorkingGroup Report 135F Benavides, T Rulicke, J-B Prins, J Bussell, F Scavizzi, P Cinelli, Y Herault and D Wedekind

Editorial

Collection on score sheets, severity assessment and humane end points: Invitation to submit 149P Jirkof, G Jarvis and B Riederer

Severity Assessment in Animal-based Research

Impulse for animal welfare outside the experiment 150L Lewejohann, K Schwabe, C Hager and P Jirkof

Original Articles

Diet-regulated behavior: FVB/N mice fed a lean diet exhibit increased nocturnal bouts ofaggression between littermates 159MM Murph, S Liu, W Jia, H Nguyen, MA MacFarlane, SS Smyth, SS Kuppa and KK Dobbin

Combination of ketamine and xylazine with opioids and acepromazine in rats: Physiologicalchanges and their analgesic effect analysed by ultrasonic vocalization 171J Aleman-Laporte, LA Bandini, MSA Garcia-Gomes, DA Zanatto, DT Fantoni, MAA Pereira, PE Navas-Suarez,

TB Kirsten, RR Jimenez, G Alvarado and CC Mori

Examining compliance with ethical standards for animal research: is there a need for refinement?A qualitative study from northern Europe 183A Brønstad and P Sandøe

News

3Rs – Reduce Reuse Recycle 194J-P Mocho

El XV Congreso de la SECAL (Sociedad Espanola para las Ciencias del Animal de Laboratorio) tuvolugar en Sevilla del 6 al 8 de noviembre 196E Hevia and CO Pintado

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Thanks to Reviewers 198

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Laboratory Animals

Editorial BoardEditor-in-Chief B RiedererDeputy Editors G Jarvis, P Jirkof

Section Section EditorsAnaesthesia, Analgesia,Pain & Stress

M Leach, P Foley,P Hedenqvist

Anatomy and Neuroscience B Riederer, S Wells (neuro)Aquatic Organisms K Finger-Baier (fish),

JP Mocho, M CrimBehaviour D Preissmann, M Gyger,

L LewejohannBiostatisics & ExperimentalDesign

R-D Gosselin, H Wurbel

Education P Vergara, C Thone-ReinekeImaging Techniques L van der Weerd, J TremoledaLarge Animal Models M Jensen-Waern, D Anderson,

T MorrisManagement of Animal Facilities J-B Prins, M DennisMolecular & Genetic Engineering T Ruelicke, P CinelliNutrition and Diets G Tobin, T NorteyPathology & Microbiology P Clements, D Salvatori,

A BleichPhysiology & Clinical Chemistry M SommersPrimates G Rainer, P Honess, C Witham3Rs & Ethics G Griffin, A OlssonReproductive Biology H Hedrich, B Pintado,

C GilbertSmall Animal Models M Berard, J-B Prins

(temporary), S WellsSurgical Procedures D Bouard, R TolbaSystematic Review M Ritskes-Hoitinga,

BS KousholtToxicology F RuttenVeterinary Medicine E Rivera, J Sanchez-Morgado,

L Whitfield,N Kostomitsopoulos

Special Issue Microbiota guest editor:Axel Kornerup-Hansen andCraig Franklin

Comment and correspondence relating to editorial matters may besent to the Chairman of the Editorial Board by email: [email protected]; or post: LAL, PO Box 373, Eye, Suffolk, IP22 9BS,UK.See also http://www.lal.org.uk

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125

AFSTALAssociation Francaise des Sciences etTechniques de I’Animal de Laboratoire

PresidentSebastian Paturance

Vice PresidentElodie Bouchoux

Secretariat: 28, rue Saint Dominique, 75007,Paris, France(www.afstal.com)

DALASDutch Association for Laboratory AnimalScience

PresidentCatriene Thuring

SecretaryLinda DerksEnergieweg 196541 CW Nijmegen([email protected])

ECLAMEuropean College of Laboratory AnimalMedicine

PresidentPatricia Hedenqvist

Secretariat: Janet Rodgers, 266Banbury Road, No. 314 OxfordOX2 7DL, UK

ESLAVEuropean Society of Laboratory AnimalVeterinarians

PresidentPeter Glerup

Honorary SecretaryMassimiliano Bardotti

Honorary SecretaryFrederic Decrock

Secretariatc/o Decrock, 78,bd Gallieni92130 Issy les Moulineaux, France(http://eslav.org)

FELASAFederation of EuropeanLaboratory Animal Science Associations

PresidentHanna-Marja Voipio

President-electAna Santos

Past PresidentHeinz Brandstetter

Hon. SecretaryJean-Philippe Mocho

Secretariat: PO Box 372,Eye, IP22 9BR, UK(www.felasa.eu)

GV-SOLASGesellschaft fur Versuchstierkunde(Society for Laboratory Animal Science)

PresidentBettina Kraenzlin

SecretaryNicole LinklaterFaculty of BiologyPhilipps UniversityKarl-von-Frisch Str. 835043 MarburgGermany(www.gv-solas.de)

ILAFIsraeli Laboratory Animal Forum

PresidentAmir Rosner

SecretaryDavid CastelNeufeld Cardiac ResearchInstituteSheba Medical CenterTel Hashomer 52621Israel(www.ilaf.org.il)

LASALaboratory Animal Science Association

PresidentAnne-Marie Farmer

Secretary GeneralMiles Maxwell

PO Box 524, Hull,HU9 9HE, UK(www.lasa.co.uk)

NVPNederlandse Vereniging voorProefdierkunde(Dutch Association for Laboratory AnimalScience)

PresidentMartje Fentener van Vlissingen

SecretaryJan LangemansBPRCLange Kleiweg 1392288 GJ Rijswik

The Netherlands(www.proefdierkunde.nl)

SECALSociedad Espanola para las Ciencias delAnimal de Laboratorio(Spanish Society for Laboratory AnimalScience)

PresidentIsabel Blanco Gutierrez

Vice PresidentJuan Rodriguez Cuesta

SecretaryJulia M. Samos Juarez

TreasurerViviana bisbal Velasco

Secretariat: c/Maestro Ripoll, 8,28006 Madrid,Spain(www.secal.es)

SGVSchweizerische Gesellschaft furVersuchstierkundeSociete Suisse pour la Science des Animaux deLaboratoire (Swiss Laboratory Animal ScienceAssociation)

PresidentDr. Birgit Ledermann

SecretaryDr. med. vet. Andrina ZbindenFaculty of Science and MedicineUniversity of FribourgCh. du Musee 8CH-1700 Fribourg, Switzerland(www.naturalsciences.ch/organisations/sgv)

SPCALSociedade Portuguesa de Cienciasem Animais de Laboratorio(Portuguese Society for Laboratory AnimalScience)

PresidentIsabel Vitoria Figueiredo

Vice-PresidentRicardo Afonso

SecretaryCatarina Pinto Reis

Secretariat: Laboratorio deFarmacologiaFaculdade de FarmaciaLargo de D. Dinis3000 CoimbraPortugal(www.spcal.pt)

126...............................................................................................................................................................

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Verify authenticity and uniformity of inbred strains and substrains Detect potential genetic contamination Describe precise genetic composition of genetically modified lines

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backcrossing projects Quantify and control genetic diversity of outbred strains

Working Party Report

Genetic quality assurance and geneticmonitoring of laboratory mice and rats:FELASA Working Group Report

Fernando Benavides1 , Thomas Rulicke2 , Jan-Bas Prins3,4,James Bussell5, Ferdinando Scavizzi6, Paolo Cinelli7,Yann Herault8,9 and Dirk Wedekind10

AbstractGenetic quality assurance (QA), including genetic monitoring (GeMo) of inbred strains and background char-acterization (BC) of genetically altered (GA) animal models, should be an essential component of any QAprogramme in laboratory animal facilities. Genetic quality control is as important for ensuring the validity ofthe animal model as health and microbiology monitoring are. It should be required that studies using labora-tory rodents, mainly mice and rats, utilize genetically defined animals. This paper, presented by the FELASAWorking Group on Genetic Quality Assurance and Genetic Monitoring of Laboratory Murines, describes theobjectives of and available methods for genetic QA programmes in rodent facilities. The main goals of anygenetic QA programme are: (a) to verify the authenticity and uniformity of inbred stains and substrains, thusensuring a genetically reliable colony maintenance; (b) to detect possible genetic contamination; and (c) toprecisely describe the genetic composition of GA lines. While this publication focuses mainly on mouse andrat genetic QA, the principles will apply to other rodent species some of which are briefly mentioned within thecontext of inbred and outbred stocks.

Keywordsanimal facilities, genetics, quality assurance/control, refinement, rodents

Date received: 19 March 2019; accepted: 13 July 2019

Standardized laboratory rodents

Inbred strains

The International Committee on Standardized GeneticNomenclature for Mice and The Rat GenomeNomenclature Committee considers a strain inbred

if it has been propagated by systematically mating

brothers to sisters (or younger parent to offspring) for

20 or more consecutive generations, and individuals of

the strain can be traced to a single ancestral pair at the

twentieth or subsequent generation.

At this point, animals within the population will aver-age�2% residual heterozygosity, and the individualsmay be regarded as genetically identical (isogenic).1

However, it has been estimated that 24 generations of

1Department of Epigenetics and Molecular Carcinogenesis, TheUniversity of Texas, MD Anderson Cancer Center, USA2Institute of Laboratory Animal Science, University of VeterinaryMedicine, Vienna, Austria3The Francis Crick Institute, London, UK4Leiden University Medical Centre, Leiden, The Netherlands5Biomedical and Veterinary Services Department, University ofOxford, Oxford, UK6National Research Council (IBCN), Rome, Italy7Department of Trauma Surgery, University of Zurich, Zurich,Switzerland8Universite de Strasbourg, CNRS, INSERM, Institut de GenetiqueBiologie Moleculaire et Cellulaire, IGBMC, Illkirch, France9Universite de Strasbourg, CNRS, INSERM, Institut Clinique de laSouris, CELPHEDIA-PHENOMIN-ICS, Illkirch, France10Institute of Laboratory Animal Science, Hannover MedicalSchool, Hannover, Germany

Corresponding author:Fernando J. Benavides, Department of Epigenetics and MolecularCarcinogenesis, The University of Texas MD Anderson CancerCenter, 1808 Park Road 1C, Smithville, TX, 78957, USA.Email: [email protected]

Laboratory Animals

2020, Vol. 54(2) 135–148

! The Author(s) 2019

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sib-mating are needed to reach a heterozygosity rate-< 1% and 36 generations to reach (almost) completeisogeneity.2

Isogeneity implies histocompatibility, meaning thestrains are syngeneic. Syngeneic animals will perman-ently accept tissue transplants from any individual ofthe same strain and sex. Unlike cloned animals andmonozygotic twins (which are 100% identical for allgenomic loci), inbred rodents, besides being isogenic,are also homozygous at almost all genomic loci.Overall, each inbred strain represents a unique,although fortuitous, assortment of alleles.3 If a strainwere to be remade from scratch, using the same foun-ders, after the same 20 generations of inbreeding itwould create a genetically distinct strain due to therandom assortment and fixation of alleles. Baselinephenotypic data for the most common inbred mousestrains are available through a coordinated inter-national effort initiated by The Jackson Laboratoryand implemented through The Mouse PhenomeDatabase (http://phenome.jax.org/).4 An example ofbaseline phenotypic data is presented inSupplementary Tables 1A and 1B. The MouseGenome Informatics (MGI) website5 provides a list,compiled by Dr Michael Festing (http://www.informat-ics.jax.org/external/festing/search_form.cgi), of 420inbred mouse and 230 inbred rat strains (some ofwhich have been lost or terminated), along with briefdescriptions. The list includes widely used inbred mousestrains: A/J, BALB/c, C3H/He, C57BL/6, DBA/2,FVB/N and others; and rat strains: ACI, BN, F344,LE and WKY.

Outbred stocks

Outbred stocks are populations of laboratory animalsthat differ from inbred strains in that they are genetic-ally heterogeneous. Compared with inbred strains orF1 hybrids, the genetic constitution of a given animal,taken randomly from an outbred stock, is not known apriori. However, all of the animals in the group sharegroup characteristics (identity), such as being albino(although not all outbred mice or rats are albino),good breeders and relatively tame compared to otherstrains; features that make these animals very popularas foster mothers for assisted reproductive techniques.Examples of outbred stocks of mice are ICR (CD-1),CFW and NMRI (all derived from the original ‘Swiss’mice imported to the USA by Clara J. Lynch in 1926)and (non-Swiss) CF-1. Examples of outbred rat stocksare Sprague Dawley (SD), Wistar (WI) and Long-Evans (LE). Since outbred stocks are not geneticallydefined, quality control is commonly based on assessingexpected phenotypic traits, such as coat colour, growthand reproductive characteristics, based on data from

the large colonies of commercial breeders. Becauseoutbred colonies, like human populations, are hetero-geneous, they are frequently used in toxicology andpharmacology research.6 However, several geneticistshave disputed this use and have criticized studies inwhich outbred mice were used inappropriately, wastingboth animal lives and precious resources in suboptimalexperiments.7

Other standardized strains of mice and rats

F1 hybrids result from the outcross of two separateinbred strains and are heterozygous at all loci forwhich the parental strains harbour different alleles. F1littermates are genetically identical and are histocom-patible. Congenic strains are produced by crossing twostrains: the donor strain that carries the allele orchromosomal region of interest, and the recipient orbackground strain that will receive the locus of interest.F1 offspring generated by crossing donors and recipi-ents are then backcrossed to the recipient strain.Offspring that carry the allele of interest are identifiedand again backcrossed to the background strain. Thisprocess is typically repeated for 10 or more successivegenerations (Figure 1), unless marker-assisted back-crosses (speed congenics) are used. Repeated backcross-ing results in the chromosomes of the backgroundstrain progressively replacing those of the donorstrain, except for a chromosomal region that carriesthe allele of interest.

Genetically altered (GA) rodents

Before presenting the different types of GA rodents, itis worth mentioning that there are basically two differ-ent approaches to characterizing gene function.Forward genetics (from phenotype to genotype) aimsto characterize the gene alteration that is responsiblefor a specific mutant phenotype (typically from spon-taneous or chemically-induced mutations). Reverse gen-etics is the opposite approach and aims to characterizethe function of a gene by analysing the consequences (atthe phenotypic level) of alterations normally engineeredby researchers at the DNA level. This section intro-duces the four basic types of GA rodents, those createdby: (a) pronuclear microinjection, (b) vector- mediatedtransgenesis (c) homologous recombination in embry-onic stem (ES) cells, (d) gene editing nucleases, and (e)either chemically induced or spontaneous mutations.Detailed descriptions of the technologies used tocreate GAs have been published.8 Before selecting agene-editing technique to create a genetically modifiedanimal, it is important to check an appropriate data-base such as those hosted by The Jackson Laboratoriesand the International Mouse Phenotyping Consortium

136 Laboratory Animals 54(2)

http://phenome.jax.org/

http://www.informatics.jax.org/external/festing/search_form.cgi

http://www.informatics.jax.org/external/festing/search_form.cgi

as to whether a suitable animal model already exists(see Supplementary Table 2 for the complete list ofonline resources for laboratory mouse and rat strains).

Transgenesis by pronuclear microinjection

Transgenic mice were introduced in the early 1980s9

and were the first transgenic animals. It is advisable touse the term ‘transgenic’ only for animals whose gen-omes have been altered by the random insertion ofDNA. (There are numerous terms used to describe gen-etic changes in animals: genetically engineered mice(GEM) or genetically modified mice (GMM) are typic-ally used to describe any type of genetic modification inthe mouse. We use the term GA rodent here to alsoinclude those carrying spontaneous or chemicallyinduced mutations, and ‘line’ instead of ‘strain’ forGA rodents.) Transgenic rodents are almost exclusivelycreated by the pronuclear microinjection of foreignDNA fragments directly into one of the two pronucleiof one-cell embryos (zygote), a technique that is stillwidely used. In this process of additive transgenesis,the microinjected transgene randomly integrates intothe genome as a single copy or more often as a conca-temer with variable copy number. The mouse and ratmodels created with this system typically express or, inthe resultant concatemer, overexpress a transgeneplaced under the control of a tissue-specific, develop-mental-stage-specific, or ubiquitous promoter (alongwith other regulatory elements), all contained in thetransgene DNA construct.

The recommended generic symbol for a transgenicinsertion is Tg. The founder transgenic animals arehemizygous for the DNA segment and are designatedTg/0. Transgenes are extra segments of DNA that haveno corresponding ‘wild-type’ sequence in the unmodi-fied homologous chromosome in hemizygous animals,that is why the use of ‘0’ instead of ‘þ’ (typically used todenote wild-type alleles) is recommended. Each trans-genic line generated via random integration creates aunique animal model and each putative founder mustbe developed independently. Traditionally, to distin-guish between homozygous (Tg/Tg) and hemizygous(Tg/0) mice, the mouse of interest was crossed to anon-transgenic partner and the progeny were statistic-ally analysed for Mendelian segregation of thetransgene. A more modern technique uses quantitativereal-time polymerase chain reaction (qPCR) to distin-guish hemizygous from homozygous transgenic mice.10

In order to achieve a pure genetic background (recom-mended), the transgene must be introduced intoembryos derived from an inbred strain.

A later improvement on the constructs used in thetransgenesis approach was the introduction of induciblesystems in which transgene expression can be turned on

and off. Examples of this strategy are the Tet-on andTet-off expression systems. In these systems, transcrip-tion of a given transgene is placed under the control ofa tetracycline-controlled trans-activator protein, whichcan be regulated, both reversibly and quantitatively, byexposing the transgenic mice to either Tetracycline (Tc)or one of its derivatives, such as Doxycycline (Dox).Both Tet-on and Tet-off are binary systems that requirethe generation of double transgenic (bigenic) mice.11

Vector-mediated transgenesis

Alternative methods for transgenesis by random inte-gration are based on vectors of different origin. Mostimportant and very efficient are retroviral/lentiviral vec-tors12 and transposons.13 Also pre-treated spermatozoahave been successfully used as vectors in combinationwith ICSI (intracytoplasmic sperm injection).14 Eachtechnique has advantages and disadvantages and thecorresponding principle of transgene integration mayaffect the quality of the resulting GA models. Viral vec-tors and transposons for instance integrate as a singlecopy, however multiple integrations, randomly distrib-uted in the genome, are not uncommon. Major con-cerns exist regarding the impact of sperm-mediatedgene transfer on the sperm genetic material, possiblyinduced by the pre-treatment of spermatozoa.15

Targeted mutagenesis by homologousrecombination using ES cells

Another important technology utilizes murine ES celllines. ES cells are undifferentiated, pluripotent, embry-onic cells derived from the inner cell mass of pre-implantation blastocysts that can participate in formingthe germ-cell lineage of chimeric mice, an indispensablestep in generating founder mice carrying the targetedmutation. Historically, the first ES cell lines werederived from embryos of the 129 family (129S2,129P3, etc.), that is inbred strains originally bred forthe isolation of embryonic carcinoma (EC) cells. TodayES cell lines are available from many mouse strains andthose of the C57BL/6N origin have become widespreadand are often selected for trans-national projects (e.g.EUCOMM).

In cases where constitutive null alleles lead to com-plex phenotypes, reduced viability, or have other draw-backs, conditional alleles may be used, allowing one tocontrol the time and tissue where a gene is turned off,typically using the Cre/loxP system.16 Production ofconditional KOs requires two independent lines: oneproviding a source of Cre recombinase, an enzymederived from bacteriophage P1, in the tissue understudy, and another containing loxP (locus of X-ingover P1) sites flanking the DNA segment of interest

Benavides et al. 137

that needs to be crossed to generate double mutantmice. The Cre enzyme cuts and recombines the‘floxed’ DNA at loxP sites. The Cre transgene can bemade inducible, adding more sophistication to thesystem. The tamoxifen-inducible CreERT2 which canbe activated in a spatio-temporal manner by adminis-tration of tamoxifen, is widely used.17 The Cre-loxPstrategy can also be used to regulate the expression ofreporter genes. For example, the lacZ gene can bedriven by a ubiquitous promoter (e.g. Rosa 26) with afloxed stop sequence, containing several terminatorcodons inserted between the promoter and the lacZcoding sequence.

Gene editing using nucleases

Over the last 10 years, a number of new techniques havebeen developed for the production of targeted muta-tions using engineered nucleases. These techniques,

briefly described here, provide ES cell-independentmethods to create targeted mutations in laboratorymice, rats and other species.

To make mutations using zinc-finger nucleases(ZFN), two complementary and sequence-specificmulti-finger peptides containing the FokI nucleasedomain must be designed. Each peptide is designed torecognize a specific DNA sequence spanning 9–18 basepairs (bp) on either side of a 5–6 bp sequence, whichdefines the targeted region. When injected into a pro-nucleus or cytoplasm of zygotes, the ZFN assembliesbind tightly, one on each strand, on both sides of thetarget site. The dimerized FokI endonuclease then cre-ates double strand DNA breaks (DSBs) triggering cel-lular mechanisms to repair the damage. Damage isnormally repaired by either homology-directed repair(HDR) or non- homologous end joining (NHEJ).HDR requires a homologous template to guide therepair and thus re-establishes the original sequence.

Figure 1. This scheme represents the successive steps in the establishment of a congenic strain. The initial step is across between the donor strain (albino in the example) carrying the gene of interest (e.g. a targeted gene or a transgene)and a recipient or background strain (black in the example). At each generation, a breeder carrying the gene of interest (*)is backcrossed to a partner of the recipient strain (genetically linked genes are transferred with it and the size of theintrogressed fragment can be many thousands or millions of bases, and include many genes). The degree of grey colourindicates that, after each backcross generation, the offspring have an increased amount of the background genome(average percentage is indicated in each N generation). When the modified gene is not resulting in an easily recognizablephenotype (e.g. skin or behavioural changes), molecular genotyping is necessary to select the carrier (heterozygous) mice.


NHEJ is much less precise and cause nucleotide dele-tions that lead to frame shifts that create potential loss-of-function or truncation mutations. Mice and ratscarrying null alleles or sequence-specific modificationshave already been produced using ZFN technology.18

Like ZFNs, transcription activator-like effector nucle-ase (TALEN) technology involves the combination of anonspecific DNA endonuclease fused to a DNA-bind-ing domain, but can be more easily engineered (com-pared to ZFN) to target a particular DNA sequence.

The CRISPR (clusters of regularly interspaced shortpalindromic repeats)/Cas system, commonly imple-mented as CRISPR/Cas9, is based on a primitivedefence mechanism that allows bacteria and archaeato fight against infection from viruses, plasmids andphages.19 CRISPR-based guide RNAs (gRNAs) aredesigned to target a Cas endonuclease to cut DNA atthe desired site through RNA-guided DNA cleavage.The RNA-guided endonucleases can be engineered tocleave virtually any DNA sequence by appropriatelydesigning the gRNA, for example to generate KOmice.20 CRISPR/Cas technology has several advan-tages over ZFNs and TALENs. The main advantageis the ease of design and the flexibility of using asequence-specific RNA interacting with the Casenzyme instead of a complex sequence-specific protein(DNA-binding domain) fused to a nuclease. Also,mutations in multiple genes can be generated in asingle step by injecting mice with multiple gRNAsthat simultaneously target different genes.21 Such multi-plex gene editing has been successful in cells, as well asmouse and rat embryos.20 CRISPR/Cas9 has been usedto create insertions, deletions and point mutations. Thesystem is highly flexible, fast and efficient, and is revo-lutionizing genomic engineering in mammals.22 Itallows making KO and KI lines in any genetic back-ground. DNA can be electroporated (with size restric-tions) or injected into either the cytoplasm or pronucleiof 1-cell or 2-cell stage embryos, thus avoiding the useof ES cells and chimeras. However, as each engineeredanimal is unique, this technology requires extensivesequence analysis to characterize multiple putativefounders to ensure the presence of the desired mutationand the absence of undesired on- and off-target muta-tions or unpredictable larger genome alterations,23,24

while also identifying mosaic founders (G0). Once iden-tified, the selected founder should be bred with wild-type animals to evaluate transmission of the mutation.

Spontaneous and chemically-inducedmutations

A list of GA rodent types is not complete withoutincluding both spontaneous and chemically-inducedmutations. Spontaneous mutations, generally identified

through the observation of an abnormal phenotype,present several advantages. First and foremost, theyare produced at virtually no cost and are generallyfreely available. Second, they usually have an obviousphenotype, as they are identified based on observation.Third, spontaneous mutations represent a great varietyof molecular events, such as deletions, insertions andpoint mutations, generating not only loss-of-functionalleles but also hypomorphic and hypermorphic alleles.Finally, mutations arise in a variety of backgroundsincluding inbred strains and outbred stocks. Severalspontaneous mutations have provided rodent modelsfor human conditions. These include classical muta-tions such as, nude (Foxn1nu), scid (Prkdcscid), hairless(Hrhr), diabetes (Leprdb), obese (Lepob) and X-linkedmuscular dystrophy (Dmdmdx) in the mouse; and themutations behind the Rowett nude (Foxn1rnu) andZucker diabetic fatty (Leprfa) models in the rat.

The discovery of the extraordinary virtues of thealkylating agent N-ethyl-N-nitroso urea (ENU) as amutagen was a milestone in the history of mouse gen-etics. Researchers using ENU have generated and pro-pagated numerous mutant alleles for protein-codinggenes, thus establishing a precious tool for genomeannotation. Because ENU typically creates point muta-tions, it has been widely used in forward geneticscreens. The major drawback of ENU-induced muta-genesis is that it creates random mutations rather thantargeted mutations. Several projects have been under-taken to systematically and extensively phenotype theoffspring of ENU-mutagenized males. Large ENUmutagenesis programmes have been conducted inGermany, England and the USA.25

Quality assurance and exchangeof GA-rodents

What to ensure after (in-house) generation or uponarrival? The possibility of crossing different GA linescombined with the increasing complexity of targetingapproaches has greatly increased the number of avail-able GA models. The need to cross different GA linestogether for a particular study generates additionalcomplexity, especially at the genetic background level.Many mutants have been and are still generated on ahybrid genetic background. Therefore, it is essential tokeep adequate records of detailed information for allgenetically modified strains. This information must betransferred with the strain to all collaborators andusers. The most important information includes thecorrect strain name, a complete description of themutation, the genetic background of the animals, agenotyping protocol and observed phenotypic changes.Together, these provide the minimum information forthe recommended ‘rodent-passport’, and several forms


have been designed for cataloguing this information.We recommend the data sheet developed by theFELASA Working group on the refinement of methodsfor genotyping genetically modified rodents.26

Every mutant strain name must provide preciseinformation on the affected gene, the type of mutationand the genetic background. For in-house generatedstrains, one must provide a specific Institute forLaboratory Animal Research Laboratory (ILAR)Code Registration for the laboratory where themutant originated. An overview on the importance ofnomenclature can be found in the ‘FELASA guidelinesfor the production and nomenclature of transgenicrodents’.27 A name designed according to the inter-national nomenclature rules is the only means to unam-biguously distinguish strains from each other. This isimportant when the same strain is held in different facil-ities around the world and/or they are listed in archivesand databases. Further, it is imperative that strains beproperly described in publications using a universalnomenclature. Without a common nomenclature, itbecomes impossible to accurately communicate scien-tific results. Vague or incomplete names create errorsrendering experiments irreproducible.

Origin and consequences of geneticvariation

A serious challenge facing rodent animal facilities iskeeping inbred strains genetically pure and GA lineson a defined background. Changes in the genetic con-stitution of inbred strains can be produced by (a) con-tamination by accidental outcrosses and (b) geneticdrift due to residual heterozygosity or fixation of denovo spontaneous mutations.

Genetic contamination

The accidental mating of individuals from one inbredstrain with animals of another origin is by far the mostimportant source of genetic profile alteration in inbredstrains. Genetic contamination of this type, whichalways results in a sudden and massive exchange ofalleles, is more likely between strains that have similarcoat colour (i.e. albino (Tyrc/Tyrc), agouti (A/A), ornon-agouti (a/a)). Where lines have the same coatcolour alleles, extra care must be taken when housingthem in close proximity of each other.

Spontaneous mutations and polymorphisms

Spontaneous mutations are a source of uncontrolledgenetic variation that is often impossible to detect bysimple phenotypic observation or routine genetic moni-toring (GeMo). Genetic polymorphism is the presence of

alternative DNA sequences (alleles) at a locus amongindividuals, groups, or populations, at a frequency>1%. Two types of genetic markers are commonlyused in association studies and genetic quality control:microsatellites and single nucleotide polymorphisms(SNPs) (see ‘Marker systems’ below).

Genetic drift and the generationof substrains

While permanent inbreeding effectively eliminates aproportion of new mutant alleles, another undetectedfraction may become progressively fixed in the homo-zygous state, replacing the original allele, a processknown as genetic drift. Genetic drift contributes inex-orably to strain divergence and the generation of sub-strains when the same strain is propagatedindependently in different places.28 Examples ofmouse substrains are abundant, for example there arec. 10 documented BALB/c substrains and c. 15 C57BL/6 substrains including the J and N substrains from TheJackson Laboratory (Jax) and the National Institutesof Health (NIH), respectively.29 In the same way, manyrat inbred strains present at least two substrains, forexample SHR has at least four substrains (includingSHR/Ola and SHR/NCrl), and WKY and F344 haveat least three substrains each. Substrain variability hasbeen confirmed by sequencing analysis for these ratsubstrains,30 with WKY showing the highest degreeof substrain variation (this is in part due to thesupply of the model prior to the prescribed 20 gener-ation inbreeding requirement).

Undesirable passenger mutations

Mutations that are hidden in the genomes of substrainsor GA lines and can affect the outcome of an experi-ment are sometimes referred to as passenger muta-tions.31 There are many examples in the literaturewhere substrains originating from the same inbredstrain have acquired new phenotypes as a consequenceof genetic drift.32 For example, mice of the C57BL/6JOlaHsd substrain are homozygous for a deletion ofthe a-synuclein (Snca) and multimerin (Mnrn1)genes.33,34 Likewise, some spontaneous mutations dif-ferentially segregate in C57BL/6J and C57BL/6N, themost common substrains of C57BL/6, separated in1951. These include a retinal degeneration mutationin the Crb1 gene (Crb1rd8), present only in the N sub-strain, and a deletion in the Nnt gene, present only inthe J substrain.35,36 Berghe and colleagues recentlyreported that passenger mutations are also commonin most GA lines derived from 129 ES cells, and thatthese mutations persist even after the creation of fullycongenic strains.37 This is not trivial; Berghe et al.


estimated that close to 1000 protein-coding genes couldbe aberrantly expressed in the 129-derived chromo-somal segments that are still segregating in these con-genic lines. This finding emphasizes the need forproperly chosen control animals to identify phenotypesdue to background mutations or the combination ofbackground mutations and the genetic modification ofinterest, rather than the modification itself.

Importance of using standardnomenclature

Rules guiding nomenclature were established by theInternational Committee on Standardized GeneticNomenclature for Mice and Rats and are continuouslyupdated. These rules, last revised in January 2016, aredescribed on the MGI webpage under ‘Guidelines forNomenclature of Mouse and Rat Strains’ (http://www.informatics.jax.org/mgihome/nomen/strains.shtml). Ahelpful and visual Mouse Nomenclature Quick Guideis available at https://www.jax.org/jax-mice-and-ser-vices/customer-support/technical-support/genetics-and-nomenclature#. For more details on nomenclaturerefer to the Supplementary material.

Genetic quality control programmes

The current gold standard for genetic quality control oflaboratory rodents depends on polymorphic geneticmarkers to distinguish between different genetic back-grounds. Genetic markers are specific DNA sequenceswith a known location on a chromosome and are essen-tial tools for genetic quality control. Genetic qualitycontrol is essential to determine the genetic compos-ition of an animal and to screen for uniformity andauthenticity of a strain. Please note that outbred colo-nies cannot be tested for authenticity. Instead, thecolony is screened for its level of genetic heterogeneityto detect genetic contamination and to monitor the pro-gress of breeding programmes and to select futurebreeders.

Marker systems

Many polymorphisms have been described in themouse and rat; however, only microsatellites andSNPs are used as genetic markers in current QA pro-grammes. Microsatellite markers, also known as SimpleSequence Length Polymorphisms (SSLPs) or ShortTandem Repeats (STRs), are still used in modernGeMo programmes because they are inexpensive andeasy to type.38,39 Animals are genotyped by analysingPCR-products amplified from short, tandemlyarranged, repeating DNA sequences. These repeatsare typically 2–6 bp long and are repeated a few to

dozens of times creating allelic diversity among stains.Genomic DNA primers are designed to uniquesequences flanking the repeats. The PCR products, typ-ically around 100–300 bp in size, are analysed usingagarose or polyacrylamide gel electrophoresis. TheMGI webpage has comprehensive SSLP information,including primer sequences and size variations in bpfor several inbred mouse strains (http://www.informat-ics.jax.org/marker). A collection of mapped, highlypolymorphic, SSLP markers for inbred laboratory ratstrains is available in The National BioResourceProject – Rat database and is linked to the MapReport of the Rat Genome Database (RGD) (http://rgd.mcw.edu). See Supplementary Table 2 for the com-plete list of online resources for laboratory mouse andrat strains.

SNP genotyping is an alternative to microsatellitesthat is now widely used for GeMo. SNP genotyping isinexpensive and can be performed in most researchinstitutions or outsourced. SNPs are the mostcommon genetic variation and exist in both codingand non-coding regions. Almost all SNPs are bi-allelic,presenting one of only two possible nucleotides (e.g.homozygous G/G or T/T), or both (e.g. heterozygousG/T) in an individual. Petkov and co-workers from TheJackson Laboratory have described the allelic distribu-tion of 235 SNPs in 48 mouse strains and selected apanel of 28 SNPs sufficient to characterize the majorityof the c. 300 inbred, wild-derived, congenic, consomicand recombinant inbred strains maintained at TheJackson Laboratory.40 Several publications havereported useful SNPs for the rat. For example,Zimdahl and colleagues described a map with>12,000 gene-based SNPs from transcribed regions.41

GeMo of inbred strains and outbred stocks

Most GeMo techniques used currently are based onmicrosatellites or SNPs. However, GeMo should notrely solely on molecular techniques, but should take abroader view that includes phenotypic parameters suchas coat colour, behaviour and breeding performance.Commercial breeders are extremely sensitized to therisk of genetic contamination and regularly monitortheir strains for genetic contamination, but not neces-sarily genetic drift, by using different sets of SNPs tomonitor their nucleus colonies. The JacksonLaboratory incorporated a unique, patented, GeneticStability Program42 designed to effectively limit cumu-lative genetic drift by rebuilding their foundation stocksfrom pedigreed, cryopreserved embryos every five gen-erations. For example, starting in 2005, they began sell-ing only C57BL/6J mice derived from two chosen micethrough hundreds of frozen embryos of the duo’sgrandchildren (enough to last for 25–30 years). It


http://www.informatics.jax.org/mgihome/nomen/strains.shtml

http://www.informatics.jax.org/mgihome/nomen/strains.shtml

https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genetics-and-nomenclature#



http://www.informatics.jax.org/marker

http://www.informatics.jax.org/marker

http://rgd.mcw.edu

http://rgd.mcw.edu

should be noted that when recovering strains fromfrozen stocks good GeMo should be carried out toassure oneself that genetic contamination or wronggenotypes were not present prior to freezing.

For outbred stocks, GeMo helps preserve the geneticheterogeneity and allele pool of a colony. This complexprocess requires analysing a large number of animalsand comparing this data with historical data document-ing the alleles present, their frequency and the level ofheterozygosity in that particular colony. In some cases,the results can reveal a loss of genetic variability result-ing in a colony with very low heterogeneity. The degreeof genetic heterogeneity in outbred colonies depends ontheir history. Low heterogeneity can result from poorselection of future breeding stock, deviation fromapproved (rotational) breeding systems or the bottle-neck effect caused by a small breeding pool, as iscommon when a small group of breeders is importedor being used to rederive a colony. In contrast, highheterogeneity can result from a recent outcross. In gen-eral, outbred stocks are characterized by measuringphenotypic traits and calculating the correspondingmean and standard deviations. Essentially, genetic con-trol of outbred stocks is directed at avoiding inbreedingand stabilizing genetic diversity over many generations.

GeMo of inbred mice and rats bred in-house. Thebest recommendation here is to purchase animalsfrom reliable vendors and replace the breeding stockwith animals from the same vendor after 10 gener-ations, rather than to maintain independent coloniesof classical inbred strains. As an additional benefit,using animals from the same vendor prevents the for-mation of substrains harbouring potential mutationsand maintains a similar microbiome. Nevertheless, in-house colonies should always be tested with a small setof informative microsatellite markers or SNPs to con-firm integrity.

Using a small panel of microsatellites(SSLPs). Microsatellites can be used to verify that theanimals in an inbred colony are essentially pure, withno traces of genetic contamination. This is especiallyimportant in facilities that maintain strains with thesame coat colour in the same room, a particularly dan-gerous practice especially when not using individuallyventilated cage (IVC) systems. Microsatellite testingcan normally be performed in-house. The number ofmarkers to use for testing has not been standardized:each situation and facility differs in how many andwhich strains are kept. Nonetheless, a panel of 40 poly-morphic SSLPs, evenly distributed across the auto-somes, will rule out recent genetic contamination, ifthe markers can distinguish among the strains beinganalysed. Supplementary Table 3 presents a small set

of mouse SSLPs that could be used to authenticatesome classical inbred strains.

Interpreting SSLP data is straightforward. Becauseinbred animals are isogenic and homozygous, they willpresent only one band in the electrophoresis gel, repre-senting a single allele, when genotyped for a particularSSLP. The presence of any heterozygosity, indicated bytwo bands, or bands that do not coincide with those ofthe strain control DNA, should be considered as indi-cating potential strain contamination (Figure 2). Howfrequently colony strain identity should be evaluateddepends on the size of the colony, the generation inter-val, etc. Generally, testing once every two years is rea-sonable for a facility maintaining a small number ofcolonies well-separated in terms of coat colour, andwith low numbers of importations.

Using a small panel of SNPs. For GeMo purposesonly, 40 polymorphic SNPs, evenly distributed acrossthe chromosomes is a reasonable number for detectingrecent genetic contamination (this suggestion should bemodified dependent on the conditions or risks in eachfacility). SNP genotyping is currently available on dif-ferent platforms, that vary in cost and automation cap-abilities. Kompetitive Allele Specific PCR (KASP), avariation on allele-specific PCR, uses allele-specificoligo extension and fluorescence resonance energytransfer,43 has the advantage that it can be automatedusing 96- or 384-well plates and pipetting robots for thePCR reactions (Supplementary Figure 3). Anotheroption, real-time PCR (TaqMan�) technology, usesspecific primers coupled with a sequence-specific, fluor-escent TaqMan probe, is effective and easy to auto-mate; however, the cost per individual assay isexpensive compared with KASP assays, and requiresa more costly real-time thermocycler. Finally, microar-ray-based SNP genotyping is not typically used forsmall scale, in-house GeMo, but may be an optionfor vendors of inbred mice. When using or requestingmicroarray genotyping services, be aware that only apercentage of the SNPs will be polymorphic betweenthe strains under analysis (e.g., c. 40% for some clas-sical inbred strain combinations). Information regard-ing which alleles (C, G, A or T) to expect for aparticular SNP/strain combination, and their genomiclocation are available for hundreds of thousands ofSNPs and for the common mouse and rat inbred strainsin easily accessed databases and genome browsers(Supplementary Table 2).

GeMo of outbred colonies. GeMo of outbred stocks ismuch more complex, because these animals are not gen-etically uniform. Outbred colonies are essentially agroup of closely related animals, with shared ancestorsand group identity, but that exhibit some level of


genetic heterozygosity. Since outbred colonies form apopulation rather than a strain, it is difficult to establisha standard GeMo programme with only a few geneticmarkers. However, with an adequate number of SNPsor SSLPs, allele frequencies within the populationcould indicate the identity of the stock.44 One of themain problems of in-house outbred stocks is that theyare often maintained with a very small number of

animals in the breeding colony, causing a reductionof alleles represented in the population. This mayimpact genetic drift and increase the inbreedingcoefficient. Such colonies are neither truly outbrednor inbred. Although SSLPs or SNPs can be used toestimate the level of heterozygosity within the colony,if it is not possible to keep an appropriate numberof breeders, it is better to purchase outbred

Figure 3. This chart explains the typical timeline for a marker assisted (speed congenic) backcross process. The pre-diction of >98% recipient genome at N5 is based on the use of 20 best breeders (carriers) at each generation,55 however,this number is not always available and fewer breeders can be used, with disparate results, depending also on chance. PI:Principal Investigator (laboratory). Service: the laboratory providing the genome scan with SNP markers.

Figure 2. Example of genetic contamination detected by SSLP PCR. The picture shows a 4% agarose gel with thecharacteristic bands obtained after PCR amplification using genomic DNA from four mice supposedly belonging to theBALB/c strain (first four lanes), plus a standard DNA control for BALB/c (last lane). In this example, only five SSLP loci areshown, located in chromosomes 1 to 5. Note the presence of heterozygosity (two bands) and homozygosity for bands thatdo not match the standard for BALB/c. This is a clear case of loss of authenticity due to genetic contamination. The PCRproducts are compared with a 100 bp DNA ladder.


rodents from vendors that maintain a very large colonyand use recommended breeding schemes to reduceinbreeding.

Background characterization (BC) for GAand mutant lines

The explosion in the number of GA lines is exacerbat-ing the problem of undefined ‘mixed backgrounds’ inexperimental rodents. This is particularly worrisomefor inducible and conditional models that require thecrossing of two independent lines (e.g. Cre-expressinglines crossed with floxed lines). Given that geneticbackground influences phenotype, especially throughthe influence of modifier genes; mutations, both spon-taneous and induced, transgenes, and targetedalleles that are introgressed into a new backgroundmay not exhibit the expected phenotype.45,46 One ofthe first cases reporting this phenomenon involved theclassical diabetes (Leprdb) mutation that presentedtransient diabetes in a C57BL/6 background butovert diabetes in C57BLKS.47 Other examplesinclude background effects on survival rate in Egfr(epidermal growth factor receptor) KO mice48 andtumour incidence in Pten KO mice.49 For this reason,every GA line should be characterized in terms oftheir genetic background. Moreover, the knowledgeof the genetic background of a mutation is also import-ant for the selection of the corresponding controlanimals.50

Genetic markers evenly distributed and covering theentire genome can be used in a genome scan to estimatethe percentages of genome coming from differentinbred origins. This process of BC is provided bysome commercial enterprises and institutional corefacilities. A typical BC employs polymorphic markersto distinguish between the suspected inbred strains. Inmost mouse cases, these strains are C57BL/6 and 129substrains because, historically, the ES cells used for thedevelopment of KO and KI mice through homologousrecombination (section ‘‘Targeted mutagenesis byhomologous recombination using ES cells’’ above)were derived exclusively from 129 substrains51 whereasWT C57BL/6 females were typically used to provegermline transmission from the chimeras. Without sub-sequent backcrosses, this scheme resulted in a B6;129mixed background. However, the availability of ES celllines derived from other strains (particularly fromC57BL/6) and the arrival of genome editing techniques(section ‘‘Gene editing using nucleases’’) that allowdirect production of targeted alterations in any mouseor rat strain52 is slowly changing this scenario. In anycase, the problem of mixed background can be circum-vented altogether by (a) injecting transgenes or nucle-ases (Cas9-sgRNA) into inbred embryos from the

strain of choice; (b) modifying the gene of interest inES cells from the preferred background strain (e.g.using C57BL/6 ES cells); and (c) crossing chimerasand KO/KI founders with mice of the same strain asthe ES cells used for the targeting. Finally, if the GAline has already been developed or acquired from acollaborator or repository, a BC should be per-formed, and if needed, a fully congenic strain shouldbe developed, either by classical or marker-assistedbackcrossing. Periodic backcrossing of a congenicstrain to the background strain (of reputable source)also minimises divergence and keeps the congenicstrain genetically close to the strain background of con-trol animals.

Marker-assisted backcrossing for qualityassurance and refinement

The use of DNA markers has allowed for a much morerapid and rigorous process of congenic strain develop-ment called marker-assisted backcrossing or speed con-genics.53 This process relies on using polymorphicgenetic markers covering the whole genome to deter-mine the percentage of donor genome present in theanimals, then selecting the animals with the lowestpercentage of donor DNA for the next backcross tothe recipient strain. This relies on the regions betweenthe polymorphic genetic markers being those of thedonor genome: the denser the number of markers thehigher the donor genome can be inferred. Commonpractice is the use of 100–300 markers. This processreduces the number of generations to reach full con-genicity (e.g. from N10 to N5), and therefore straindevelopment time, by approximately half. Usingmarker-assisted backcrosses and the right number ofanimals we can obtain c. 80% recipient background atN2, c. 94% at N3, and c. 99% at N4 (compared to theclassical mean values of 75.0, 87.5 and 93.7%). Aflowchart depicting a standard speed congenic proto-col is shown in Figure 3. Ideally, the backcross pro-cedure is started with a donor female and a recipientmale. Then, F1 mutant males will carry the correctY-chromosome and after mating to a recipientfemale, males of the N2 generation will carry the cor-rect X- and Y-chromosome of the recipient strain(avoiding the use of genetic markers on these chromo-somes).54 It was predicted by Markel et al. that if 20best breeders (carriers) are used at each generation ofthe speed congenics protocol >98% recipient gen-ome can be attained at N5.55,56 However, the chromo-somal segments flanking the selected locus tend toremain associated with it and this is a limitation ofthe congenic lines due to the potential presenceof modifier genes in this segments, the so-called‘flanking gene problem’.57


Genetic stability and cryopreservationprogrammes

For inbred, co-isogenic and congenic strains, breedingmethods and genetic stability programmes help to min-imize substrain divergence due to genetic drift, and also toprevent genetic contamination by accidental crosses withother strains. To reduce genetic drift, the number of gen-erations of in-house breeding should be minimized, andthe lines submitted to repositories such as, JAX, EMMA,MMRRC, IMSR or RIKEN, to be archived as frozenembryos and/or sperm. This secures the line and providesa means of replacing the breeding stock every 10 gener-ations as recommended by The Jackson LaboratoryGenetic Stability Program (GSP) in order to slow downcumulative genetic drift.42 For outbred stocks, the intentis to minimize inbreeding, maintain heterozygosity andmanage genetic drift that would otherwise lead tocolony divergence. Ideally, outbred colonies should bemaintained with �25 breeding pairs, all of which haveto contribute to the next generation, in order to avoidan increase of the inbreeding coefficient per generationof more than 1%. Smaller colonies drift fast towardhomozygosity because breeders are closely related.58

Cryopreservation strategies have been adopted forlong-term storage of embryos and gametes in severallarge centralized repositories including the EMMA/INFRAFRONTIER (European Mutant MouseArchive), the Knock Out Mouse Project (KOMP)Repository, The Jackson Laboratory Repository, TheCenter for Animal Resources and Development(CARD) and the Riken Bio Resource Center, whichcan provide cryopreserved material or live mice tolaboratories. These repositories facilitate the availabil-ity of these strains to the worldwide scientific commu-nity and provide a backup for a potential loss of astrain. The International Mouse Strain Resource(IMSR) is a searchable online database of mousestrains, stocks and mutant ES cell lines available world-wide, including inbred, mutant and genetically engin-eered strains (http://www.findmice.org/).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of this

article.

Funding

The author(s) received no financial support for the research,authorship, and/or publication of this article

Supplementary Material

The full report, including 160 references, is available asSupplemental Material to this publication online via https://journals.sagepub.com/doi/full/10.1177/0023677219867719.

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43. Myakishev MV, Khripin Y, Hu S, et al. High-throughputSNP genotyping by allele-specific PCR with universalenergy-transfer-labeled primers. Genome Res 2001; 11:

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6J strains. Biochem Genet 1972; 7: 1–13.48. Threadgill DW, Dlugosz AA, Hansen LA, et al. Targeted

disruption of mouse EGF receptor: effect of genetic back-

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ground controls tumor development in PTEN-deficientmice. Cancer Res 2006; 66: 6492–6496.

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Resume

L’assurance qualite (AQ) genetique, dont la surveillance genetique (SG) des souches consanguineset la caracterisation des souches (CS) de modeles animaux genetiquement modifies (GM) devraitconstituer un aspect essentiel de tout programme d’AQ dans les installations de recherche animale. Lecontrole qualite genetique est aussi important que la surveillance sanitaire ou microbiologique pour assurerla validite du modele animal. Il devrait etre obligatoire que les etudes utilisant des rongeurs de laboratoire,principalement des souris et des rats, utilisent des animaux genetiquement definis. Le document presentepar le groupe de travail FELASA sur l’assurance qualite et la surveillance genetiques des rats et souris delaboratoire, decrit les objectifs et les methodes disponibles pour les programmes d’AQ menes dans lesinstallations utilisant des rongeurs. Les objectifs principaux d’un programme d’AQ sont les suivants :(i) verifier l’authenticite et l’uniformite des souches consanguines et des sous-souches, assurant ainsi lamaintenance d’une colonie genetiquement fiable; (ii) detecter les contaminations genetiques eventuelles;et (iii) decrire precisement la composition genetique des lignees GM. Bien que cette publication se concentresur l’AQ genetique des souris et des rats, les principes s’appliqueront a d’autres especes de rongeurs,dont certaines sont brievement mentionnees dans le contexte des animaux issus de lignees consanguinesou croisees.

Abstract

Die genetische Qualitatssicherung (QA), einschließlich des genetischen Monitorings (GeMo) vonInzuchtstammen und der Hintergrundcharakterisierung (BC) von genetisch veranderten (GA) Tiermodellen,sollte generell ein wesentlicher Bestandteil aller QA-Programme in Versuchstiereinrichtungen sein. Diegenetische Qualitatskontrolle ist zur Gewahrleistung der Validitat von Tiermodellen ebenso wichtig wie dieUberwachung ihrer Gesundheit und mikrobiologischen Qualitat. Fur Studien mit Labornagern, hauptsachlichbetrifft es Mause und Ratten, sollte ausschließlich die Verwendung von genetisch definierten Tieren vorge-sehen werden. Dieses Dokument, das von der FELASA Arbeitsgruppe uber genetische Qualitatssicherung undgenetisches Monitoring von Labormausen und -ratten prasentiert wird, beschreibt die Ziele und verfugbarenMethoden fur genetische QA-Programme in Labortierhaltungen. Die Hauptziele eines jeden genetischen QAProgramms sind: (i) Uberprufung der Authentizitat und Uniformitat von Inzuchtstammen und derenSubstamme, um so eine genetisch zuverlassige Erhaltung der Kolonie zu gewahrleisten, (ii) Erkennungmoglicher genetischer Kontaminationen, und (iii) prazise Beschreibung der genetischen Beschaffenheit vonGA-Linien. Diese Veroffentlichung konzentriert sich hauptsachlich auf die genetische QA von Maus und Ratte,wobei die Prinzipien auch fur andere Nagetierarten, von denen einige im Zusammenhang mit Inzucht- undAuszuchtstammen kurz erwahnt werden, zutreffen.

Resumen

La garantıa de calidad genetica (QA), incluidos el monitoreo genetico (GeMo) de las cepas consanguıneas y lacaracterizacion de fondo genetico (BC) de los animales geneticamente modificados (GA), deberıa ser uncomponente esencial de cualquier programa de QA en los animalarios de roedores. El control de la calidadgenetica es tan importante para asegurar la validez del modelo animal como lo es el control de calidadsanitaria y microbiologica. Deberıa exigirse que los estudios que utilicen roedores de laboratorio, principal-mente ratones y ratas, utilicen exclusivamente animales geneticamente definidos. Este manuscrito, presen-tado por FELASA Working Group on Genetic Quality Assurance and Genetic Monitoring of Laboratory Murines,describe los objetivos y metodos disponibles para los programas de calidad genetica en instalaciones de


roedores de laboratorio. Los principales objetivos de cualquier programa de calidad genetica son: (i) verificarla autenticidad y uniformidad de las cepas (y sub-cepas) consanguıneas; (ii) detectar una posible contamina-cion genetica; y (iii) describir con precision la composicion genetica de las lıneas geneticamente modificadas.Si bien esta publicacion se centra principalmente en los controles de calidad genetica de ratones y ratas, losmismos principios se aplican a otras especies de roedores de laboratorio, algunas de las cuales se mencio-nan brevemente en el contexto de las cepas consanguıneas y los grupos exocriados de ratones y ratas.


Editorial

Collection on score sheets, severityassessment and humane end points:Invitation to submit

Paulin Jirkof1, Gavin Jarvis2 and Beat Riederer3

Laboratory Animals publishes original papers andreviews on the care and use of animals in biomedicalresearch with the aim of promoting the welfare oflaboratory animals. An important tool for evaluatingand promoting animal welfare in experiments is severitymonitoring. EU Directive 2010/63/EU introducedrequirements for classifying the severity of procedures,not only for project authorisation but also for reportingactual severity experienced by the individual animals.1

Score sheets standardise and formalise severityassessment. This makes it possible to record the effectof scientific procedures on animals, and therefore toinform decisions on remedial measures or experimenttermination. Additionally, well-defined humane endpoints reduce suffering and minimise unnecessarydeaths of animals. Data collected in score sheets candirect the development of refinements, for exampleoptimised analgesic protocols, and facilitate their effect-ive implementation, thereby improving the welfare ofanimals used in research.2

As part of the implementation of EU Directive 2010/63/EU, extensive efforts are being made to improvemethods for severity assessment (see Special Issue‘‘Severity assessment in animal-based research’’Laboratory Animals 2020; 54(1); https://journals.sage-pub.com/toc/lana/54/1). However, there is no one-size-fits-all approach to scoring severity and animal welfare,and practical, evidence-based, established and availablescore sheets are absent for many experimental proced-ures. Further development is therefore essential toincrease the accuracy, specificity and effectiveness of scor-ing and grading systems for severity and animal welfare.

As a contribution to this process, LaboratoryAnimals aims to collate recently published articles on

the development and evaluation of severity assessmentmethods, along with example score sheets from differ-ent scientific fields (https://journals.sagepub.com/page/lan/collections/score-sheets). We hope that this collec-tion will encourage further research in this field,improving sharing of the knowledge gained from theiruse and subsequent improvements in laboratory animalwelfare.

We invite scientists to submit original papers onseverity assessment methods and humane end-point cri-teria, reviews on the use and suitability of currentassessment methods and scoring schemes and shortreports on example score sheets to Laboratory Animals.


The author(s) declared no potential conflicts of interest withrespect to the research, authorship and/or publication of thisarticle.

Funding

The author(s) received no financial support for the research,authorship and/or publication of this article.

References

1. Smith D, Anderson D, Degryse AD, et al. Classification

and reporting of severity experienced by animals used inscientific procedures: FELASA/ECLAM/ESLAVWorking Group report. Lab Anim 2018; 52: 5–57.

2. Golledge H and Jirkof P. Score sheets and analgesia. LabAnim 2016; 50: 411–413.

1Department Animal Welfare and 3R, University of Zurich,Switzerland2Department of Physiology, Development and Neuroscience,University of Cambridge, UK3Platform for Morphology and Department of FundamentalNeurosciences, Faculty of Biology and Medicine, University ofLausanne, Switzerland

Corresponding author:Paulin Jirkof. Department Animal Welfare and 3R, University ofZurich, Zurich, Switzerland.Email: [email protected]

Laboratory Animals

2020, Vol. 54(2) 149




DOI: 10.1177/0023677220906399


https://journals.sagepub.com/toc/lana/54/1

https://journals.sagepub.com/toc/lana/54/1


https://doi.org/10.1177/0023677220906399


Severity Assessment in Animal-based Research

Impulse for animal welfare outsidethe experiment

Lars Lewejohann1,2 , Kerstin Schwabe3, Christine Hager4 andPaulin Jirkof5

AbstractAnimal welfare is a growing societal concern and the well-being of animals used for experimental purposes isunder particular scrutiny. The vast majority of laboratory animals are mice living in small cages that do notoffer very much variety. Moreover, the experimental procedure often takes very little time compared to thetime these animals have been bred to the desired age or are being held available for animal experimentation.However, for the assessment of animal welfare, the time spent waiting for an experiment or the time spentafter finishing an experiment has also to be taken into account. In addition to experimental animals, manyadditional animals (e.g. for breeding and maintenance of genetic lines, surplus animals) are related to animalexperimentation and usually face similar living conditions. Therefore, in terms of improving the overall wel-fare of laboratory animals, there is not only a need for refinement of experimental conditions but especiallyfor improving living conditions outside the experiment. The improvement of animal welfare thus depends to alarge extent on the housing and maintenance conditions of all animals related to experimentation. Given thecurrent state of animal welfare research there is indeed a great potential for improving the overall welfare oflaboratory animals.

Keywords3Rs, animal use, environmental enrichment, housing, laboratory animal welfare

Date received: 7 April 2019; accepted: 10 November 2019

Introduction

There is no reliable, let alone official, number of labora-tory animals used worldwide. Even for highly regulatedareas like the EU, comprehensive data is published witha considerable delay. Moreover, the data availablecomprises a fuzziness as there is no uniform conventionon what exactly to count. The latest numbers availablefor the EU are for the year 2017, with 9.4 million ani-mals used for animal experimentation.1 Many countriesprovide yearly statistics on animal use, allowing somegeneral conclusions to be drawn and enabling morecurrent estimates. For example, the numbers for 2018published by the German Federal Ministry of Foodand Agriculture,2 as well as the numbers of proceduresin the UK in 2018,3 can help to conclude some generaldirections. Overall, the latest numbers published werecomparable to preceding years, and again, the mostwidely used species was the mouse with roughly 1.54

million individuals in Germany and 2.57million pro-cedures carried out with mice in the UK. For the year2017, for the first time, not only the number of experi-mental animals but also the number of animals used forbreeding and maintenance as well as the number of

1German Federal Institute for Risk Assessment (BfR), GermanCentre for the Protection of Laboratory Animals (Bf3R), Berlin,Germany2Institute of Animal Welfare, Animal Behavior and LaboratoryAnimal Science, Freie Universitat Berlin, Germany3Department of Neurosurgery, Hannover Medical School, Germany4Institute for Laboratory Animal Science, Hannover MedicalSchool, Germany5Department Animal Welfare and 3Rs, University of Zurich,Switzerland

Corresponding author:Lars Lewejohann, Freie Universitat Berlin, Konigsweg 67, Berlin,14163 Germany.Email: [email protected]

Laboratory Animals

2020, Vol. 54(2) 150–158




DOI: 10.1177/0023677219891754


https://orcid.org/0000-0002-0202-4351

https://orcid.org/0000-0002-6971-9780

https://orcid.org/0000-0002-7225-2325


https://doi.org/10.1177/0023677219891754


animals bred but not used was assessed throughoutthe EU. Overall almost 14 million additional animalswere counted in 28 EU countries indicating that forevery two experimental animals additionally three sur-plus animals have to be counted.1 Assuming that themajority of the additional animals are mice as well, wewill focus on mice for the most part of this impulsepaper, but the discussion that is being fostered herewill of course also apply to other experimental animalspecies.

Additional animals are held available for breedingand maintenance of certain genetic lines, are killed fororgan or tissue samples, or are considered surplus ani-mals, which will not be used for experimental purposesdue to wrong sex, age, or genotype (Table 1). In prin-ciple, the same legislative rules for housing and main-tenance apply for the additional animals as for theexperimental animals. Animal experimentation is con-ducted for a wide range of different scientific purposes,4

and in many studies animals spend the longest time oftheir lives not in the respective experiment itself. This isespecially true for laboratory mice which are often con-sidered as ‘‘disposable goods’’ in science,5 and newexperiments are usually carried out with new animalsbeing bred in sufficient numbers in local facilities as wellas by commercial breeders. While waiting for theexperiment or after finishing a non-lethal experiment,laboratory mice are usually living in customary stan-dardized laboratory housing conditions. In most coun-tries these housing conditions fall under a variety ofrestrictions with regard to meeting the minimumrequirements (e.g. for mice in the EU the minimumcage floor size is 330 cm2, bedding, nesting material,and social company shall be provided; see EU guide-lines 2010/63/EU). Above the minimum requirements,according to the 3Rs – which are anchored in manystatutory provisions – refinement of living conditionsshall be taken into consideration. Therefore, maximiz-ing potential welfare by improving living conditions ofexperimental animals is not limited by legislations, butrather by experimental and economic reasons.

What is animal welfare?

Animal welfare much alike human welfare is a termthat is notably hard to access and disentangle andthere is no unambiguous consensus.6–10 An earlyapproach in defining animal welfare was raised by apress release of the Farm Animal Welfare Council in1979, tracing back to the ‘‘Brambell Committee1965,’’11 and is referred to as the five freedoms (i.e.freedom from 1. hunger and thirst, 2. discomfort,3. pain, injury, and disease, 4. fear and distress, and5. restrictions to express normal behavior). The fivefreedoms, however, state what has to be avoided in

order to prevent poor animal welfare rather thandefining what constitutes animal welfare per se.Consequently, the later literature emphasizes subjectiv-ity for animal welfare and incorporates the view of ani-mals as perceived animal welfare.12,13 The five freedomslately have been advanced to the ‘‘five provisions’’focusing on what should be provided to achieve goodwelfare.10 It was also recognized that animal welfare isnot static and thus concepts include adaptive capabil-ities in terms of coping with environmental challengesand/or being able to achieve certain goals.9,14–16

Today’s view on animal welfare also largely includesa quality of living approach,17,18 focusing on goodliving rather than mere avoidance of unfavorable con-ditions. Quality of living inherently reflects a muchmore holistic view over an animal’s life and is thusless affected by short timed events (e.g. pleasurablemoments, few minutes of fear).17 Our definition ofanimal welfare is based on the current literature anddeliberately reflects the difficulties that come alongwith defining as well as with assessing animal welfare.

Animal welfare describes (objectively verifiable) thestatus of a subjectively perceived quality of life of anindividual at a given period and is measured on anordinal (nonlinear), multidimensional scale.

The core unit of animal welfare is the subjective per-ception of an individual. This makes welfare especiallyhard to measure, as individual perception seems tonotoriously elude scientific quantification. However,recent advances in theoretical concepts and method-ology increasingly allow to quantify for example affect-ive states.16,19–21 The multidimensionality results fromthe different levels on which animal welfare can beaffected: An individual might suffer from an injury(an obvious indicator for bad welfare) but might beengaged in positive social interaction (indicator forpositive welfare) at the same time. Naturally, this com-plicates the assessment of animal welfare as calculationof potential compensatory and/or additive effectsbetween different dimensions is inherently difficult.Moreover, the difference between categories such aspoor and very poor welfare is not necessarily thesame as the difference between good and moderate wel-fare, thus the scale has to be considered ordinal. In thesame vein, nonlinearity owes to the fact that physicaland physiological parameters as well as descriptors ofaffective states do not follow simple mathematical addi-tive rules in relation to their impact on animal welfare.Although physiological parameters like heart rate orstress hormonal levels are measured on an intervalscale they are not linearly related to animal welfare.15,22

Although the status of animal welfare is usuallyassessed at a given point in time, the measurementreflects a period of unknown length preceding theassessment. In addition, preceding lifetime events

Lewejohann et al. 151

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affect a current state of animal welfare differentiallydepending on frequency of occurrence, length, andintensity, for example.

Assessing animal welfare

It is obvious from the above that measuring animal wel-fare is not an easy endeavor. It is, however, feasible tomeasure animal welfare on an ordinal scale and to anno-tate labels ranging from very poor to very good welfarewith reasonable precision. Poor welfare can be measuredby evaluating to what extent the first four freedoms aremet. In a broad sense, being free from pain, discomfort,hunger and thirst, fear, and disease can be considered aminimum standard that should be expected to be thenormal state a laboratory animal is in. Although notalways overly trivial, these parameters are generally con-sidered to be measurable reasonably well.10,14,23 Aboveobvious signs for poor welfare like sickness behavior,wounds, signs of starvation or dehydration, physio-logical parameters (e.g. stress hormones, heart rate)may be indicative for how well an animal is able tocope with the challenges introduced by the environmentprovided under laboratory conditions. The fifth freedomto be free to show normal behavior is far more difficultto assess. A wide spectrum of species specific behavior isrelated to coping with challenging situations in the wildthat one can reasonably assume not to be applicable forlaboratory animals (e.g. extensive foraging, predatoravoidance, exaggerate aggressive encounters).Therefore, it is not unequivocally established what con-stitutes ‘‘normal behavior’’ in a laboratory animal.Nevertheless, monitoring day-to-day behavior of labora-tory animals and comparing time budgets allocated todifferent behavioral domains is a feasible approach toanalyze normal behavior in laboratory animals. In asimilar vein, disturbed circadian rhythm or other behav-ioral deviations such as stereotypic behavior or hair pull-ing are considered to be associated with impaired animalwelfare.24–27 Finally, post mortem analysis (e.g. ulcers,adrenal weights) can also help to retrospectively assesspoor animal welfare.28

Measuring good animal welfare on the other hand, isgenerally considered to be more complicated althoughnot impossible.29 Play behavior and affiliative behav-iors, as well as some vocalizations,30 appear to be pro-mising measurable indicators for assessing positiveanimal welfare.31 In addition, recently newly developedapproaches in human animal interaction were alsorelated to positive emotions, e.g. clicker-training andtunnel handling might indeed be perceived as positiveinteractions by the animals.32,33 Finally, with regard tothe quality of living, which would reflect a more holisticview of animal welfare, an ideal assessment should takeinto account that welfare throughout an animal’s life

(within and outside the experiment) has to be con-sidered as well.

Specific conditions in animal experiments

For many years, it has been fostered to try keepingexternal physical, social, and internal physiologicalstates as constant as possible. This was meant notonly in order to standardize experimental conditionsbut also to guarantee the fulfillment of animal needs.Unfortunately, this approach falls short in regard toanimal welfare as biological systems have evolved tocope with transience of external stimuli and thereforeallostasis (‘‘stability through change’’) rather thanhomeostasis is a key element of animal welfare.15,34

This has been partially addressed by improving housingconditions especially by introducing environmentalenrichment over the last decades. On the other hand,experimental set ups naturally require a standardizationstrategy (including, e.g., systematic variation toincrease external validity35) to minimize animal useand maximize test sensitivity. Still, boredom as a nat-ural consequence of under stimulation should be con-sidered a major concern with regard to animal welfareof laboratory animals.36 Sensation seeking is reflectingsuch a need for change and has been measured forexample as proneness to sensory stimuli in mice,37

self-administration of glucocorticoids in rats,38 or seek-ing even aversive stimuli in mink.39 Consequently, bal-ancing standardization against boredom along with theanimals’ ‘‘need for change’’ will remain a challenge infuture experimental designs. It would be fallacious toexpect that any individual animal (or human) could bein a superior welfare state at all times. Thus fluctuationin welfare states is an inherent part of an animal’s lifeand also contributes to a life worth living.14 Overall,transience between welfare states within the range ofvery good, good, neutral, and even lightly aversive ismost likely part of an interesting life worth living. This,however, is not at all easy to be realized for laboratoryanimals. Even if we assume that there were no restric-tions with regard to financial shortage, qualified per-sonnel, and available space, at least some categoriesof laboratory animals will be less eligible for the fullrange of possible welfare enhancement (see Table 1).

Enhancing animal welfare

Although assessing animal welfare is coming along witha number of problems with regard to accuracy, specifi-city, and generalizability, there is also a pragmaticapproach when the goal is to increase animal welfareof laboratory animals. A positive welfare state can bederived by being able to engage in activities that areperceived as rewarding. Such behaviors are expected to


be capable to elicit positive affects which are related toanticipation of achieving goals, achieving the goal itself,and retrospectively eliciting the memory of having pre-viously achieved a goal.10 Consequently, any measuresthat enable laboratory animals to engage in rewardingactivities, as well as states associated with anticipation ormemory of rewarding activities are likely to enhanceanimal welfare. A classical reward is the provision oftreats, which is very common, e.g. in companion ani-mals. In laboratory animals, however, treats are oftenrestricted to experiments of operant and classical condi-tioning where special food items are provided as areward to increase their performance. If paying attentionto nutritional needs, there should be no principle objec-tions against providing special treats to other laboratoryanimals as well. However, one should bear in mind thatif anticipated rewards are suspended the mismatchbetween expected reward and the reality check possiblyleads to frustration.40 Therefore, withholding treats orother positive stimuli that previously have been grantedcan also negatively affect animal welfare.

Positive affective states are also elicited in positivesocial interactions. Social interactions concern the entirelife of social mammals and incisive experiences in earlylife also affect later social behavior. For example, it hasbeen shown that delayed weaning increased social behav-ior later in life.41 However, the weaning age is usuallydesigned to maximize breeding success and does notnecessarily correspond to the natural breeding behaviorof the species. Social housing for laboratory animals laterin life is generally recommended except for solitary spe-cies. However, group housing for animals that frequentlyengage in aggressive encounters, e.g. as observed in malegroups of many mouse strains,42,43 is sometimes not feas-ible. This is something that has to be taken into accountwhen planning experiments and choosing the right modelspecies, strain, or sex. As already outlined above, bore-dom due to a lack of stimuli and missing opportunities toengage in rewarding activities in laboratory housing sys-tems is a growing concern.36 This can be partially ame-liorated by means of environmental enrichment andproviding materials to perform species typical behavior(e.g. for rodents, nesting material, burrowing and gnaw-ing substrate). For laboratory mice nesting material andshelters were slowly introduced over the last three dec-ades and can nowadays be found in almost all Europeananimal facilities as this is required by the EU directive2010/63/EU. Still there is much room for improvementwith regard to entertaining enrichment and providingopportunities to engage in rewarding behaviors. Thiscan be realized by providing novel stimuli (e.g. newenrichment items that can be explored44), by introducingcognitive training (e.g. puzzle boxes, clicker training32)into the home environment, or by measures of occupa-tional therapy (e.g. running wheels, or letting the animals

work in order to get access to water or food). In addition,home environments could be improved by providingbetter opportunities for play behavior. Although playbehavior is most prominent in juveniles and adolescents,adults of many species, including mice and rats,45–47 doalso play. Play behavior is usually considered to be anindicator for positive animal welfare,48 but sometimeseven elicited when coping with negative affectivestates.49 Nevertheless, the absence of play behavior inan otherwise playful species certainly is an example fora deviation from ‘‘normal’’ behavior and thus shouldgenerally considered to be an indicator for disturbedanimal welfare. Adult mice engage frequently in loco-motor play if provided with enough space.45,46 Indeed,more than 85% of play behavior in mice involves loco-motor play.50 Therefore providing more space (e.g. largercages, connecting several small cages with tubes) or otheropportunities to engage in locomotory activity should betaken into account to improve housing conditions forlaboratory mice. Noteworthy, there is an ongoingdebate with regard to the costs and benefits of changing‘‘established’’ housing conditions with regard to size, typeof nesting and bedding material, or different forms ofenrichment. For example, excessive usage of runningwheels might resemble stereotypic behavior in some indi-viduals,51,52 but in group housed mice no signs of stereo-typic running wheel behavior were found.53 Moreover,several behavioral as well as morphological, and physio-logical parameters can be affected by introducing envir-onmental enrichment.54 Also it is known that housingconditions can have interaction effects with pharmaco-logical treatments.55 However, concerns that enrichmentgenerally increase variation in experimental results couldnot be substantiated.56 Overall, possible interferences ofimproved as well as of restricted housing conditions withthe experimental design, reproducibility, and external val-idity should be kept in mind. Enrichment is generallythought to enhance animal welfare although sex differ-ences might apply (i.e. aggressive behavior42), and it isnot always clear how different items are perceived by theanimals themselves and thus animal centric strategies likepreference tests will help to assess and rate differentitems.57

Improving animal welfare in- and outsidethe experiment

Generally speaking, we should aim to maximize animalwelfare of laboratory animals owing to the fact that weare responsible for their well-being. As animal experi-mentation is under special scrutiny there is a high eth-ical standard ruling animal experimentation and it hasbecome mandatory to consider refinement measures inthe experimental design. Moreover, it is widelyaccepted that above the ethical concerns there is also


a scientific need for improving the welfare of laboratoryanimals.58 However, restrictions affecting the welfare ofexperimental animals cannot always be overcome ifthey are directly related to the experimental aims. Inour view, relating to historical data or established hous-ing conditions alone does not suffice to refuse enhan-cing the living conditions. One of the main lessons to belearnt from the reproducibility crisis should be thatonly data that can be replicated in other contexts aretruly biologically meaningful.35 Experimental animalsspend much of their lives outside the experiment anda large number of animals are not even used for experi-ments but held available for breeding or other mainten-ance related purposes. Table 1 summarizes the potentialof enhancing animal welfare for different categories oflaboratory animals. For each category of animals oneshould ask what can reasonably be done to maximizetheir welfare. Experimental animals and animals wait-ing for an experiment are probably more restricted withregard to maximizing their welfare as many measurespotentially counteract the experimental purpose.Nevertheless, quality and size of measures to increasewelfare depend on the experimental design and shouldbe evaluated accordingly. On the other hand, for post-experimental animals there is basically no limit on whatcould be done to increase their welfare, even if it mightonly be for a short time compared to the life expect-ancy.5 All in all, the time outside the experiment can beconsidered a special opportunity to improve the overallwelfare of laboratory animals.



article.

Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship, and/or publication of thisarticle: This work was funded by the DFG (grant numbers

FOR2591, LE 2356/5-1, and JI 276/1-1).

ORCID iDs

Lars Lewejohann https://orcid.org/0000-0002-0202-4351Christine Hager https://orcid.org/0000-0002-6971-9780

Paulin Jirkof https://orcid.org/0000-0002-7225-2325

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https://orcid.org/0000-0002-0202-4351

https://orcid.org/0000-0002-0202-4351

https://orcid.org/0000-0002-6971-9780

https://orcid.org/0000-0002-6971-9780

https://orcid.org/0000-0002-7225-2325

https://orcid.org/0000-0002-7225-2325

https://ec.europa.eu/transparency/regdoc/rep/1/2020/EN/COM-2020-16-F1-EN-MAIN-PART-1.PDF



https://www.bmel.de/DE/Tier/Tierschutz/_texte/Versuchstierzahlen2017.html

https://www.bmel.de/DE/Tier/Tierschutz/_texte/Versuchstierzahlen2017.html

https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/835935/annual-statistics-scientific-procedures-living-animals-2018.pdf




https://www.worldcat.org/title/report-of-the-technical-committee-to-enquire-into-the-welfare-of-animals-kept-under-intensive-livestock-husbandry-systems/oclc/475844139




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Resume

Le bien-etre des animaux est une preoccupation societale croissante et le bien-etre des animaux utilises ades fins experimentales fait l’objet d’une attention particuliere. La grande majorite des animaux de labor-atoire sont des souris qui vivent dans de petites cages n’offrant pas beaucoup de variete. En outre, laprocedure experimentale prend souvent tres peu de temps par rapport a la periode d’elevage de ces animauxjusqu’a l’age desire ou leur mise a disposition pour l’experimentation animale. Toutefois, pour l’evaluation dubien-etre animal, le temps passe en attente d’une experience ou le temps passe apres avoir termine uneexperience doit egalement etre pris en compte. En plus des animaux de laboratoire, de nombreux animaux(par exemple, pour l’elevage et l’entretien des lignes genetiques, les animaux excedentaires) sont lies al’experimentation animale et font generalement face a des conditions de vie similaires. Par consequent, entermes d’amelioration du bien-etre general des animaux de laboratoire, il n’existe pas seulement un besoind’amelioration des conditions experimentales, mais surtout d’amelioration des conditions de vie en dehors del’experience. L’amelioration du bien-etre des animaux depend donc dans une large mesure des conditions delogement et d’entretien de tous les animaux lies a l’experimentation animale. Etant donne l’etat actuel de larecherche sur le bien-etre des animaux, il existe en effet un grand potentiel d’amelioration du bien-etregeneral des animaux de laboratoire.

Abstract

Der Tierschutz ist ein zunehmend wichtiges gesellschaftliches Anliegen, und das Wohlergehen von furVersuchszwecke dienenden Tieren muss besonders strenger Uberprufung unterzogen werden. Die uberwie-gende Mehrheit von Versuchstieren sind Mause, die in kleinen Kafigen ohne viel Abwechslung leben. Hinzukommt, dass die Dauer der Zuchtung der Tiere bis zum erforderlichen Alter bzw. der Haltung in Vorbereitungauf Versuche oft lang ist, wahrend die Versuche selbst nur sehr wenig Zeit in Anspruch nehmen. Bei derBewertung des Tierschutzes ist jedoch auch die den eigentlichen Versuchen vorausgehende Zeit ebenso wiejene nach Abschluss eines Experiments zu berucksichtigen. Neben den Versuchstieren selbst sind vieleweitere Tiere (z. B. solche zur Zuchtung und Erhaltung von genetischen Linien, uberzahlige Tiere) mitTierversuchen verbunden, die in der Regel unter ahnlichen Lebensbedingungen gehalten werden. ImHinblick auf die Verbesserung des allgemeinen Wohlergehens von Versuchstieren besteht daher nicht nurdie Notwendigkeit einer Verbesserung der Versuchsbedingungen, sondern insbesondere auch derLebensbedingungen außerhalb der Versuche. Die Verbesserung des Tierschutzes hangt daher in hohemMaße von den Haltungs- und Unterbringungsbedingungen aller im Zusammenhang mit Versuchen stehendenTieren ab. Nach dem derzeitigen Stand der Tierschutzforschung gibt es in der Tat großes Potenzial, das zurVerbesserung des allgemeinen Wohlergehens von Versuchstieren ausgeschopft werden sollte.

Resumen

El bienestar animal es una creciente preocupacion social y el bienestar de los animales utilizados paraexperimentos esta siendo analizado detenidamente. La gran mayorıa de animales de laboratorio son roedoresque viven en jaulas pequenas que no ofrecen gran variedad. Asimismo, el procedimiento experimental amenudo es de poca duracion en comparacion con el tiempo en que estos animales han sido criados hastaalcanzar la edad deseada o estan disponibles para experimentar con ellos. Sin embargo, para la evaluaciondel bienestar animal, el tiempo de espera para un experimento o el tiempo transcurrido tras finalizar un


experimento son factores que tambien tienen que considerarse. Ademas de los animales para experimentos,muchos otros animales (p. ej., para criar y mantener lıneas geneticas o para tener un excedente de ejem-plares) estan relacionados con la experimentacion animal y a menudo viven en condiciones similares. Portanto, en lo referente a la mejora del bienestar general de los animales de laboratorio, no solo hay unanecesidad de refinar las condiciones experimentales sino especialmente de mejorar las condiciones de vidamas alla del experimento. La mejora del bienestar animal, por tanto, depende en gran medida de las con-diciones de mantenimiento y de las jaulas de los animales de experimentacion. Dado el estado actual de lainvestigacion sobre el bienestar animal, existe gran potencial para mejorar el bienestar general de losanimales de laboratorio.


Original Article

Diet-regulated behavior: FVB/N micefed a lean diet exhibit increased nocturnalbouts of aggression between littermates

Mandi M Murph1 , Shuying Liu2,3, Wei Jia1, Ha Nguyen1,Megan A MacFarlane1, Susan S Smyth4, Sudeepti S Kuppa1

and Kevin K Dobbin5

AbstractThe hyperactive FVB/N inbred mouse strain is widely used for transgenic research applications, althoughrarely for behavioral studies. These mice have visual impairments via retinal degeneration, but are consideredhighly intelligent and rely largely on olfaction. While investigating diet-induced obesity in autotaxin transgenicFVB/N mice, we observed an increase in the necessity for male, but not female, cage separations. Based onthe observations, we hypothesized that feeding FVB/N mice a lean diet increases nocturnal bouts of aggres-sion between male littermates. The diets of adult littermates were switched from normal chow to either adlibitum high-fat (45% fat) or lean (10% fat) matched diets for 27 weeks, whereby the mice reached an averageof 43 g versus 35 g, respectively. Then, cage separations due to nocturnal bouts of aggression became man-datory, even though littermates peacefully cohabitated for 10–16 weeks previously. Since the data was ofan unusual nature, it required uncommon statistical methods to be engendered to evaluate whether andwhere significance existed. Therefore, utilizing the randomization and population models, we establisheda methodology and postulated that either testosterone, the autotaxin transgene or diet alteration was thecausal factor. Statistical evaluation demonstrated a significant correlation between cage separations andaggressive behavior associated with the lean-diet-fed mice, not autotaxin. Biochemical data did not appearto explain the behavior. In contrast, energy metabolism highlighted differences between the groups of nor-mally hyperactive mice by diet. This characteristic makes FVB/N male mice unsuitable subjects for long-termstudies with lean-diet modifications.

KeywordsFVB/N, behavior, mathematical computation, energy, lean diet

Date received: 21 August 2018; accepted: 6 February 2019

The FVB/N inbred mouse strain is widely used inlaboratory research applications. It is spontaneouslyhyperactive, only slightly more anxious when comparedto C57BL/6 mice, which are also widely used forresearch.1,2 FVB/N mice possess unique characteristicsthat make them suitable to generate transgenic animalsfor experiments, including their high mating frequency,their consistent ability to produce large, healthy littersand their zygote survivability.3 Female FVB/N miceexhibit strong maternal behavior whereby moms dis-play exceedingly low rates of infanticide or cannibal-ism, which allows robust numbers of pups to survive,even with a litter size of 12 and an average of 9.5.4

1Department of Pharmaceutical and Biomedical Sciences, TheUniversity of Georgia, College of Pharmacy, Athens, GA, USA2Department of Breast Medical Oncology, The University of Texas,Houston, TX, USA3Department of Systems Biology, The University of Texas, Houston,TX, USA4Division of Cardiovascular Medicine and Department ofPharmacology, The University of Kentucky, Lexington, KY, USA;Department of Veterans Affairs Medical Center, Lexington, KY,USA5College of Public Health Epidemiology and Biostatistics, TheUniversity of Georgia, Athens, GA, USA

Corresponding author:Mandi M Murph, University of Georgia, 240 West Green Street,Athens, GA 30602, USA.Email: [email protected]

Laboratory Animals

2020, Vol. 54(2) 159–170




DOI: 10.1177/0023677219834582


http://orcid.org/0000-0002-5152-4977


https://doi.org/10.1177/0023677219834582


Although FVB/N mice are described as ‘unsuitablefor behavioral studies’ because they have visual impair-ments via retinal degeneration, that conclusion is basedlargely on the reliance of commonly used tests thatrequire acute vision to perform the function, not olfac-tion.5,6 For example, Morris water navigation tasks tofind a visible platform and novel object recognition aresignificantly easier without impaired vision. However,more recent studies show FVB/N mice are superior inlearning and memory performance over C57BL/6 miceusing an olfactory tube maze test.7 This suggests thatvision, not intelligence, has led to false impressionsabout what constitutes normal behavior in mice.

There are somewhat conflicting studies surroundingthe degree of aggressive behavior in FVB/N males. Onestudy concluded that FVB/N males have only ‘some-what higher levels of aggressiveness’ when compared toC57BL/6 males.1 In contrast, another study concludedthat in comparison to C57BL/6 male mice, FVB/Nmale mice display a higher duration of offensive behav-ior and significantly longer attack durations againstintruder mice, which might result from their inabilityto maintain regular circadian rhythm patterns.8 Studieson aggression typically focus on behavior among males,not females.

Initially, we set out to conduct a study aboutthe interaction between diet-induced obesity with auto-taxin transgene expression. Transgenic mice overex-pressing autotaxin have larger adipocytes, biologicaldifferences with obesity and develop spontaneouscancers after 12 months of age.9,10 Herein, we observedan unexpected and striking increase in FVB/N maleaggression among certain cages, which necessitated per-manent separations. This required incessant cagepatrolling and routine separations throughout thecourse of the experiment.

Due to the unusual nature of the data, we requiredthe creation of a new statistical method to evaluatewhether statistical significance existed within theseobservations. Several aspects of these data combineto prevent it from fitting neatly into any existing dataanalysis paradigm. The data are both clustered andpartially missing. The clustering is due to the treatmentassignment being received by cages of animals, ratherthan individual animals. The resulting data structureis similar to a group randomized clinical trial. The par-tial missing-ness is due to the way aggressive behavior isobjectively measured by cage separation – aggressiveanimals are isolated in individual cages; however,animal separation is an imperfect surrogate foraggressiveness.

Herein we describe the study and the statisticalcomputations along with the data that inspired themathematical exploration. Based upon these param-eters, we conclude that violent behavior ensues among

cohabitating groups of adult male FVB/N micethrough diet modification to lean chow with 10% fat.To our knowledge, this is the first description of sucha scenario in FVB/N, largely due to the non-relianceon the strain for behavioral research. Therefore, thebehavior of this model makes the males unsuitable forlong-term studies using lean-diet modifications; how-ever, this model could be ideal to study male mouseaggression that is not reliant on visual stimulation.

Materials and methods

Animals

All protocols for the use of vertebrate animals at theUniversity of Georgia were approved by TheInstitutional Animal Care and Use Committee. TheGuide for the Care and Use of Laboratory Animals isendorsed in this study. Transgenic mice were the kindgift of Drs Gordon B Mills and Susan S Smyth and, asdescribed previously, generated on an FVB/N geneticbackground.11 Pups were weaned after 21 days and fedad libitum water and standard rodent chow containingkcal% of 30% protein, 57% carbohydrate and 13% fat(Lab Diet, #5001, St Louis, MO) prior to experimentsemploying the diet modifications. There were no restric-tions on the quantity of food or water available andcages were kept well stocked with pellets. Polymerasechain reaction (PCR) analysis was used to confirm thegenotyping and expression from ear clips with anAgilent 2100 bioanalyzer (Agilent, Santa Clara, CA)and the following primers: 5’-GATCCCAGCCAGTGGACTTA-30 (forward) and 50-TCTGACACGACTGGAA CGAG-30 (reverse). In total, 140 male mice(106 AT-ATX transgenic and 34 wild-type) were fedwith standard rodent chow, for which the data areincluded in this study.

After approximately 10–16 weeks of age, transgenic(39 male) and wild-type (17 male) mice were thenre-assigned to a different diet, from initially eatingstandard chow. Mice were fed either a high-fat dietwith kcal% of 20% protein, 35% carbohydrate and45% fat (Research Diet D12451, New Brunswick, NJ)or the matched control, lean diet that included 17%sucrose, lean diet of 20% protein, 70% carbohydrateand 10% fat (Research Diet D12450H). Male cagesrequired significant management and cage separationsfor fighting and wounding, as governed by the AnimalUse Protocol and recommendations of the veterinarianattendant. The diet study in male mice lasted 27 weeks.

The mouse facility automates the lighting in therooms on a 12-hour cycle, whereby the lights come onat 7:00 am EST and then go off at 7:00 pm EST.The temperature is maintained at 72�F� 4�F. Themice are housed in Individually Ventilated Cages


(IVC) mouse DGM (digital ready green line mouse)racks and Sealsafe Plus GM500 cages measuring15.40� 7.83� 6.30 in (Tecniplast, West Chester, PA).The area of the cage floor is 77.66 in2. The mouse cagesinclude Bed-o’Cobs� bedding and one Bed-r’Nest�

portion-controlled nesting material (The Andersons,Inc, Maumee, OH). For enrichment, each cage includesone plastic red mouse house that allows only red wave-length light to pass through.

Aggressive behavior was flagged by our veterinarystaff. The animal cages are checked daily for healthand hygiene monitoring. Tail handling is the maintype of handling. The facility also performs full changesof the cages every other week. During the daily moni-toring, they observed heightened and unexpected fight-ing without cessation, wounding, chasing and bitingwithin the cages. Most often this was concurrent withbloody bedding, visual wounds and open cuts on vic-tims, whereas perpetrators perched on top of mousehouses and lacked extensive bleeding wounds. Cageswere cleaned on Monday mornings, every other week,whereas separations and deaths from aggressive boutsoccurred randomly throughout the week. All of theseobservations required intervention and cage separ-ations per guidelines for care of the animals. As theseresults were unexpected, there was no blinding.

Humane endpoints were based on an overlappingstudy.10 Prior to this study, we did not foresee aggressivebouts occurring to this degree. The humane endpointsgoverning our animal use protocol included: excessivetumor burden, inability to move, seizures, abdominal dis-tension that interferes with mobility, inability to feed,severe lethargy with little/no spontaneous movement, dif-ficult or labored breathing, blue mucus membranes,bleeding from deep wounding or severe ulcerationwhere there is an absence of an epidermal layer overlyingthe skin that is >50% of the wound size. At the conclu-sion of the study, animals were euthanized with a methodconsistent with the guidelines of the American VeterinaryMedical Association.

Statistical methodology

The treatments and control in this experiment were ran-domly assigned to the cages. The effects of the treat-ments on aggressiveness can, therefore, be evaluatedusing two different statistical modeling approaches.We examine both approaches to analyze these data.The first approach is based on a randomization modeland a sharp null hypothesis.12 The second approach isbased on a population model and a null hypothesisabout population parameters.13 For our purposes,the randomization model has the advantage that thelimited sample size will not invalidate a randomiza-tion-based permutation test (unlike a central limit

theorem-based test), so that reliable p-values can begenerated with this approach. On the other hand, thepopulation model has the advantage of permitting amore detailed description of the effect size and uncer-tainty estimates for comparisons of treatment groupsand control.

The unusual data structure

Unlike all the humane endpoints governing this proto-col, the endpoint of aggression has an unusual statis-tical structure. First, aggressiveness cannot be observedin isolation, but is a behavior that requires the presenceof other mice in the same cage. When no other mice arepresent, then it is no longer possible to observe aggres-siveness for the remaining mouse. This gives rise to aspecific type of right censoring that must be taken intoconsideration during the analysis. Second, the data arealso clustered by cages, so that proper analysis requirestaking into account the clustering structure and pos-sible correlations of observations in the same cage.Third, the number of cage separations can be modeledas a discrete random variable taking on values between0 and the total number of animals in a cage minus one.The induced distribution due to the right censoring willbe non-standard and must be mathematically calcu-lated (see Supplemental Figure 2 for calculations).

Randomization model methodology

The sharp null hypothesis used for the randomizationmodel states that there is no effect on the response(aggressiveness) for any of the treatments. Any observeddifferences are, therefore, attributable to the randomiza-tion itself – that is, to the proverbial ‘luck of the draw.’Therefore, every other possible randomization wouldhave resulted in the exact same set of observations. The‘unusualness’ of the observed data can, thus, be quanti-fied by a permutation test. For this experiment, there isone control condition and four different treatment con-ditions. The number of cages per condition were 20 forthe control, and 4, 4, 3, 3 for the different treatments, fora total of 34 cages. Therefore, the total number of dis-tinct permutations of the cages is as follows:

34

20

� �14

4

� �10

4

� �6

3

� �� 6� 1015

This number suggests that we can obtain a verysmall p-value for the test.

Population model analysis

For the population model approach, the natural popu-lation parameter of interest is the proportion of animals

Murph et al. 161

in the target population of mice that turn aggressivewhen exposed to the control or a specific treatment.Let p0 be the population proportion for the control,and p1,p2,p3,p4 be the population proportions foreach of the active treatments. We assume that beingseparated is an adequate surrogate for turning aggres-sive.14 As noted previously, the data are partially miss-ing because if all the mice end up separated one doesnot know whether all turned aggressive, or all but oneturned aggressive. Also, there may be cage conditionsthat impact the proportion of animals that turn aggres-sive, so different cages in the same condition are per-mitted to have different proportions.

Biochemical analysis

Testosterone content in serum was determined using acolorimetric competitive enzyme immunoassay ELISAkit (Enzo Life Sciences, Farmingdale, NY) followingthe protocol according to the manufacturer’s instruc-tions. A calibration curve was created using the testos-terone standard included in the kit, which has a rangefrom 2000 pg/mL to 7.81 pg/mL. The absorbance wasread at 405Nm using a SpectraMax M2 plate reader(Molecular Devices, Sunnyvale, CA).

The adenosine diphosphate/adenosine triphosphate(ADP/ATP) ratio was determined using single-cell sus-pensions with a luciferin–luciferase bioluminescentassay kit (Abcam, Cambridge, MA) according tomanufacturer’s instructions. The ratio was calculatedbased on a calibration plot made with a standardATP solution with a range from 1nM to 10 nM (ATPDisodium Trihydrate, Amresco, Solon, OH). Single-cellsuspensions were created using 200mg of flash-frozentissues and dissociated with collagenase type I (StemcellTechnologies, Vancouver, Canada) and suspended inphosphate buffered saline (PBS). Luminescence wasdetermined using a Molecular Devices SpectraMaxM5 plate reader (Sunnyvale, CA). The data are plottedas a scatterbox using GraphPad Prism to display theaverage and range of the data points.

Immunoblotting

For protein resolution, liver tissue from mice washomogenized in 500 mL of lysis buffer (RIPA bufferwith a protease inhibitor in a 1:100 ratio). Bufferedtissue was sonicated, centrifuged and the supernatantextract was obtained. Protein concentrations weredetermined by a bovine serum albumin (BSA) proteinassay (Pierce BSA protein assay kit, Thermo FisherScientific, Rockford, IL). Approximately 30 mg persample was resolved using sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) for120min at 110V. Proteins were then transferred to

nitrocellulose membranes on ice for 90min at 100V.Membranes were blocked in milk buffer at room tem-perature for 1 h and incubated on a shaker plate withAndrogen receptor AN4441 (MA5-13426, ThermoFisher Scientific, Rockford, IL) overnight at 4 �C.Membranes were washed with Tris-buffered saline withTween 20 (TBST) three times, 10min each, then incu-bated with secondary for 120min at room temperatureand washed again, as stated previously.Chemiluminescent substrate Dura (SuperSignal WestDura extended duration substrate, Thermo FisherScientific, Rockford, IL) was applied to the membranefor 5min and then imaged for 6min. A glyceraldehyde 3-phosphate dehydrogenase (GAPDH) image wasobtained the same way with a regular chemiluminescentsubstrate (SuperSignal West Pico, Thermo FisherScientific, Rockford, IL) applied for 3min and imagedfor 2min. Imaging was obtained with a Li-cor OdysseyFc (Li-cor Biosciences, Lincoln, NE).

Software and calculations

Calculations were carried out in RStudio with R ver-sion 3.4.1. GraphPad Prism was used to create graph-ical presentations and assess the statistical significanceamong some of the data, where indicated.

Results

Diet-induced aggression in maleFVB/N mice

During a diet-induced obesity study using FVB/Ninbred mice, we observed an unexpected manifestationof aggression among male, but not female, cages thatencumbered the investigation. In the colony containingseveral hundred FVB/N mice, cages were provided witha diet of standard rodent chow ad libitum. The femalecages required no separations for aggressive behavior(fighting, wounding, chasing, biting, blood discoveredin the cage, etc.). During the same period, the malecages required only a few separations (Figure 1(a)).

Diets were altered in a small group of the animals after10–16 weeks to measure weight changes leading to adult-onset, diet-induced obesity. In this regard, some malecages were provided ad libitum high-fat (45%) or lean(10% fat) diets over a period of 27 weeks. Males wereassigned a cage based on their genotype (SupplementaryFigure 1) and littermate status before the food was altered,with a stringent prerequisite to keep siblings together andencourage peaceful cohabitation (Figure 1(b)).

Over the 27 weeks of the diet experiment, the weightof the males in the respected cages began to increase orstabilize, based on the food provided (Figure 2(a)).The final average weights of the lean-diet-fed cages


were approximately 35 g compared with 43 g for high-fat diet cages (Figure 2(b); comparing lean with high-fatdiets with the same genotype). The changes in weightsranged from 5 g to 18 g (Figure 2(c)). This was reflectedin the fur of high-fat-diet-fed mice as it changed inappearance due to the oily red food and the propensityof the males to eat and nest in it, creating a greasy coat(Figure 2(d)). The study was terminated when one ofthe largest males, who exceeded 60 g, suddenly died forunknown reasons, although obesity and/or fatty liverdisease were suspected by the veterinarian upon nec-ropsy (data not shown).

Initially the experiment commenced with peacefulcohabitation among the groups, and this continued inthe male high-fat diet cages (Figure 3(a)).Unexpectedly, males in the lean diet cages began toshow wounds during morning observations (Figure3(b)). Cage separations were necessary to stop fighting,with some unfortunate victims of nocturnal bouts ofaggression, during the time of day when rodents areactive. Although there were more victims of aggressionin the lean diet-fed cages, over the course of the experi-mental period, there was not a significant difference inoverall survival between groups (Figure 3(c)).

At first we attributed this to male FVB/N behaviorand hypothesized that either the transgene, colony sizeor diet was the source. Initially, each male cage had anaverage of 3–5 littermates in it when the diet studycommenced, dependent on the litter size and sex atbirth. However, the number per cage dropped to anaverage of 1.4 (among wild-type, lean diet cages) toan unchanged 3.3 (among wild-type, high-fat dietcages) by the study’s end (Figure 3(d)). Once fightinghad occurred exclusively in nearly every lean cage, itbecame apparent that the behavior was abnormal.

The most striking reduction in the number of miceper cage occurred in the transgenic lean diet cages,where the average number started at 5.0 and endedwith 1.6 (p< 0.01). Although we hypothesized thatthe transgene, autotaxin, may be related to the aggres-sive behaviors observed, the following statisticaldata did not support that. Among the high-fat diettransgenic cages, the average number of mice per cagestarted at 4.75 mice and finished at 3.2 mice. Similarly,the wild-type lean diet cages started with an average of3.4 mice and finished with 1.3 mice. This suggested thatdiet was the causal factor.

Randomization model results

Thus, it became necessary to design a strategy to quan-tify the effect of each group. In the beginning of thestudy, six lean and seven high-fat diet cages were cre-ated, and at the end of the study, 17 lean and nine high-fat diet cages were recorded. To obtain the p-value, astatistic must be found that gives some measure of thedifferences between the treatment groups and the con-trol. A natural cage-level response variable is thenumber of separations in a cage divided by the totalnumber possible. A corresponding statistic typicallyused is an F-test statistic. Note that in this case thedata need not be normal, and the F-statistic need nothave an F distribution, because our results will not relyon any asymptotic, large-sample theory. There is a totalof 156 mice, with an average of 4.5 per cage.The maximum number of possible separations for acage is the number of animals in that cage minusone. The observed F-statistic based on the proportionof separations was 3.72 and the p-value from100,000 permutations was 0.025 (estimated

Figure 1. FVB/N males, but not females, are more likely to require cage separations. (a) Bar graphs showing the numberof total cages required overall with a standard, chow diet. This is reflective of typical cage separations over a four-monthtime period with FVB/N mice and standard conditions of sterile bedding, supplemental nesting material, mouse houses,ad libitum food and water. Males began with �5 littermates in each of the 20 cages (n¼ 100) and ended with 26 cages.Females began with �5 littermates in each of the 28 cages (n¼ 140) and ended without any changes. (b) Four malelittermates fed a high-fat diet peacefully cohabitating in their cage. The fifth mouse is just outside of the image and slightlyaway from the group.

Murph et al. 163

standard error 0.0005). The histogram of the permu-tations is shown in Figure 4 and can also be foundat: https://github.com/dobbinke/Rmarkdown4cagesep.Since the p-value is less than 0.05, we reject the nullhypothesis at the 0.05 significance level and concludethat the separations differ by treatment assignment forat least some of the treatment groups and control.

Population model results

Therefore, for cage i assigned to conditionk 2 0,1,2,3,4f g, we denote the probability of turningaggressive as pki, where E pki½ � ¼ pk. We mathematicallyderived (Supplementary Figure 2) both the method ofmoments estimator and the maximum likelihood

estimator for the proportion of aggressive animals(Supplementary Figure 2). We chose to use the max-imum likelihood estimator, which for cage i in condi-tion k with mki separations, is

pki ¼mki=nki : mki � nki 2

1 : mki ¼ nki 1

�

Then pk ¼1rk

Prkk¼1 pki, where rk is the number of

cages assigned to condition k. A one-way analysis ofvariance results in an F-statistic with four numeratorand 29 denominator degrees of freedom, a value of 4.18and a p-value of 0.009. Since the p-value is below 0.05,we reject the null hypothesis. But, because there areonly three or four replicates per active treatment, one

Figure 2. Male FVB/N mice show significant increases in weight with high-fat diets. (a) Males were fed either a lean orhigh-fat diet for 27 weeks and then weighed. Box plots shows the range of (b) the final average weight or (c) the change inweight, in grams, per group at the end of the study. The association herein is related to diet, not the transgene, which hasno affect. (d) Image comparing a mouse fed a lean diet (bottom) with a mouse fed a high-fat diet (top). Note the differencein coat appearances between the mice due to the fat content of the diets.***p< 0.001.**p< 0.01; one-way ANOVA, multiple comparisons test, followed by Bonferroni’s post-test.


https://github.com/dobbinke/Rmarkdown4cagesep

may have concern that these small replicate numbersmean that the distribution of this F-statistic may notbe close to normal. A more robust alternative is thenon-parametric Kruskal Wallis test. A non-parametricKruskal Wallis one-way analysis of variance produces achi-square statistic of 9.0172 with 4 degrees of freedomand a p-value of 0.06. In this case, the p-value is notsignificant at the 0.05 significance level. However, theKruskal Wallis test has low power and that may explainthe discrepancy. Moreover, since the permutation testused for the randomization model did not rely onasymptotics, the p-value from that analysis argues

strongly against the null hypothesis. The maximumlikelihood estimates pk are p0 ¼ 0:06, p1 ¼ 0:10, p2 ¼0:00, p3 ¼ 0:60, p4 ¼ ,0:33. One can see that group 3appears to have a higher probability of aggressionthan the others.

The Dunnett test15 is appropriate for comparing mul-tiple treatments to a single control. For the Dunnett test,only group 3 is significantly different than the control,with a corrected t-test statistic of 3.759 and a p-valueof 0.0031. The Dunnett test output for all compari-sons is shown in Table 1. The estimate of p3 p0 is0.54 and a 95% confidence interval for the difference is

Figure 3. Males fed a lean diet significantly increase aggressive behaviors. (a) Bar graphs displaying the overall numberof cage separations over the entire diet experiment. The study was required to be terminated due to spontaneous deathamong an obese male mouse. (b) Image of mice from a lean-diet-fed cage showing a victim (left) that suffered a seriouswound as a result of the perpetrator (right) from nocturnal activities that were discovered the next morning. The per-petrator is seemingly sniffing the opponent, even after inflicting horrific wounds overnight. The veterinarians requestedthat both animals be euthanized using a method consistent with the guidelines of the American Veterinary MedicalAssociation. (c) A Kaplan–Meier statistic was used to estimate the overall survival of the mice in the study, based on diet.The solid red line indicates those on the high-fat diet versus the dashed line for the lean diet. Results were not statisticallysignificant. (d) Group cage separations that were required during the diet study.**p< 0.01; preliminary permutation p-value of 0.00432, based on 100,000 Monte Carlo.

Murph et al. 165

(0.26,0.82). Despite the statistically significant p-value,more experiments would be needed to narrow downthis range.

Biochemical analysis

To biochemically explain the reason why lean-diet-fedmice exhibit more aggression towards littermates, weinitially hypothesized that the concentration of testos-terone was variable between the groups. However, acomparison of lean-diet-fed mice versus high-fat-diet-fed mice demonstrated no significant difference in tes-tosterone (Figure 5(a)). In contrast, when the ratio ofADP/ATP was assessed in tissue, the lean-diet-fed micedemonstrated a significant decrease in comparisonto high-fat-diet-fed mice (Figure 5(b)). This suggeststhat energy status and metabolism are the crux of theenhanced nocturnal activity,16–18 which is consistent

with this strain. Immunoblotting of the tissues fromthe animals showed no apparent differences in theexpression of the androgen receptor between groups(Figure 5(c)).

Discussion

In this study, we observed unexpected, heightenedmanifestations of aggressive nocturnal bouts amongFVB/N male cages fed a lean diet. To evaluate thisphenomenon, we developed statistical modeling andcomputation methods to analyze the data using ran-domization and population models with a censoredlikelihood. We conclude that male cage separationsdiffer significantly due to a lean diet (i.e. treatmentassignments) and are unrelated to transgene, autotaxin,status and the testosterone level of the mice.

The logical conclusion of our study is that energyamong FVB/N males is intensified by feeding males alean diet because this is a normally hyperactivestrain,1,2 which then has amplified activity for noctur-nal bouts of aggression. The FVB/N strain is defined insome circumstances by their aggressive nature,although this is almost exclusively assigned to males,not females.8 Previous studies with crosses of inbredCBA and outbred CFLP mouse strains determinedthat lean mice exhibit more nocturnal wheel runningand higher food intake, compared to fat mice, withlean mice expending substantially more energy towardsactivity.18

The opposite is also true – feeding hyperactiveFVB/N males a high-fat diet mitigates active behaviorbecause they become sedentary and are less likely toengage in vigorous nocturnal movements. Fat inbredCBA and outbred CFLP mice are also less likely to par-ticipate in activities and will steadily decrease partici-pation in nocturnal behaviors, such as wheel runningand vertical rearing.19 Taken together, a lean diet withfour-fold less fat enhances active behaviors and keepsthe mice agile so that they are more likely to engage innocturnal bouts of aggression.

Therefore, the contributing factor that causesthe hyperactive, aggressive FVB/N mice to increasenocturnal activity is a lean diet with 17% sucrose andtwice the amount of carbohydrates. This diet heightensnocturnal behaviors, whereby FVB/N male energy isexpended inappropriately on aggressive behavior. Tothis point, FVB/N mice have a significantly highertotal glucose production compared with C57BL/6J,129S1/SvImJ and ICR mouse strains.20 Since FVB/Nmice exhibit reduced anxiety levels in comparison toother strains,21 it is unlikely that anxiety contributesto this phenomenon.

We also tested testosterone levels and androgenreceptor expression between groups, but the results

Figure 4. F-statistic histogram of the data permutations.The F-statistic shows the ratio of the variances. Becausethe p-value is less than 0.05, this resolves that it isappropriate to reject the null hypothesis at the 0.05 sig-nificance level. Further, the statistics conclude that theseparations differ by treatment, for at least some of thetreated groups and control. The vertical line is observed.

Table 1. Multiple comparison of means with Dunnettcontrasts.

EstimateStandarderror

T-teststatistic p-value

Group 1 vs control 0.0400 0.1436 0.278 0.997

Group 2 vs control –0.0600 0.1624 –0.370 0.992

Group 3 vs control 0.5400 0.1436 3.759 0.003

Group 4 vs control 0.2733 0.1624 1.683 0.338

Dunnett contrasts used for comparing the proportions in eachgroup to the control.Group 0 is the control.


were not significant (Figure 5(a) and (c)), suggestingthese were not contributing factors for this study.However, this is in contrast with a study showing thatthe highly aggressive CD1 strain ceases fighting uponcastration.22 Nevertheless, castration has major ethicalconsiderations and is an unnatural scenario; and, yet,an adult male living among a group of other adult malemice is an unnatural scenario, too. Most wild mice ter-ritories have one adult male, several females and theirpups, which is not practical in the laboratory.23

When planning the study, we sought to mitigateFVB/N aggressive tendencies by housing males withlittermates, which facilitates stable social groups andhierarchy from an early age, and by maintaininggroup housing since periods of single housing canexacerbate aggression.24 Other studies show thatstress can be induced in male mice with olfaction expos-ure to human male experimenters25; however, all of thehuman experimenters in this study were female. Theonly male in the research group was the statisticianwho did not handle the mice.

On the other hand, cages did have structural enrich-ment, in the form of a mouse house, that was notablyused as a male marking post. Sometimes these types ofstructures provide opportunities for dominant perpet-rators to ambush a victim or attack them as they enterand exit the mouse house.24 Although nesting materialwas provided and is supposed to decrease aggression,26

this did not prevent aggression among the FVB/Nmales fed a lean diet, but we did not test what occursin the absence of nesting material.

Prior to this study, we did not anticipate an elevationin overnight wounding to arise in male cages fed lean

diets. To control the fighting, cages with wounded micerequired separation to avoid future occurrences. By theend of the study, we had an entire housing rack filledwith singly housed males in the facility, which requireswritten justification for each circumstance and commu-nication with the veterinary staff. Studies have shownmale mice prefer conspecific housing with another maleversus an empty cage, even in the presence of aggres-sion.27 Therefore, group housing is our standard prac-tice and deviations must be explicitly justified. Weavoid solitary housing unless it is absolutely necessary,a position which coincides with the general consensus infavor of group housing for male mice.23 Recently, Weberet al. strongly recommended limiting necessary malegroup housing to three animals in a standard cage andnot providing shelter enrichments under scenarios thatare known to increase aggression.28

In summary, we developed a novel statistical methodfor analyzing an abnormal set of biological data, stem-ming from a study about diet-induced obesity.Although unexpected, we observed a rise in male noc-turnal bouts of aggression among cages with ad libitumlean food. Using the randomization and populationmodels, we concluded significance was present insome of the data. Cage separations were not random;rather, they were resultant from a lean diet consumedby FVB/N adult males that enhanced energy status andresulted in nocturnal bouts of aggression. In conclu-sion, the hyperactive and subsequent aggressive behav-ior of the FVB/N adult males them an unsuitable modelfor long-term studies requiring lean-diet modifications;however, they could be utilized to study male mouseaggression that only requires a modification in diet.

Figure 5. Biochemical analysis of male mouse tissue. (a) Serum was collected at necropsy and tested for the concen-tration of testosterone (ng/mL). (b) The ratio of ADP/ATP was measured in the male tissues to assess the energy status ofthe groups. (c) Immunoblotting of androgen receptors did not reveal any striking trends between the groups and quan-tification of the bands revealed no differences.*p< 0.05 using student’s t-test to measure lean versus high-fat diet groups.

Murph et al. 167

Acknowledgement

Our thanks to Charnel Byrnes for proofreading this

manuscript.


The author(s) declared no potential conflicts of interest withrespect to the research, authorship and/or publication of this

article.

Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship and/or publication of this

article: The research was supported by a grant (to MMM)from the National Institutes of Health (grant number1R15CA176653).

ORCID iD

Mandi M Murph http://orcid.org/0000-0002-5152-4977

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18. Comelli M, Pretis I, Buso A, et al. Mitochondrial energy

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19. Simoncic M, Horvat S, Stevenson PL, et al. Divergentphysical activity and novel alternative responses to highfat feeding in polygenic fat and lean mice. Behav Genet

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http://orcid.org/0000-0002-5152-4977

Resume

La souche de souris consanguine hyperactive FVB/N est largement utilisee pour les applications de recherchetransgenique, bien que rarement pour des etudes comportementales. Ces souris ont une deficience visuelledue a leur degenerescence retinienne, mais sont considerees comme tres intelligentes et se basent engrande partie sur l’olfaction. Lors d’une enquete sur l’obesite induite par le regime alimentaire meneechez des souris FVB autotaxines transgeniques, nous avons observe une necessite accrue de separer lesmales, mais pas les femelles, en utilisant des cages separees. Sur la base de nos observations, nous avonsemis l’hypothese que l’alimentation maigre des souris FVB/N augmentait les episodes d’agression nocturneentre les males de la meme portee. L’alimentation des animaux adultes d’une portee etait changee de lanourriture normale a des aliments a haute teneur en matieres grasses (45% de matiere grasse) ad libitum oumaigres (10% de matieres grasses) pendant 27 semaines, les souris parvenant ainsi a un poids moyen de 43 gcontre 35 g, respectivement. Du fait des combats nocturnes qui eclataient, la separation des animaux en lesmettant dans des cages differentes s’est imposee meme si les souris de la portee avaient cohabite demaniere pacifique pendant les 10-16 semaines precedentes. Les donnees etant de nature inhabituelle,elles necessitaient d’elaborer des methodes statistiques peu communes afin d’evaluer si elles revetaientde l’importance et si cette importance etait significative. Par consequent, nous avons etabli une methodologieen nous appuyant sur la randomisation et les modeles de population, et emit l’hypothese que la testosterone,l’autotaxine transgenique ou alteration du regime alimentaire constituait le facteur causal. Les evaluationsstatistiques ont demontre une correlation significative entre le comportement agressif et la separation dansdes cages differentes associes aux souris suivant une alimentation faible en lipides, pas avec l’autotaxine. Lesdonnees biochimiques ne semblent pas expliquer le comportement. En revanche, le metabolisme de l’energiea mis en lumiere des differences entre les groupes de souris normalement hyperactives selon l’alimentation.Cette caracteristique fait des souris FVB/N males des sujets inappropries pour les etudes a long termeimpliquant des modifications alimentaires basees sur un faible taux de lipides.

Abstract

Der hyperaktive FVB/N Inzucht-Mausstamm wird haufig in der transgenen Forschung verwendet, seltenjedoch fur Verhaltensstudien. Diese Mause sind sehbehindert aufgrund von Netzhautdegeneration, geltenaber als hochintelligent und sind weitgehend auf ihren Geruchssinn angewiesen. Bei der Untersuchung vonernahrungsbedingter Adipositas bei transgenen Autotaxin-FVB/N-Mausen beobachteten wir eine verstarkteNotwendigkeit der Kafigtrennung von mannlichen, nicht jedoch von weiblichen Tieren. Aufgrund derBeobachtungen vermuteten wir, dass die Futterung der FVB/N-Mause mit magerer Kost nachtlicheAggressionsschube zwischen mannlichen Wurfgeschwistern verstarkt. Die Ernahrung erwachsenerWurfgeschwister wurde ad libitum 27 Wochen lang von normalem Futter auf fettreiche (45% Fett) odermagere (10% Fett) Ernahrung umgestellt, wobei die Mause durchschnittlich 43 g bzw. 35 g erzielten.Daraufhin wurden Kafigtrennungen aufgrund nachtlicher Aggressionsschube unvermeidlich, obgleich dieWurfgeschwister 10-16 Wochen zuvor friedlich zusammengelebt hatten. Da die Daten ungewohnlich waren,mussten ungewohnliche statistische Methoden entwickelt werden um festzustellen, ob und inwiefernSignifikanz bestand. Deshalb entwickelten wir unter Einsatz der Randomisierungs- undPopulationsmodelle eine Methodik und postulierten, dass entweder Testosteron, das Autotaxin-Transgenoder die Ernahrungsumstellung der Kausalfaktor war. Statistische Auswertungen zeigten eine signifikanteKorrelation zwischen Kafigtrennungen und aggressivem Verhalten bei den mit Magerkost gefuttertenMausen, nicht Autotaxin. Biochemische Daten schienen das Verhalten nicht zu erklaren. Im Gegensatzdazu zeigte der Energiestoffwechsel Unterschiede zwischen den Gruppen von normalerweise hyperaktivenMausen je nach Ernahrung auf. Diese Eigenschaft macht mannliche FVB/N-Mause ungeeignet furLangzeitstudien mit Ernahrungsumstellung auf Magerkost.

Resumen

La cepa consanguınea hiperactiva del raton FVB/N se utiliza ampliamente para aplicaciones de investigaciontransgenica, aunque pocas veces para estudios del comportamiento. Estos ratones tienen dificultades devision a traves de la degeneracion de la retina, pero se consideran muy inteligentes y dependen mucho delolfato. Al investigar la obesidad por dieta en ratones FVB/N transgenicos autotaxin, observamos un aumento

Murph et al. 169

de la necesidad de separar ratones macho, no ası con los ratones hembra. Basandonos en la observacion,hemos teorizado sobre el hecho de que el dar una dieta saludable a los ratones FVB/N aumenta las peleasnocturnas entre los machos de la camada. Las dietas de los machos adultos de la camada pasaron de comidanormal a dietas altas en grasas a voluntad (45% de grasa) o dietas saludables equiparables (10% de grasas)durante 27 semanas, durante las cuales los ratones alcanzaron una media de 43 g frente a 35 g, respecti-vamente. Entonces, la separacion de las jaulas debido a las peleas nocturnas tuvo que ser obligatorio, a pesarde que los miembros de la camada habitaron pacıficamente durante las 10-16 semanas anteriores. Ya que losdatos eran poco usuales, se requirieron metodos estadısticos distintos para poder evaluar si habıa datossignificativos y donde los habıa, en caso de existir. Por tanto, utilizar los modelos de poblacion y aleatoriedad,establecimos una metodologıa y postulamos que el factor causal era la testosterona, el transgene autotaxin ola alteracion de la dieta. La evaluacion estadıstica demostro una correlacion significativa entre las separ-aciones de jaulas y el comportamiento agresivo asociado a una dieta saludable de los ratones, no a laautotaxin. Los datos bioquımicos no parecıan explicar el comportamiento. Por contraste, el metabolismoenergetico destaco las diferencias entre los grupos de ratones con una hiperactividad normal segun ladieta. Esta caracterıstica hace que el raton macho FVB/N sea un modelo inadecuado para estudios a largoplazo con modificaciones de dietas saludables.


Original Article

Combination of ketamine and xylazinewith opioids and acepromazine in rats:Physiological changes and their analgesiceffect analysed by ultrasonic vocalization

Jilma Aleman-Laporte1,2 , Luciana A Bandini1,Mariana SA Garcia-Gomes1, Dennis A Zanatto1,Denise T Fantoni3, Marco A Amador Pereira3,Pedro E Navas-Suarez1, Thiago Berti Kirsten4,Randall R. Jimenez5, Gilbert Alvarado1,6

and Claudia Cabrera Mori1

AbstractIn this study, the effect of four anaesthetic protocols that included the combination of xylazine (X) and keta-mine (K) with acepromazine (A) and opioids (methadone (Me), morphine (Mo) or tramadol (T)) was evaluated inlaboratory rats of both sexes. Ultrasonic vocalization (USV) was used as an indicator of pain during therecovery period. The objective was to evaluate the physiological parameters and the analgesic effect ofeach protocol to determine which protocol was the safest and fulfil the requirements of a balanced anaes-thesia. The better protocols were the XKA protocol for both sexes and the XKMe protocol for females becausethe combinations achieve surgical plane of anaesthesia in rats. However, pain assessment during the formalintest revealed that rats anaesthetized with XKA produced more numbers of USV, suggesting that it is not agood protocol for the control of immediate postoperative pain. All protocols produced depression in bodytemperature and respiratory and heart rates, and had important effects, such as micturition and maintenanceof open eyes. Only rats anaesthetized with XKA protocol did not present piloerection. These results demon-strated that good monitoring and care during anaesthesia must be included to prevent complications thatcompromise the life of the animal and to ensure a good recovery. The inclusion of analgesia in anaesthesiaprotocols must be used routinely, ensuring minimal presence of pain and thus more reliable results in theexperimental procedures.

KeywordsAnaesthetic protocol, ultrasonic vocalization, analgesia, physiological measures, refinement

Date received: 27 June 2018; accepted: 18 April 2019

Rats are commonly used in research, and the use ofanaesthesia is required and recommended for manyexperimental procedures. Well-controlled anaesthesiais essential to reduce potential surgical complications

1Department of Pathology, University of Sao Paulo, Brazil2Laboratorio de Docencia en Cirugıa y Cancer (DCLab), Universityof Costa Rica, Costa Rica3Department of Surgery, University of Sao Paulo, Brazil

4Environmental and Experimental Pathology, Paulista University,Brazil5Institute of Evolutionary Ecology and Conservation Genomics,University of Ulm, Germany6Laboratory of Experimental and Comparative Pathology(LAPECOM), School of Biology, University of Costa Rica, Costa Rica

Corresponding author:Jilma Aleman Laporte, Department of Pathology, School ofVeterinary Medicine and Animal Science, University of Sao Paulo(USP), Av. Prof. Dr Orlando Marques de Paiva, 87, CidadeUniversitaria, CEP 05508-270, Sao Paulo Brazil.Email: [email protected]

Laboratory Animals

2020, Vol. 54(2) 171–182




DOI: 10.1177/0023677219850211


https://orcid.org/0000-0003-4466-512X


https://doi.org/10.1177/0023677219850211


and ensures safety and success in the experimental pro-cedures.1 Anaesthesia in laboratory rodents is particu-larly challenging due to the problems related to theirsize, for example, accelerated metabolism, easy devel-opment of hypothermia and difficulty in determiningcardiorespiratory functions.2 Therefore, the goal is toestablish a balanced anaesthetic protocol that includesa combination of several drugs to obtain good controlof unconsciousness, analgesia and myorelaxation tominimize anaesthetic risks.3

The anaesthetic protocols that mix two or threecomponents have better effects compared to a singleanaesthetic because one product cannot produce allthe effects that are sought in anaesthesia (hypnosis,muscle relaxation and analgesia).4 Ketamine is one ofthe most commonly used parenteral drugs in laboratoryrats and is normally combined with other drugs such asxylazine.5,6 The use of this combination with acepro-mazine has been described in the literature,3,5 but it isnot commonly used in many laboratories. In addition,there is not much information on anaesthetic protocolsused in laboratory rodents that include the combin-ation of these anaesthetics with opioids, such as trama-dol, morphine and methadone. The preoperativeadministration of opioids simultaneously with theanaesthetic protocols has advantages such as the possi-bility of reducing the required dose of anaesthetics toachieve surgical anaesthesia as well as reducing the needfor postoperative analgesics.7 In view of these benefitsthe present study is important to clarify the interactionof the combination of XK with different opioids in aninjectable anaesthetic protocol.

Vocalization serves as an indicator of emotional andaversive states in rats.8 Juvenile and adult rats emitbasically two types of ultrasonic vocalizations (USV)that are distinguished on the basis of the frequency:the 22 kHz vocalizations produced in response to aver-sive and dangerous situations and the 50 kHz vocaliza-tions that are produced in response to appetitivesituations, such as copulation and play behavior.9

Jourdan et al. also demonstrated that monitoringrodent USV is a potential method of measuring thenegative affective component of pain by a brief elec-trical pulse applied to the tail.10

The effects of opioids have been extensively studiedusing the formalin test, and it has been shown thatmorphine, methadone and tramadol influence pain con-trol in rats.11,12,13 However, there are no studies thathave been performed yet with the opioids that wereused in this experiment in the context of anaesthesiain rats. For this reason, it is important to evaluateintraoperative pain and recovery from anaesthe-sia.10,14,15 The objective of this study was to evaluatethe effect of different anaesthetic protocols on thephysiological parameters (heart and respiratory rate,

temperature and clinical signs) and their intraoperatoryanalgesic power to determine the most effective and safeprotocols for surgical procedures and to ensure thewell-being of laboratory rats.

Materials and methods

Animals

In total, 64 SPF Wistar-Han rats (Rattus norvegicus),28 females and 36 males aged from eight to 12 weekswere used. Animals were obtained from the animalfacility of the Institute of Biomedical Science of theUniversity of Sao Paulo, and they were free of ecto-and endoparasites, Mycoplasma pulmonis, Pasteurellapneumotropica, Bordetella bronchiseptica, Helicobacterspp., Klebsiella oxytoca, Klebsiella pneumoniae,Pasteurella multocida, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus �-hemolytic spp.,Streptococcus pneumoniae and Salmonella spp., KilhamRat Virus, Pneumonia Virus of Rat and Reovirus. Atmost, 4 animals were housed per cage on corn-cob bed-ding (Granja R.G., SP, Brazil) in polypropylene cages(41� 34� 16 cm) that were changed once a week. Paperrolls were added as environmental enrichment. All theanimals were maintained under climate-controlled con-ditions of 12:12-h light/dark cycle, temperaturerange from 22 to 24�C, relative humidity of 45–65%,ad libitum access to drinking water, and a standardfood-pellet diet (Nuvital�-Quimtia, PR, Brazil).Before the beginning of the experiments, the animalswere housed in the animal facility of the Departmentof Pathology of the School of Veterinary Medicine andAnimal Science, University of Sao Paulo, where theystayed for at least one week for adaptation and hand-ling before the start of the experiment.

All animal procedures were performed in accord-ance with the guidelines of the Ethics Committee ofthe School of Veterinary Medicine and AnimalScience of the University of Sao Paulo, Brazil(no.9635260116).

Anaesthetic procedure

Four different protocols were established for thisstudy16–18 (see Supplementary Material 1).

For males:

1. XKA: Xylazine 7.5mg/kg (Anasedan�-Ceva, 20mg/ml,SP, Brazil)þKetamine 60mg/kg (Dopalen�-Ceva,100mg/ml, SP, Brazil), þAcepromazine 2mg/kg(Acepran�-Vetnil, 2mg/kg, SP, Brazil);

2. XKMe: Xylazine 5mg/kgþKetamine 60mg/kgþMethadone 5mg/kg (Mytedom�-Cristalia, 10mg/ml,SP, Brazil);


3. XKMo: Xylazine 7mg/kgþKetamine 60mg/kg,þMorphine 1mg/kg (Dimorf�-Cristalia, 10mg/ml,SP, Brazil); and

4. XKT: Xylazine 7.5mg/kgþKetamine 65mg/kg,Tramadol 5mg/kg (Tramadon�-Cristalia, 50mg/ml,SP, Brazil).

For females:

1. XKA: Xylazine 5mg/kgþKetamine 60mg/kgþAcepromazine 1mg/kg;

2. XKMe: Xylazine 5mg/kgþKetamine 60mg/kgþMethadone 5mg/kg;

3. XKMo: Xylazine 5mg/kgþKetamine 60mg/kgþMorphine 1.5mg/kg; and

4. XKT: Xylazine 5mg/kgþKetamine 60mg/kg,Tramadol 5mg/kg.

Rats were previously weighed to determine the exactdose of each drug. To avoid any interference of circa-dian rhythm, the experiment was always performedfrom 6.00 a.m. to 12.00 p.m. The administration of theanaesthetic protocol was done by intraperitoneal route.The drugs were mixed in a sterile plastic tube immedi-ately prior to the administration and were given in oneinjection to minimize handling stress with a maximuminjection volume of 0.5–0.7ml. Then, the rat was placedin a cage alone to observe its behaviour, and when theanimal lost the righting reflex, it was laid in dorsalrecumbency on a preheated thermal blanket (between35 and 37�C) to minimize the loss of body temperature.Sterile ocular lubricant (Vidisic�-Gel-BauchþLomb,2mg/kg, SP, Brazil) was administered to both eyes.

During the anaesthesia, the following periods wererecorded:

1. Induction time: the period from the administrationof the anaesthesia until the loss of the righting reflex.

2. Non-surgical anaesthesia: the period between theloss of the righting reflex and the loss of most orall reflexes (blink, pedal and tail withdrawal reflex).

3. Surgical anaesthesia: the period between the loss ofall the reflexes (considering surgical tolerance theloss of the pedal withdrawal reflex) and theirrecuperation.

4. Recuperation time: the period between the recuper-ation of all the reflexes and the return of the abilityto walk.

The total duration time of the anaesthesia, theperiod from the administration of the anaestheticprotocol until the withdrawal of the ability to walk,was also measured.

The rats initially breathed room air but 7 minutesafter losing the righting reflex, they were supplied with

100% oxygen using a nose cone until the end of theanaesthesia (Supplementary Material 2).

After the induction time, a pulse-oximeter(NT1A-V�-Solaris Medical Technology, Inc., CA,USA) was positioned on the pad of the left hind limb,and then the physiologic parameters and reflexes weremeasured every 10 minutes until the righting reflexreturn. The heart rate was determined by the pulse-oximeter and the respiratory rate was counted by obser-ving thoracic or abdominal movements. The bodytemperature was measured by a digital thermometerplaced into the animal’s rectum. The reflexes were eval-uated as follows: the pedal withdrawal reflex by press-ing the paw with an atraumatic forceps, alternatingboth hind limbs; the palpebral reflex by a slight touchwith the atraumatic forceps in the margin of the eyelid;and the tail pinch reflex by a compression of the tailwith the atraumatic forceps;19 any negative or positiveresponse was classified as the loss or the return of thereflex, respectively.

Pain evaluation by ultrasonic vocalization

When the rats recovered the pedal reflex or their whis-kers started to move, a subcutaneous injection of 60 mlof formalin solution (formaldehyde 37%, wt/wt,diluted to 10% in 0.9% saline wt/wt)8 was performedin the dorsal surface of the right hind paw of each rat.14

After 5 minutes of the formalin administration, theUSV were recorded using a USV detector monitor(Ultravox 2-0, Noldus Professional System forAutomatic Monitoring of Ultrasonic Vocalization,USA). Each rat was tested individually by placingthe USV detector 2 cm above the head of the ani-mal in dorsal recumbency without any kind of restric-tion. The USV detector was set to detect frequenciesof 22 kHz with an amplitude filter setting of 4 tominimize background noise. To avoid environmentalnoise, the experiment was performed in a soundproofroom.

The total number and duration of USV for eachrat were recorded during four sessions, starting 5 min-utes after the application of formalin, for atotal period of 25 minutes or until the righting reflexrecovery. Each session consisted of 3 minutes ofrecordings with 2-minute lapses between them.During the recording, the rats received a stimulusevery 20 seconds by compressing the area where theformalin was injected using a bulldog clamp.The intensity of the clamp tightening was always thesame, letting it completely close for a second andimmediately open.

At the end of the study all animals wereeuthanized by an overdose of X (30mg/kg) and K(300mg/kg) IP.

Aleman-Laporte et al. 173

Statistical analysis

A power calculation was carried out with a total powerof 0.8 and a two-sided significance of <�0.05.Estimates of variability for power calculation werebased on data from a pilot project. The study wasappropriately powered to detect a mean difference of20% in the total duration of surgical anaesthesia, sincethis was considered clinically relevant. In males, weused up to 10 rats because the administration of forma-lin caused an immediate recovery from anaesthesia insome animals, and this did not allow the USV record-ing of them. The rats of each treatment were distributedrandomly in different cages by sex.

Linear mixed effects models (LMMs) were used totest the effect of drug treatment and time (predictorvariables) on the response variables: body temperature,cardiac frequency and respiratory frequency in maleand female rats. The rat identity was used as arandom factor to account for repeated measures ineach rat. Linear models (LMs) were used to investigatethe effect of drug treatment on the response variables:induction time, non-surgical anaesthesia, recovery andtotal duration of anaesthesia. Generalized linear mixedmodels with Poisson distribution were conducted toinvestigate whether the number of vocalizations(response variable) differed according to the predictorvariables: drug treatment and time. The random factorwas rat identity. A candidate set of models were con-structed for each analysis previously mentioned andused an information-theoretic model selection (I-T)to determine which model(s) was best fit and well sup-ported by the data.21 The I-T model selection providesa strength of evidence for a set of a priori modelsbased on Akaike’s Information Criterion values(AIC) and do not rely on null hypothesis testing.21

The best fit model of each candidate set was deter-mined based on AIC adjusted for small sample size(AICc); and delta AICc (�AICc) and Akaike weights(AICcw) were used to evaluate the support of themodels (for a complete treatment of I-T, seeBurnham and Anderson).20 Here, we considered amodel having the lowest AICc, �AICc <2 andAICcw approaching 0.90 or higher the best fit andwell-supported model for the containing predictor(s).We took the best fit model in each case and reportedthe effects of predictor(s). When a main effect wasdetected we conducted post-hoc pairwise comparisons.Specifically, we used mean difference and Cohen’s deffect size for conducting post-hoc tests on LMMs andLMs, respectively, and 95% confidence intervals ofestimates (95% CIs) to measure precision and ‘statis-tical significance’. If 95% CIs overlapped, 0 indicates‘statistical non-significance’ in a pairwise comparison.The rules of thumb defined in Sawilowsky21 were usedfor interpreting Cohen’s d values. We used descriptive

statistics to describe the patterns of vocalization dur-ation due to small size to fit a statistical model. Thedata are shown as the mean � SE, unless otherwisestated. We decided not to report statistical significance(p values) in order to focus attention on the bio-logical relevance of effect sizes.22 Statistical analyseswere conducted in Rv.3.3.2 (R Development CoreTeam 2017).

Results

Physiological parameters

The best fit and well-supported model for explainingvariation in the body temperature and heart andrespiratory rates in male and female rats was the timeonly model (Supplementary material 3). The significantdifferences between the parameters over time usingmean differences and 95% CIs can be observed inSupplementary material 3–5.

Considering all the anaesthetic protocols, the meanbody temperature of the males ranged from 35.66�0.19�C (at 10 minutes) using XKA to 37.62� 0.24�C(at 60 minutes) using XKMe (Figure 1(a)). In females,the mean body temperature ranged from 36.30�0.07�C (at 10 minutes) using XKMe to 37.78�0.54�C (at 50 minutes) using XKMo (Figure 1(b)). Inmales, all protocols presented a decrease in the meanheart rate, which reached the lowest rate at minute 20(approximately 258 bpm); after this time, there was anincrease in the mean heart rate in the four anaestheticprotocols (Figure 2(a)). In the case of the females, inwhich the protocol of XKMe was administered, after10 minutes, the mean heart rate remained approxi-mately 250 bpm until minute 40 (Figure 2(b)). Allprotocols for both sexes presented means lower than70 rpm at minute 10 in the respiratory rate. However,females had a marked increase in respiratory rate after20 minutes, except in the XKMo and XKMe protocols(Figure 3). In both males and females, there was adecrease in all the physiological parameters evaluatedin the first 10 minutes.

Clinical signs, reflexes andanaesthetic times

In both sexes, more than 86% of the rats presentedmicturition in all the protocols, and 100% of the ratspresented opening of the eyes during the anaesthesia.Only the rats that were anaesthetized with XKA did notpresent piloerection (Table 1).

The protocol in which acepromazine was used hadbetter surgical tolerance in both sexes, and females alsohad good tolerance with the protocol XKMe. The ratsfrom both sexes from protocol XKT did not achieve


surgical tolerance, as well as males from protocolXKMo (Table 1).

The drug treatment model was the best fit and wellsupported model for explaining variation in the dur-ation of nonsurgical anaesthesia and recovery of malerats (Supplementary material 7). Male rats anaesthe-tized with XKA showed a lower duration of non-surgical anaesthesia compared to the other protocols(Table 1), and differences were large between themaccording to Cohen’s d effect sizes (Supplementarymaterial 8). In relation to duration of recovery, therewere large and medium differences between protocols,except between XKT and XKMe, where the differencewas negligible (Supplementary material 8). Regarding

duration of anaesthesia, the model with drug treatmenteffect was not well supported but provided the best fit(Supplementary material 7). The protocols XKA andXKMo showed a higher duration compared to XKMeand XKT, and their differences were large according toCohen’s d (Supplementary material 8).

In females, the duration of non-surgical anaesthesiadiffered between protocols (Supplementary material 7).The protocols XKA and XKMe showed a lower dur-ation compared to the protocols XKMo and XKT(Table 1). Drug treatment models were not wellsupported but provided the best fit to examine the dur-ation of recovery and total duration (Supplementarymaterial 6). Recovery was the lowest in rats

Figure 1. Variation in body temperature (�C) in male (a) and female (b) rats submitted to different anesthetic associations.(X: Xylazine, K: Ketamine; A: Acepromazine; Me: Methadone; Mo: Morphine; and T: Tramadol.) The points represent fits tothe model’s predicted values, and the lines indicate the SE of model fits.

Figure 2. Variation in heart rate (beat per minute (bpm)) in male (a) and female (b) rats submitted to different anaestheticassociations. (X: Xylazine; K: Ketamine, A: Acepromazine, Me: Methadone, Mo: Morphine, and T: Tramadol.) The pointsrepresent fits to the model’s predicted values, and the lines indicate the SE of model fits.


anaesthetized with XKMe (Table 1) and showed somesignificant differences with other protocols according toCohen’s d effect sizes (Supplementary material 8). Totalduration was the lowest in female rats anaesthetized.with XKT (Table 1) with some significant mediumand very large differences with other protocols(Supplementary material 8).

Ultrasonic vocalization

For the number of male vocalizations, the best fit andwell-supported model of the four possible modelsexamined through AICc criteria was the drug treatmentby time interaction model; the same result was observedin female rats (Supplementary material 9). These inter-actions indicate that the effect of the protocol on thenumber of times rats conducted a vocalization was notconstant through time. The painful stimulus increasedthe number of vocalizations with a longer duration pat-tern in the XKA protocol for both sexes (Figure 4and 5). In the case of male rats, after the applicationof formalin some animals recovered the righting reflexbefore the USV recordings started, so to evaluate theformalin test, the number of animals was increased inorder to reach at least seven animals per group. Only inthe 20–23 interval, in males, was an increase in thenumber of vocalizations observed with the XKMoprotocol, but with a lower duration pattern in compari-son to the XKA protocol (Figure 4(a)).

Discussion

The doses recommended in the literature for ketamineand xylazine are very varied and depend on whetherthey are applied alone or in combination with other

drugs. Because the combinations of XK with opioidsused in this study were not described in the literature,the doses were based on a pilot study conducted prior tothis experiment (unpublished data). There is evidencethat rats female and male can have differences in theresponse to the same anaesthetics,23 so the sex of the ani-mal is a variable that should be considered for the estab-lishment of the doses to be used. Although the doseswere not identical for both sexes, the similar behaviourin the dynamics of the physiological parameters of eachprotocol allows us to conclude that the doses used in thisstudy were adequate. For this same reason, the primaryobjective was not to compare the effect between sexes.

Physiological parameters

The reduction in all physiological parameters in the firstminutes observed in all anaesthetic protocols has beenpreviously described with the XK protocol.6,24,25 Thereduction in body temperature observed in rats may berelated to the depressant effect of XK on thermoregula-tory mechanisms, in addition to the fact that smallrodents easily lose heat when anaesthetized, given thehigh proportion of body surface in relation to theirweight.25,26

The heart rate depression observed in all protocols inmale rats and in the XKMe protocol in females can beproduced mainly by the action of xylazine and metha-done. Ketamine usually causes a stimulation of cardio-vascular function causing an increase in heart rate andblood pressure. Thus, the reduction in heart rate maybe a side effect of a2-agonists as a result of an increasein vagal reflex activity and decrease in norepinephrinerelease in the sympathetic nervous system.26

Methadone can cause calcium channel blockage

Figure 3. Variation in the respiratory rate (breaths per minute) in male (a) and female (b) rats submitted to differentanaesthetic associations. (X: Xylazine; K: Ketamine; A: Acepromazine; Me: Methadone; Mo: Morphine; and T: Tramadol.)The points represent fits to the model’s predicted values, and the lines indicate the SE of model fits.


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producing bradycardia,27 an effect that was observed inboth sexes with the XKMe protocol.

In addition to cardiac depression, respiratorydepression was observed in all protocols. Opioids canproduce this effect in high doses or in combination withother CNS depressants.17 Opioid receptors are abun-dant in the respiratory control center. Moreover,m-opioid receptor agonists, such as morphine andmethadone, bind to these receptors, activating themand causing respiratory rate depression.28 Xylazinecan also cause CNS depression by stimulation ofa2-adrenoreceptors.29

All protocols in this study follow the same pattern ofphysiological parameter decrease at the first 10 minutes.The clinical support of the animal is critical mainly inthis period of time. A better recovery and a reduction ofmortality can be enhanced by monitoring, heating andoxygenating the animals.

Clinic signs, reflexes and anaesthetic times

A marked micturition (86% or more) was observed inall protocols, which was also recorded in rats treatedwith xylazine.25,26,30 This drug produces inhibition of

Figure 4. Number of ultrasonic vocalizations during the formalin test in male (a) and female (b) rats at different timeintervals (A: 5–8 minutes; B: 10–13 minutes; C: 15–18 minutes; and D: 20–23 minutes), submitted to different anaestheticassociations. (X: Xylazine; K: Ketamine; A: Acepromazine; Me: Methadone; Mo: Morphine; and T: Tramadol.) The barsrepresent fits to the model’s predicted values, and the lines indicate the 95% confidence interval of model fits.

Figure 5. Duration of ultrasonic vocalizations (seconds) during the formalin test in male (a) and female (b) rats atdifferent time intervals (A: 5–8 minutes; B: 10–13 minutes; C: 15–18 minutes; and D: 20–23 minutes), submitted todifferent anaesthetic associations. (X: Xylazine; K: Ketamine; A: Acepromazine; Me: Methadone; Mo: Morphine; andT: Tramadol.) The bars represent mean values, and the lines indicate�SE.


the antidiuretic hormone release, which is the reasonwhy the use of combinations with xylazine are not rec-ommended in animals with urinary tract obstruction,dehydration or hypovolemia.31 Only the rats that wereanaesthetized with XKA did not present piloerection.Xylazine can cause piloerection as a side effect, butwhen combined with acepromazine, which has relaxantproperties, this effect can be counteracted.32

All animals anaesthetized in this study kept theireyes open during anaesthesia; besides the loss of blinkreflex, the combination of XK induces a mild proptosisof the globe and the retraction of the eyelid.33

According to Turner and Albassam, it is possible thatthe blink reflex is depressed to a greater degree in ratsanaesthetized with the combination of XK versus otherinjectable or inhaled anaesthetic combinations.34

Corneal lesions predisposed by the use of this anaes-thetic combination may occur due to trans-cornealwater loss due to aqueous humour alteration when con-tinuous corneal exposure occurs,35 or by the vasocon-striction of the ciliary and iridial vessels, and the localand systemic hypoxemia caused by xylazine, which mayculminate in cell death.34 For this reason, the use ofprotocols with XK is not recommended for ocular stu-dies in which corneal evaluation is important. However,if there is no other option to use a different anaestheticcombination, eye drops, 100% oxygen supplementationand the administration of an adequate dose of yohim-bine (to reverse the action of xylazine) are recom-mended to minimize the incidence of corneal lesions.

The XKA protocol resulted in a better anaestheticeffect in males. This protocol allows procedures with aduration time of approximately 25 minutes with a goodmargin of safety. Based on Guedel’s stages of anaesthe-sia, the loss of the pedal withdrawal reflex indicates thatthe animal achieved the second plane of the third stageof anaesthesia.36 This means that those rats that lostthis reflex achieved surgical tolerance. The XKMeprotocol had a longer duration of surgical anaesthesiacompared to XKA protocol having a recovery timethree times faster; however, fewer animals achievedthis level of anaesthesia. None of the animals achievedsurgical anaesthesia with the XKMo and XKT proto-cols. Therefore, the use of these doses for surgical pro-cedures is not recommended. For females, XKMe andXKA were the most effective anaesthetic protocols. TheXKMe protocol had a longer surgical anaesthetic dur-ation and a much faster recovery compared to XKA.Since methadone is an opioid that has great analgesiceffect, the XKMe combination may be a good optionfor procedures that are invasive and painful.

When using the XKMo protocol, only two out ofseven animals achieved surgical anaesthesia and therecovery period was very prolonged. These results sug-gest that this protocol is not the best recommendation

for surgical procedures. As in males with the XKTprotocol, no female reached anaesthesia, which indi-cates that in this dose interval, it is not a good protocolfor use in surgery either.

The only animal that died in the experiment (withthe XKA protocol), presented hyperthermia andincreased heart rate. This could be associated to anindividual reaction to anaesthesia.

Ultrasonic vocalization

Although there was great individual variability amongthe treated individuals, in both sexes, the XKA proto-col presented the highest number of ultrasonic vocal-izations with a frequency of 22 kHz with the longestduration, but these vocalizations did not exceed 0.3seconds classified as short call. Two biologically signifi-cant subtypes of 22 kHz vocalizations have been iden-tified, both expressing negative emotional sates: longcalls (more than 300ms) that serve as alarm calls andsignal external danger or potential danger; short callsthat express a state of discomfort or distress withoutexternal source of danger.37 Thus, although XKAprotocol seemed to be a safe anaesthetic protocol, weshowed that these rats may be feeling pain.

The great difference of vocalization observed fromthe protocol XKA with the other protocols can bebecause acepromazine is a phenothiazine that can pro-duce moderate sedation but has no analgesic effectalone.27 On the other hand, opioids are analgesicsthat interact with m-opioid receptors that inhibit pain.It has been demonstrated that the emission of 22 kHzUSVs by rats subjected to experimental pain can beattenuated by the administration of drugs that possessclinically relevant analgesic properties.8

The increase in the number of vocalizations in the20–23-minute interval with the XKMo protocol inmales could be because morphine can generate an exci-tatory effect in rats39 that could generate the animalsvocalizing more.

Contrary to what was described by Wallace et al.,40

these results suggest that the USV can be used as acomplementary behavioural parameter in experimentalmodels of pain. Nevertheless, this study was a pioneerin using USV to assess pain in anaesthetized rats in arecovery phase, so additional research is required toverify possible anaesthesia influences on the USV pat-tern of rats.

In conclusion, the XKA anaesthetic protocol was thebest protocol for male rats and was also suitable forfemales, considering only the intraoperative period.Indeed, the analgesic effect of this protocol is low,and the use of an intraoperative analgesic is recom-mended. The XKMe protocol resulted in better resultsfor females due to its high analgesic effect that can be


used for more invasive surgical procedures. USV meas-urement was revealed to be a useful tool to evaluate thepotential analgesic effect of drugs used in rats subjectedto anaesthesia for surgical procedures, and it couldbe used in other behavioural models used inneuropharmacology.

Acknowledgements

We thank Maria Martha Bernardi, Aline Magalhaes andYame Miniero for earlier discussions about the topic. We

thank the anonymous reviewers for their thoughtful com-ments that improved the manuscript. We would like tothank Carol Valenzuela for the English language review.



article.

Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship, and/or publication of thisarticle: this work was supported by the Coordenacao de

Aperfeicoamento de Pessoal de Nıvel Superior-Brazil(CAPES) Finance Code 001.

ORCID iD

Jilma Aleman-Laporte https://orcid.org/0000-0003-4466-512X

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vocalization by rodents does not correlate with behav-ioral measures of persistent pain. Eur J Pain 2005; 9:445–452.

Resume

Dans cette etude, l’effet de quatre protocoles anesthesiques dont l’0association de xylazine (X) et de ketamine(K) avec l’acepromazine (A) et les opiaces [methadone (Me), morphine (Mo) ou tramadol (T)] a ete evalue chezdes rats de laboratoire des deux sexes. La vocalisation ultrasonique (USV) a ete utilisee comme indicateur de ladouleur pendant la periode de retablissement. L’objectif etait d’evaluer les parametres physiologiques et l’effetanalgesique de chaque protocole pour determiner le protocole le plus sur qui reponde aux exigences d’uneanesthesie equilibree. Le protocole XKA s’est avere le meilleur pour les deux sexes, tandis que le SKMe etaitpreferable pour les femelles parce que ces associations permettaient d’atteindre un niveau d’anesthesie chir-urgicale chez les rats. Cependant, l’evaluation de la douleur au cours du test a la formaline a revele que les ratsanesthesies a l’XKA produisaient davantage d’USV, suggerant qu’il ne s’agissait pas d’un bon protocole pourcontroler la douleur postoperatoire immediate. Tous les protocoles ont induit une baisse de la temperaturecorporelle et du debit respiratoire et cardiaque et ont eu des effets importants, les rats urinant et gardant lesyeux ouverts. Seuls les rats anesthesies selon le protocole XKA n’a pas presente d’horripilation. Ces resultatsont demontre qu’une bonne surveillance et des soins de qualite au cours de l’anesthesie doivent etre prevuspour prevenir les complications qui compromettent la vie de l’animal et lui assurer un bon retablissement.L’inclusion de l’analgesie dans les protocoles d’anesthesie doit etre utilisee de facon routiniere en minimisantla douleur et en assurant donc des resultats plus fiables au cours des procedures experimentales.

Abstract

In dieser Studie wurde die Wirkung von vier Anasthesieprotokollen, die die Kombination von Xylazin (X) undKetamin (K) mit Acepromazin (A) und Opioiden (Methadon (Me), Morphin (Mo) oder Tramadol (T)) umfassten,bei Laborratten beiderlei Geschlechts untersucht. Ultraschallvokalisation (USV) wurde als Indikator furSchmerzen wahrend der Erholungsphase benutzt. Ziel war es, die physiologischen Parameter und die analge-tische Wirkung der einzelnen Protokolle zu bewerten, um zu ermitteln, welches Protokoll am sichersten istund die Anforderungen an eine ausgewogene Anasthesie erfullt. Die besseren Protokolle waren dasXKA-Protokoll fur beide Geschlechter und das XKMe-Protokoll fur weibliche Tiere, da die Kombinationen


chirurgisches Narkosestadium bei Ratten erreichen. Die Schmerzbeurteilung wahrend des Formalin-Testsergab jedoch, dass Ratten, die mit XKA betaubt wurden, mehr USV produzierten, was darauf hindeutet, dassdies nicht als gutes Protokoll zur Kontrolle unmittelbarer postoperativer Schmerzen zu werten ist. AlleProtokolle verursachten eine Absenkung der Korpertemperatur sowie der Atem- und Herzfrequenzen undhatten wichtige Auswirkungen, wie z. B. Miktion und Augen-Offen-Halten. Nur mit dem XKA-Protokollbetaubte Ratten zeigten keine Piloarrektion. Diese Ergebnisse belegen, dass eine gute Uberwachung undBetreuung wahrend der Narkose erfolgen muss, um das Leben des Tieres beeintrachtigende Komplikationenzu vermeiden und eine gute Erholung zu gewahrleisten. Analgesie muss in Anasthesieprotokollenroutinemaßig berucksichtigt werden, um eine minimale Schmerzbelastung und damit zuverlassigereErgebnisse in den experimentellen Verfahren zu gewahrleisten.

Resumen

En este estudio, el efecto de cuatro protocolos anestesicos que incluıan la combinacion de xilacina (X) yketamina (K) con acepromacina (A) y opioides [metadona (Me), morfina (Mo) o tramadol (T)] se evaluo en ratasde laboratorio de ambos sexos. La vocalizacion ultrasonica (USV) fue utilizada como indicador de dolordurante el periodo de recuperacion. El objetivo era evaluar los parametros fisiologicos y el efecto analgesicode cada protocolo para determinar que protocolo era el mas seguro y cumplıa con los requisitos de unaanestesia equilibrada. Los mejores protocolos fueron XKA para ambos sexos y XKMe para las hembrasporque las combinaciones conseguıan un plano quirurgico de anestesia en ratas. Sin embargo, la evaluacionde dolor durante la prueba de formol revelo que las ratas anestesiadas con XKA producıan mas numeros deUSV, lo que sugerıa que no es un buen protocolo para el control del dolor postoperativo inmediato. Todos losprotocolos producıan un descenso de la temperatura corporal y de la frecuencia cardıaca y respiratoria y,asimismo, tuvieron efectos importantes como miccion y un mantenimiento de los ojos abiertos. Solo las ratasanestesiadas con el protocolo XKA no presentaron piloereccion. Estos resultados demostraron que se debeincluir un buen control y cuidado durante la anestesia para evitar complicaciones que pongan en peligro lavida del animal y para garantizar una buena recuperacion. La inclusion de analgesia en los protocolos deanestesia debe usarse de forma rutinaria, garantizando una presencia de dolor mınima y, por tanto, unosresultados mas fiables en los procedimientos experimentales.


Original Article

Examining compliance with ethicalstandards for animal research:is there a need for refinement?A qualitative study from northern Europe

Aurora Brønstad1 and Peter Sandøe2

AbstractEthical guidelines for research on animals such as the 3Rs (Replacing, Reducing, Refining) and positive harm-benefit evaluations are anchored in EU Directive 2010/63. In this qualitative study we investigated how ethicalguidelines interact and/or compete with other considerations when animal research is planned. Four focusgroups consisting mainly of researchers involved in animal use were conducted in four Northern Europeancountries and findings were analysed thematically with the support of NVIVO. Practical issues and the import-ance of doing good science were dominant topics. Practical issues could not easily be separated from the goalof good science. Participants expressed concerns which accord with the core-values of the 3Rs, but in onegroup they explicitly referred to the 3Rs as a concept. Conflicts between reductions in animal numbers andthe risk of creating unreliable results were addressed. They also criticized the practice of using more animalsto improve statistical figures to get results published in highly ranked journals – a finding we believe is new.The main conclusion of this study is that ethical values could not easily be separated from the goal ofproducing good science. Whereas policy makers seem to expect researchers to explicitly take ethical con-siderations into account, we found that their ethical thinking is mainly manifested as an implicit part ofmethodology and design. We don’t see this as a problem as long as the underlying core values are implicitlyrespected, or promoted, in the relevant experimental practice.

Keywords3Rs, animal use, ethics, ethics and welfare

Date received: 19 July 2018; accepted: 10 March 2019

Introduction

Animal research raises ethical issues. In most societiesthere are conflicting views about under what conditionsit is ethically justifiable.1–7 In Europe, this kind ofresearch is regulated through a common directiveimplemented in different national legislations thatdefines minimum standards for animal use in research,teaching and testing and is designed to reflect andenforce widely accepted ethical standards.

Among the criteria defined in EU Directive 2010/638

are the principles that all experiments must implementthe 3Rs6 and be based on a positive harm-benefit ana-lysis.5 The 3Rs aim to reduce negative impacts on sen-tient animals either by Replacing animals, Reducing

their number, or by Refining procedures to minimizeor eliminate harm to them.6 Directive 2010/638 article 1explicitly employs the language of the 3Rs:

This Directive establishes measures for the protection

of animals used for scientific or educational purposes.

1Department of Clinical Medicine, University of Bergen, Norway2Department of Veterinary and Animal Sciences and Department ofFood and Resource Economics, University of Copenhagen,Denmark

Corresponding author:Aurora Brønstad, Dyreavdelingen, University of Bergen Vivarium,Haukeland sykehus Bergen, No-5021 Norway.Email: [email protected]

Laboratory Animals

2020, Vol. 54(2) 183–191




DOI: 10.1177/0023677219841080


http://orcid.org/0000-0002-5511-5425

http://orcid.org/0000-0003-0397-3273


https://doi.org/10.1177/0023677219841080


To that end, it lays down rules on the following: (a) the

replacement and reduction of the use of animals in pro-

cedures and the refinement of the breeding, accommo-

dation, care and use of animals in procedures.

The 3Rs – and especially refinement – have had a cen-tral place in the evaluation by the competent bodies(IACUC, Ethical committees).9,10 Although the direct-ive defines the role of advisory bodies on the 3Rs, theresearchers are mainly responsible for ensuring that the3Rs are properly applied. The 3Rs have been investi-gated in earlier studies. The findings were mixed,3,11–13

and differences between scientists’ views and policygoals were identified.14

Harm–benefit assessment is another import-ant,5,9,15–19 mandatory8 part of project evaluation.20–23

The harm–benefit analysis brings the particular projectinto a context where the harm to animals is justified byexpected benefits.5 Harm–benefit evaluations resonatewith the public1,2,24 as well as policymakers,8,25–27

although they can be difficult to compare and weighagainst each other.15,18

Research activities face time and resource pressures,and researchers must define priorities when they planexperiments. There is a risk that the aspiration to applyhigh ethical standards will compete with other interestsor needs. Guidelines requiring researchers to continueto adhere to ethical standards have been defined bothfor the planning phase28 and the reporting of animalresearch.29,30 Several bodies have defined codes of con-duct describing acceptable research practice.31–33

Compliance with these codes is crucial if scientific integ-rity and credibility is to be maintained.31

The aim of this qualitative study, based on inter-views with researchers in four Northern Europeancountries, was to deepen our understanding of theways in which, in practice, ethical guidance, and in par-ticular that given by the 3Rs, interacts and/or competeswith other considerations when research involvinganimal use is being planned.

Methods

Four focus group interviews were performed in fourcountries (the Netherlands, Denmark, Sweden andNorway) between 2011 and 2013. The intervieweeswere recruited through contacts who volunteered toorganize the events, and no incentives to participatewere offered. In total 21 interviewees participated,11 males and 10 females. The interviewees’ contribu-tions were anonymized. Their age was between 25 and56 years (average 35, median 34 years) with between 0and 30 years of experience in animal research (average8.4, median 5 years). Eight participants identified them-selves as PhD students, two as post-docs, five as

researchers and three as senior researchers. In addition,one identified him/herself as a research animal veterin-arian, one as a technician and one as a bioethical assist-ant. The contributions of the bioethical assistant didnot change the dynamics or the content of the discus-sion as compared with the other focus groups. Differentlevels and types of competence were represented in allfocus groups. At the start of the interviews the partici-pants introduced themselves, as they did not alwaysknow each other, and described their research projects.The interviews lasted for 60–90 minutes and wererecorded and transcribed verbatim. Two of them wereconducted in English and two in Scandinavianlanguages.

The participants were first asked what they thoughtwas important, second about their motivation, andfinally about difficult issues when planning animalresearch. The question about important factors wasasked because we assumed that the comments partici-pants spontaneously made in response to it would belikely to indicate the matters to which they gave mostattention in planning their research. For the questionabout motivation, it was assumed that these would alsoreceive much attention. The final question about diffi-culties was asked to explore if there were any ambiva-lences, or conflicts, in the planning of the animalexperiments. As a follow-up question, intervieweeswere asked how they solved their difficulties. Noattempt was made to lead the discussion towards eth-ical issues, but relevant issues came up spontaneously inall interviews, and all participants contributed by refer-ring to their own experiences or concerns. Additionalfollow-up questions were put to the informants toencourage them to further elaborate their contribu-tions. All participants contributed to the open discus-sions, bringing in new elements or commenting onother participants’ contributions. There was no needto interrupt or rein in dominant informants.

We assumed that ethical standards must be a part ofthe planning of an experiment if they are to be takeninto consideration in the performance of subsequentresearch. Further, we assumed that spontaneouslyreferring to ethical norms reflects moral sensitivityabout the use of animals in research34 which empha-sizes the researcher’s commitment to practices thatmaintain ethical standards.

The transcribed interviews were coded and themat-ically analysed using NVIVO11 software. A provisionallist of codes35 included ‘3R’, ‘replacement’, ‘reduction’and ‘refinement’. In applying these we were guided byinterpretations of the meaning of the comments, that iswe did not require explicit matches with the wordingof the comments. The codes were then sub-coded as‘3R explicit’ or ‘3R values’. In an inductive approach,additional codes were added during the analysis to


systematize issues raised during the discussion.35 Thesecondary codes also included ‘other ethical concerns’.This sub-code, which covered ethical topics that couldnot be directly related to the context of the 3Rs, wassubdivided into animal related or non-animal relatedissues. The secondary codes also included ‘operationalissues’ and ‘good science’, both of which turned out tobe major topics.

Participants signed informed consent forms beforethe interviews. Demographic data was recorded inother documents and stored in a different folder toassure anonymity. Studies such as the present onedoes not require ethical approval in Norway, but theymust be and were notified to the Norwegian SocialScience Data Service.

Results

Solutions to practical issues and the importance ofdoing good science were dominant topics in all focusgroups. The practical issues could not easily be sepa-rated from the goal of doing good science in the ana-lysis, because these themes are interrelated.

The practical issues the respondents consideredincluded the acquisition of ethical permissions, theavailability of resources, the choice of animal proced-ures, logistics and information or communication chal-lenges. The discussions of good science addressed theplanning and designing of experiments, the choice ofmodels, pilots, statistical considerations revolvingaround p values, and the reporting of animal experi-ments. There was agreement that proper design isnecessary to obtain valid, reproducible results, andthat animal use can be justified only if the design is inorder. This may, however, create a tension with thedemand for a reduction in the number of animals used.

Which means that if you are very concerned about the

ethics, or the large number of animals you are using,

trying to downsize it [animal number] in some way the

study is unethical, because you are decreasing your

chances to actually get the correct answer. (2M3 –

interview/sex/informant)

On the other hand, criticism was voiced about the ideathat more animals should be used simply to improvestatistical power – with the aim of getting results pub-lished in a more highly ranked journal.

but then there is more needed to get it published, to get

it published in a better journal. People they look at

your output, in what kind of journals you publish.

(1M1)

So it depends on impact factors, you will increase how

many animals you will need. (1M2)

You got a p value different or journal . . .The question

is, of course, is it ethical? (1F2)

Reducing animal numbers by excluding pilots or con-trols was also discussed. Pilots can provide importantinformation for further studies. Controls are importantelements of experimental design. Some respondentswondered whether historical controls should be usedmore often.

you can use historical data actually as a control. It

depends on what type of study you’re [doing]. (2M3)

Better sharing of organs and tissues from animals toreduce animal numbers, was also discussed. Respectfor the interest of healthy animals to stay alive wasconveyed in another interview, and frustration thatresearch regulations require healthy animals to bekilled was also expressed.

That is the basic rule here – all animals come in – they

don’t come out . . . so therefore we have to kill it. That is

stupid – a healthy animal! (1M2)

The dilemma between using fewer animals and refiningthe housing conditions was raised in one of the inter-views. The respondents appeared to think that thisdilemma has no clear solution, but they demonstratedethical awareness and the ability to identify and reflecton such dilemmas.

I think sometimes that there is too much focus on the

reduction bit and too little on the refinement . . .To me

it makes more sense to look at the refinement, to look

at how do we reduce the stress on the animals . . . (2F1)

Solving practical tasks was a recurrent topic in all of theinterviews. The collection of information enabling goodpractical solutions while taking animal-welfare concernsinto consideration, was an important focus. Althoughsuch strategies fall into the refinement category, theword ‘refinement’ was not often used. However, theword ‘stress’ was frequently used when negative impactson animals were being described. The importance oftreating animals well to avoid stress of the kind thatmay bias an experiment was frequently raised.

you ensure that the animals are not stressed, so you’re

getting something that is physiologically meaningful . . .

you want the animal to be happy, and to be as close to

its normal [way of life] as possible, to be sure that . . . I

mean, both to reduce the data variability, but also to, I

mean, to be sure that the data you are getting actually

reflects like a normal physiological regulation, and not

a regulation in response to stress . . . (2M2)

Brønstad and Sandøe 185

Accurate information about tolerance level for ananimal – for example by injection – makes a significantdifference to the welfare of that animal. Searches forinformation on how to improve or refine procedureswere important in the planning of experiments.

When we start with new tumors, we look up in the

literature – What can we inject in these mice, and

how should we inject it? (1F2)

Web-based literature searches were identified as usefulmethods for collecting information. However, talkingto experienced colleagues seemed to be a preferredstrategy for collecting information, especially when itcame to refinement.

There are a lot of things written down, but other things

not, and sometimes you go through the hard way,

asking people, asking people – and calling people.

And then you find out. (1M2)

I have also talked with people – I mean, a lot of people

already have experience, and with this type of model,

and what are the pitfalls. (1M3)

Talking with experts also served the purpose of build-ing good collaborative relationships – for example, withthe animal care staff.

My experience is that the more contact, the more they

look after the animals . . . (1M1)

Replacing animal research with experimentalapproaches using alternative methods is a goal ofpolicy makers.8 However, the respondents voiced con-cerns about the consequences for science if this goal isenforced when no full replacements are available. Onthe other hand, they expressed disapproval of the situ-ation where animals are still used, and where regula-tions may prohibit the use of available alternatives.

actually the in-vitro test is so much more sensitive than

the rabbit test, eh? So it’s quite astonishing that the

FDA has been so reluctant to accept that.

It’s the health authorities that actually have the last

word . . . (2F1)

When the necessity of animal models was discussed,respondents often focused on the idea that there wasno need to use animals. Already, in the planning of ananimal experiment and the choice of model, the ques-tion of alternatives was raised.

Does it [the animal model] tell you what you want to

know? Is it really needed? I mean some basic questions

can sometimes be done in vitro. (1M2)

Consideration of alternatives was not limited to theplanning phase. According to one participant, alterna-tives should be reconsidered as the research projectprogresses.

At a certain point you have to say we cannot see this in

an animal – maybe we should go back to the lab and

see if we can do some in vitro [investigations]. (1F3)

Although the informants raised concerns about alter-natives, animal numbers and strategies to refine andimprove procedures, only a few occasions they referredexplicitly to the 3Rs. When they did so they treated the3Rs as a tool for continuous improvement and changeof practices.

can we do it in a different way? And having that

policy put into the system, also with the 3Rs award

and so on, I think that’s a good instrument actually

to try to making it . . . eh . . .make it more natural, you

can say – to put your ideas actually into a format that is

also put into reality. (2M2)

Discussion

The aim of this study was to explore researchers’ atten-tion to ethical standards and concerns when they areplanning animal experiments, with a special focus onthe 3Rs.

During the analysis the practical issues anchored inethical values could not easily be separated from thegoal of producing good science because these two elem-ents are interdependent. This created some methodo-logical problems for the analysis. On the other hand,it demonstrates that the identification of practical solu-tions is a prerequisite of good science, and there is aroom for the application of ethical standards in achiev-ing this.

While the present study confirmed that scientists puta strong emphasis on scientific standards, it alsodemonstrated that researchers address values in accord-ance with ethical standards for research, including the3Rs. When the 3R issues were addressed, they wereusually connected with practical solutions. On only afew occasions, and in only one of the interviews, werethe 3Rs explicitly referred to in their own right. Therelevant EU directive, however, requires researchersto prepare an explicit statement explaining how com-pliance with the 3Rs has been ensured. It requires: Ademonstration of the compliance with the requirement ofreplacement, reduction and refinement (article 43 1.b).8

The spirit of the 3Rs is mentioned in several places inthe directive (Article 1.1 a, Article 4, Article 13.2 a-c,Article 27.1.b, ANNEX V.10, ANNEX VI.2).8


In ANNEX VI.2 the 3Rs are explicitly referred to:‘2. Application of methods to replace, reduce andrefine the use of animals in procedures’. ANNEX VI.3–5 states that the researcher must describe any meth-odological strategies that have been employed to avoidpain, suffering and to achieve humane endpoints – all ofthese are examples of refinement. Similarly, ANNEXVI.6 requires researchers to describe strategies theyhave used to minimize or reduce the number of animalsused. The ANNEX VI guidance may indeed encourageresearchers to perceive the instruction to demonstratecompliance with the 3Rs as a detached element ratherthan an integrated part of the methods they select inorder to maximize scientific benefits or reduce negativeimpact on animals.

A recent paper by Kirk37 stresses that Russell andBurch framed the 3Rs, not as an ethical tool, but as astrategy to maximize high-quality science. The 3Rs areonly one pathway among others to high-quality science,and this might explain the informants’ tendency tofocus on ‘good science’ without explicitly referring tothe 3Rs.

As the 3Rs were launched as a concept for humaneexperimental technique by Russell and Burch,6

Wurbel19 launched the 3Vs as a principle to maximizescientific validity. According to Wurbel and others, theassessment of scientific quality should be part of theethical evaluation of animal experiments.19,21,23,31,38,39

Some authors regard the scientific quality of experi-ments as a part of the harm domain.21,23,39 Otherstreat it as part of the benefit domain.40–42 By contrast,Bateson38 and Wurbel19 assess quality as a separatedomain.

In a previous study of researchers’ attitudes to the3Rs, only a minority were found to be aware of the 3Rsand able to properly name the principles.3 This accordswith findings of the present study, that is only in oneinterview were the 3Rs explicitly mentioned. However,it might be asked whether it is important to be able toname 3Rs correctly as long as the core value of limitingharm are implicitly respected, or promoted, in the rele-vant experimental practice, as they seem to be inthis study.

The same study3 showed that researchers favouredrefinement over reduction. The dilemma of reductionversus refinement was spontaneously raised in one ofthe focus groups. Here, the view that refinement wasmore important was again expressed. In our study,however, reduction issues were addressed more fre-quently than refinement issues. Concerns about usingtoo few animals to generate reliable data, were voicedand regarded as an unnecessary and unethical animaluse. The inclusion of a sufficient number of animals toassure statistical power was very important, and neces-sary to justify animal use, according to the researchers

we interviewed. This still accords with Russell andBurch’s definition of reduction:

Reduction in the number of animals used to obtain

information of a given amount and precision.6

The use of animals for a certain experimental purpose,or in a certain context, brings the benefit domain intoplay. Some benefits are more legitimate than others.Increasing animal numbers to improve the statisticalvalidity of a study is acceptable; using more animalsto strengthen a study statistically in order to get studiespublished in higher ranked journals as a personal bene-fit was regarded unacceptable – although, of course,there is a fine line between these two goals. To ourknowledge, the issue of increasing animal numbers toimprove statistical parameters in order to get resultspublished in higher ranked journals, or journals withhigher impact scores, has not been raised before. Such apractice has troubling implications for animals and canalso be criticized for bad data management and violat-ing principles of good science.31–33 Young scientistsespecially may be under pressure from senior colleaguesor journal reviewers asking for more experiments.

The preference for talking with experts ratherthan conducting a literature search is at odds with anearlier study from Canada, where web-based searcheswere the preferred method of collecting information.11

Information collection was one of the priorities men-tioned in connection with the planning of experiments,but the participants in this study preferred to talk tocolleagues rather than search databases. This accordswith van Luijk et al.,13 a study showing that 3Rs data-bases were rarely used and that other forms of know-ledge exchange were preferred.13

A web-based study by Fenwick et al.11 discoveredthat 3Rs assistance should be ‘constructive’ and ‘neu-tral’ and provided by an ‘expert’. The same study alsosuggested that such 3Rs assistance should not be man-datory but be offered when problems are identified.Results from our qualitative study indicated thatresearchers are very aware of their need to seekadvice, they seem to have a good relationship withtheir 3Rs advisors, and that advice was provided in acollegial, collaborative manner. Studies have shownthat the collaborative approach is more efficient,when it comes to compliance with the advice given,than a ‘paternal’ relationship where a superior instructon the right solution. Hence, the collaborativeapproach has been described as the best way of buildinga culture in which the 3Rs are governing values.36

Lack of reproducibility, and the validity of experi-ments more generally, is a major concern in preclinicalscience.43,44 A strong commitment to principles of goodscience, as addressed in this study, reflects a serious


emphasis on achieving this. However, the attentiongiven to principles of good science expressed in thisstudy seems to be at odds with the existence of a repro-ducibility crisis.43,44 One explanation may be that goodintentions in the planning phase are not necessarily fol-lowed up in practice. This may be because animalexperiments are complex, with many pitfalls, so thatgood intentions drown in practicalities.

Both the finding that researchers preferred to talkwith, and collect information from, colleagues and thepoint about the complexity and pitfalls of animal stu-dies suggest that it is necessary to establish strong, com-petent, updated support functions, as well as nurturinga collaborative culture around animal research activ-ities to better facilitate both good science and highstandards of animal welfare.

A qualitative method was chosen for this study,since the aim was to explore the role of ethical stand-ards in the planning of animal experiments, and toinvestigate how these standards are addressed and com-plied with – all with a focus on the 3Rs. A qualitativeapproach opens up a more nuanced picture than can beobtained by quantitative investigation11,35,45 or byusing questionnaires of the sort adopted in earlier stu-dies.3,11,46 A qualitative approach provides a betterunderstanding of the way in which concepts like thoseof replacement, reduction and refinement are perceived,interpreted, phrased and applied.45 It provides insightinto the researcher’s perspective that is useful in plan-ning as well as reporting and evaluating studies.Qualitative studies are not suitable for quantifying, ordiscovering the distribution of certain opinions andtheir results should not be generalized. The strengthof the focus group method is that participants in suchgroups share, compare and discuss different views, andgive feedback on each other’s opinions.45 There isalways a danger that some individuals will dominatethe discussion and obscure other group-members’ opin-ions, but this was not seen in the present study. Fournorthern European counties were chosen, as weassumed that they are rather similar in terms of theirlevels of legislation and more generally in the attentionthey give to animal welfare.

The data in this study were collected during theperiod of transition to full implementation of the newdirective. Although the 3Rs as such are referred to forthe first time in the 2010 directive, they are also present,albeit without being explicitly set out, in the previous1986 directive (Articles 7 2, 3 and 4). We suspect thatstronger explicit emphasis on the 3Rs in the 2010 direct-ive has affected the way researchers talk about changesand improvement in terms of the 3Rs, even if thechanges are part of a process that takes time. We there-fore believe that our findings still give valuable insightinto the thinking of researchers working with animals.

The results of this study show that researchers doindeed address values and work in accordance withthe 3Rs. However, terms such as ‘necessity’ or ‘stress’are rather used than the official terminology. This hasimplications for the way we should examine the appli-cation of the 3Rs. For example, instead of asking whatreplacement alternatives have been considered, it maybe more productive to ask a researcher: why are ani-mals necessary for this study? Similarly, instead ofasking what refinement strategies have been applied,we could ask: what decisions have been made whichreduce unnecessary stress in the animals? Talking thesame ‘language’ will probably nurture a more fruitfuland collaborative collegial relationship, and it mayalso stimulate reflection on current practices andchanges to these.

The respondents in this study were never asked dir-ectly what place ethical standards had in the planningof their experiments. Nevertheless, they spontaneouslybrought ethical issues into the discussion, and in doingthis they demonstrated an ethical awareness of theissues raised by the use of animals in research, andempathy for their animals.

Acknowledgements

We are grateful to Merel Ritskes-Hoitinga, Lars Friis

Mikkelsene and Anders Forslid for assistance in recruitingparticipants in this study; to the University of Bergen for‘Smaforsk’ funding and Charles River Laboratories fortravel grants; to Jesper Lassen and Thomas Bøker Lund for

advice on methodology and Paul Robinson for language pol-ishing; and to Joseph for being a patient listener, challengingour ideas and giving constructive feedback from a lay

perspective.



article.

Funding

The author(s) received no financial support for the research,authorship, and/or publication of this article.

ORCID iDs

Aurora Brønstad http://orcid.org/0000-0002-5511-5425Peter Sandøe http://orcid.org/0000-0003-0397-3273

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Resume

Les lignes directrices ethiques concernant la recherche menee sur les animaux, tels que les 3Rs et lesevaluations dommage-avantage, sont ancrees dans la Directive de l’UE 2010/63. Dans cette etude qualitativenous avons etudie comment les lignes directrices d’ethique interagissent avec et/ou concurrencent d’autreselements a prendre en compte lorsqu’une recherche animale est prevue. Quatre groupes de discussioncomposes principalement de chercheurs impliques dans l’utilisation des animaux ont ete organises dansquatre pays d’Europe du Nord et les conclusions ont ete analysees de facon thematique avec l’aide de NVIVO.Les questions pratiques et l’importance de pratiquer une bonne science etaient des sujets dominants. Lesquestions pratiques ne pouvaient pas etre facilement separees de l’objectif d’une bonne science. Les par-ticipants ont exprime des preoccupations en accord avec les valeurs fondamentales des 3R, mais dans ungroupe, ils ont expressement fait reference aux 3R en tant que concept. Les conflits entre la reduction dunombre d’animaux et le risque de creer des resultats peu fiables a ete aborde. Les participants ont egalementcritique la pratique consistant a utiliser davantage d’animaux pour ameliorer les donnees statistiques etobtenir la publication des resultats dans des revues tres cotees - un fait qui nous semble nouveau. Laprincipale conclusion de cette etude est que les valeurs ethiques ne pouvaient pas etre facilement separeesde l’objectif de produire une bonne science. Alors que les responsables politiques semblent s’attendre a ceque les chercheurs tiennent explicitement compte des considerations ethiques, nous avons constate que leurreflexion ethique se manifestait principalement de maniere implicite dans le cadre de la methodologie et de laconception. Nous ne voyons pas cela comme une probleme tant que les valeurs fondamentales sous-jacentessont implicitement respectees, ou promues, dans la pratique experimentale concernee.

Abstract

Ethische Richtlinien fur die Tierforschung wie das 3R-Prinzip und positive Schaden-Nutzen-Bewertungen sindin der EU-Richtlinie 2010/63 verankert. In der vorliegenden qualitativen Studie haben wir untersucht, wieethische Richtlinien bei der Planung von Tierversuchen mit anderen Uberlegungen zusammenwirken und/oder konkurrieren. Die Untersuchungen wurden mit vier Fokusgruppen, die hauptsachlich aus mit derTierverwendung befassten Forschern bestanden, in vier nordeuropaischen Landern durchgefuhrt und dieErgebnisse mit Unterstutzung von NVIVO thematisch analysiert. Praktische Fragen und die Bedeutungguter Wissenschaft waren dominierende Themen. Dabei ließen sich praktische Fragen nicht leicht vom Zieleiner guter Wissenschaft trennen. Die Teilnehmer außerten Bedenken im Einklang mit den Kernwerten der3R, wobei die 3R jedoch von einer Gruppe ausdrucklich als Konzept bezeichnet wurden. Konflikte zwischen


http://www.oecd.org/science/inno/40188303.pdf

http://www.oecd.org/science/inno/40188303.pdf

einer Verringerung der Tierzahlen und dem Risiko, unzuverlassige Ergebnisse zu erzielen, wurden ange-sprochen. Man kritisierte auch die Praxis des Einsatzes von mehr Tieren, um statistische Zahlen zu verbes-sern, damit Ergebnisse in hoch angesehenen Zeitschriften veroffentlicht werden – eine Erkenntnis, die wir furneu halten. Die wichtigste Schlussfolgerung dieser Studie besteht darin, dass ethische Werte schwer vom Zielder Realisierung guter Wissenschaft zu trennen sind. Wahrend politische Entscheidungstrager offenbarerwarten, dass Forscher ethische Uberlegungen explizit berucksichtigen, haben wir festgestellt, dass sichihr ethisches Denken hauptsachlich als impliziter Teil von Methodologie und Design manifestiert. Wir sehendies nicht als Problem, solange die zugrunde liegenden Kernwerte in der jeweiligen experimentellen Praxisimplizit respektiert bzw. gefordert werden.

Resumen

Las directrices eticas para la investigacion animal como las 3R y las evaluaciones positivas dano/beneficioestan ancladas en la Directiva de la UE 210/63. En este estudio cualitativo, investigamos como las directriceseticas interaccionan y/o compiten con otras consideraciones al planificarse una investigacion animal. Sellevaron a cabo cuatro grupos de debate compuestos principalmente de investigadores involucrados en eluso de animales en cuatro paıses de Europa del Norte y las conclusiones se analizaron tematicamente con elsoporte de NVIVO. Los temas practicos y la importancia de hacer una ciencia buena fueron los temasdominantes. Los temas practicos no podıan separarse facilmente del objetivo de conseguir una cienciabuena. Los participantes expresaron su preocupacion que concuerdan con los valores principales de las3R, pero en un grupo se refirieron explıcitamente a las 3R como un concepto. Se trataron los conflictosentre la reduccion de los numeros de animales y el riesgo de crear resultados poco fiables. Tambiencriticaron la practica de utilizar mas animales para mejorar las cifras estadısticas a fin de conseguir quesus resultados sean publicados en revistas de prestigio, un hecho que pensamos que es nuevo. La principalconclusion de este estudio es que los valores eticos no podrıan separarse facilmente del objetivo de producirbuena ciencia. Mientras los creadores de polıticas parecen esperar que los investigadores tenganexplıcitamente en cuenta consideraciones eticas, encontramos que su pensamiento etico se manifiesta prin-cipalmente como una parte implıcita de una metodologıa y diseno. No pensamos que esto suponga unproblema siempre que los valores principales subyacentes se respeten implıcitamente, o se fomenten, enla practica experimental relevante.


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News

3Rs – Reduce Reuse Recycle

Jean-Philippe Mocho

It is often shocking to see how much plastic is sent forrecycling after a single experiment. Imagine the scale ofit for your whole establishment, a country or theLaboratory Animal Science (LAS) community. Someuniversities are now taking action to significantlyreduce single-use plastics. Labs across the globe rou-tinely set their �80�C freezer to warmer temperatures(�60�C to �70�C) and save over one-third of energyconsumption. Anaesthetists have stopped using gas likedesflurane or nitrous oxide due to their very high green-house effect.

Green initiatives are flourishing in the LAS commu-nity. Establishments have joined accreditation schemes

or set up policies to reduce environmental impact.Individuals (researchers, technicians, vets) have devel-oped their own tricks. Strategic discussions now involveall stakeholders with sustainability in mind, for exam-ple, when designing a new facility and deciding on ven-tilation or disinfection and sterilisation of a barrier.

We would like to hear about all these initiatives: onthe bench in the labs, in the animal facility, or even inthe staff room! FELASA is collecting tips, good ideas,and references to peer-reviewed articles or useful newsitems that could help the LAS community in its effortto reduce environmental impact. All relevant receivedinformation will be shared on the FELASA websitehttp://www.felasa.eu/, anonymously, for all to accesseasily. Please send your input to me (details below)and to Penny, the FELASA secretariat, at [email protected].

Another challenge for you: are you ready to cycle tothe FELASA 2022 congress in Marseille http://www.felasa2022.eu/?

FELASA Honorary Secretary, FELASA, Eye, UK

Corresponding author:J-P Mocho, FELASA, PO Box 372, Eye, IP22 9BR, UK.Email: [email protected]

Contributions to the News section are not subject to peer reviewand reflect the opinion of your subscribed society.

Laboratory Animals

2020, Vol. 54(2) 194




DOI: 10.1177/0023677220909272


http://www.felasa.eu/

http://www.felasa2022.eu/?

http://www.felasa2022.eu/?


https://doi.org/10.1177/0023677220909272


News

El XV Congreso de la SECAL (SociedadEspanola para las Ciencias del Animal deLaboratorio) tuvo lugar en Sevilla del6 al 8 de noviembre

Elena Hevia1 and C. Oscar Pintado2

El evento fue todo un exito con mas de 300 participantesvenidos de todo el paıs y de algunos paıses latinoamer-icanos y europeos. 27 ponentes de reconocido prestigiode varias nacionalidades participaron en las 9 sesionescientıficas que cubrıan un amplio rango de aspectos rela-cionados con el animal de laboratorio. (Las sesiones quese impartieron en ingles contaron con traduccion simul-tanea gracias a la ayuda concedida por LAL).

Las sesiones del congreso versaron sobre:

Nuevas tecnologıas y su impacto en los animales de

laboratorio

Motivacion del personal y resolucion de conflictos

Gestion de centros de produccion animal

Avances en las 3Rs

Desafıos de la investigacion en ambientes no

convencionales.

Bases eticas de la proteccion y experimentacion animal

Reproducibilidad y traslacion

Transparencia en la investigacion animal

Cuidados veterinarios en procedimientos severos.

Durante las sesiones el nivel de participacion activa fueextraordinario lo que determino que el congreso fueraespecialmente enriquecedor.

Para la mesa redonda, cinco representantes de lasautoridades competentes discutieron con los partici-pantes la aplicacion de la legislacion en animales deexperimentacion y en la formacion del personal quetrabaja con animales. Se presentaron mas 100 postersde alta calidad lo que hizo difıcil la seleccion por partedel Comite Cientıfico de los dos trabajos ganadores. Elprograma cientıfico se completo con 4 talleres que sedesarrollaron el dıa 5 de noviembre de los que se tuvounos esplendidos comentarios.

La exposicion comercial tambien tuvo gran exito ypermitio a 24 exhibidores con mas de 30 stands mostrara todos los participantes sus ultimas innovaciones en susproductos o servicios. Ademas tuvo lugar por primeravez en la SECAL una sesion comercial para la presenta-cion corta de las empresas presentes en el Congreso.

Sevilla fue un marco incomparable para este encuen-tro. El tiempo agradable invitaba a pasear por sus callestras las sesiones o a ir a pasear, correr o montar en bici-cleta en los alrededores del rıo. El programa social incluyouna visita guiada por la ciudad ası como una cena de galadonde todos pudimos disfrutar de las vistas, la comida ydel baile que tuvo lugar al terminar la misma.

En encuentro termino con la presentacion de la sededel proximo congreso que nos reunira de nuevo a todosen la ciudad de Lleida en 2021.

XV Congress of SECAL (Spanish Society forLaboratory Animal Science) took place inthe beautiful city of Seville on 6–8November

The event was a great success, with more than 300 par-ticipants from all over the country, as well as fromLatin American and European countries. Twenty-seven renowned speakers from various nationalitiesparticipated in the nine scientific sessions that covereda wide range of laboratory animal science topics.(Sessions were delivered in English, with simultaneoustranslation available thanks to a LAL grant).

The topics included were:

. New technologies related to laboratory animals

. Staff motivation and conflict resolution

1Centro de Biologıa Molecular Severo Ochoa, Madrid, Spain2Animal Facility and Transgenic Unit. CITIUS III, University ofSeville, Seville, Spain

Corresponding author:Elena Hevia, Centro de Biologıa Molecular Severo Ochoa,c/ Nicolas Cabrera, 1 Campus de la Universidad AutonomaCantoblanco, Madrid 28049, Spain.Email: [email protected]

Contributions to the News section are not subject to peer reviewand reflect the opinion of your subscribed society.

Laboratory Animals

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DOI: 10.1177/0023677220906766



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. Efficient management of laboratory animal facilities

. Advances in the 3Rs

. Challenges in non-conventional researchenvironments

. Philosophical bases of animal protection andresearch

. Reproducibility and translation of experiments

. Transparency of animal research

. Veterinary care in severe experiments

During the sessions, the level of attendance andactive participation was extraordinary, making themeeting especially enriching.

For the round table, five representatives of theCompetent Authorities discussed with the attendeesthe application of legislation in animal experimentationand in the training of personnel working with animals.More than 100 high-quality posters were presented,making the selection of the two winning works hardfor the Scientific Committee. Four practical workshops,

which took place on 5 November, completed the scien-tific programme from which splendid feedback wasreceived.

The commercial exhibition was also very popularand allowed 24 exhibitors with more than 30 standsto show participants the latest innovations in theirproducts or services. Furthermore, a commercial ses-sion took place for the first time in a SECALConference, allowing for flash presentations of thecompanies that attended the meeting.

Seville was an incomparable setting for this meeting.The lovely weather invited us to walk through thestreets at the end of the sessions or to go jogging, walk-ing or cycling on the riverbanks. The social programmeincluded a night tour and a gala dinner in which we allenjoyed the views, the food and the dancing that tookplace next.

The meeting ended with the presentation of the nextCongress that will bring us all together again in the cityof Lleida in 2021.

Hevia and Pintado 197

Thanks to Reviewers

Thanks to Reviewers

The Editor-in-Chief, Editorial Board and Publisher would like to thank the following reviewers for theircontribution to Laboratory Animals in 2019.

Abelson, KlasAddo, PhyllisAdedeji, TemitopeAdjei, SamuelAfify, MamdouhAfonson, RicardoAldrich, GregAllen, KyleAllen, SueAnderson, DavidAntunes, LuisAppelgren, Lars-ErikArras, MargareteBaca, JustinBackhans, annetteBainbridge, DavidBarnard, alunBeckmann, NicolauBeekhuijzen, MarionBehr, RudigerBenavides, FernandoBenga, LaurentiuBergadano, AlessandraBernard, ReneBertrand, HenriBessems, JosBindelle, JeromeBleich, AndreBleilevens, ChristianBourges-Abella, NathalieBradbrook, CarlBrossard, LudovicBrown, MarkBuckmaster, CindyBuettner, ManuelaBusardo, FBussell, JamesChen, Chih-ChengChen, JichunCho, Jong KiChourbaji, SabineClutton, Eddie

Czaplik, MichaelDa Cunha, DaiseDahlborn, KristinaDavies, BenDecrock, FredericDennis, MikeDeziel, RobertDhondt, KevinDiederich, KaiDirnagl, UlrichDoe, BrendanDontas, IsmeneDorsch, MartinaDoube, MichaelDrynan, LesleyEkstrand, CarlEssex-Lopresti, AngelaEstanislau, CelioExner, ConnyFigueiredo, IsabelFindlay, amyFinnemore, PaulFlecknell, PaulFonio, EhudFrance, MalcolmFray, MartinFruijtier-Polloth, ClaudiaGalatos, ApostolosGardiner, MarkGarrels, WiebkeGaskill, BriannaGetchell, RodmanGiamberardino, MariaGiavedoni, LuisGilbert, ColinGjendal, KarenGlowka, TimGosselin, Romain-DanielGregori, MichelaGriffin, GillyGrujic-Milanovic, JelicaHabedank, Anne

Hansen, AxelHansen, KristineHatch, GrahamHawkins, PennyHecht, GilHedrich, HansHendriksen, CoenraadHermann, BrianHiebel, BernhardHilken, GeroHinkel, RabeaHolterman, ChetHood, derekHorst, KlemensHultgren, JanIdahor, KingsleyIllgen-Wilcke, BrunhildeJarvis, GavinJaubert, JeanJirkof, PaulinJolivet, GenevieveKalliokoski, OttoKane, AliceKatsimpoulas, MichalisKimmina, SarahKing, Karenklarenbeek, SjoerdKlinge, UweKnotek, ZdenekKolbe, ThomasKramer, StephanieKuiken, ThijsLangermans, JanLarsson, AndersLeenaars, CathalijnLi, zhenLines, KateLofgren, JennieLutz, ThomasMagara, FulvioMahabir, EstherManell, Elin

Laboratory Animals

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DOI: 10.1177/0023677220903579



https://doi.org/10.1177/0023677220903579


Marinelli, SaraMartinez-Salgado, CarlosMasano, YuukiMason, GeorgiaMcAllister, StacyMcCLure, FionaMerkus, DaphneMiller, AmyMogil, JeffreyMoreno-del Val, GonzaloMorgan, MichaelMorton, DavidMuro, Andres FernandoNeumann, DetlefNeunaber, ClaudiaNiimi, ManabuOlsen, LenaPacharinsak, CholawatParker-Thornburg, JanParks, SimonPekow, CynthiaPierzynowska, KaterynaPintado, BelenPloegaert, ToscaPloj, KarolinaPrins, Jan-BasPritchett-Corning, KathleenPryce, ChristopherRaschzok, Nathanael

Reichel, RudolfReinhardt, DieterRichardson, ClaireRiebel, KatharinaRiederer, BeatRobb, DavidRocio, RocioRodrigues dos Santos, NunoRulicke, ThomasRyden, AnneliSaeb-Parsy, KouroshSahine, ErhanSalvatori, DanielaSanchez-Morgado, JoseSanders, JustinSchenkel, JohannesSchiering, InaSchofield, PaulSchwiening, ChristofSena, Emily SSheeley, HeatherSlattery, DavidSmith Richards, BrendaSmith, AdrianSmith, JenniferSorzano, Carlos OscarSpergser, JoachimSteinborn, RalfStrecker, Jan

Swindle, MichaelSztein, JorgeTalbot, StevenTarigan, BernadettaThone-Reineke, ChristaVergara, PatriVerreck, FViebahn, ChristophVisser, JennyVoelkl, BernhardVoikar, VooteleVollert, JanWagner, HeikeWallgren, PerWalski, TomaszWang, DongfangWarshaw Funk, AmyWedekind, DirkWells, SaraWindschnurer, InesWolterbeek, AndreWyatt, JeffYoshiyuki, RiekoYoung, SimonZacharioudaki, ArgyroZechner, DietmarZintzsch, Anne

Thanks to Reviewers 199

Calendar of events

In light of concerns over COVID-19, some of the events below may be cancelled or postponed. Please checkindividual websites for further information.

2020

28 May 3R Symposium-Alternatives to CO2, Bern, Switzerland. For further information visithttps://www.nc3rs.org.uk/events/3r-symposium-%E2%80%93-alternatives-co2

22–26 June FELASA Laboratory Animal Science Course on Primates, Gottingen, Germany. For

further information visit https://www.nc3rs.org.uk/sites/default/files/documents/

LASCourse_Registration_Juni2020_form.pdf

2–3 July 2nd Lung In Vitro event (LIVe2020), Nice, France. For further information visit https://

www.epithelix.com/support/LIVe2020

5 July The science of animal sentience: refining experimental biology, Prague, Czech Republic.

For further information contact [email protected]–27 August 11th World Congress on Alternatives and Animal Use in the Life Sciences, Maastricht,

The Netherlands. For further information see http://wc11maastricht.org/

6–9 September Eurotox 2020, Copenhagan, Denmark. For further information visit http://www.

eurotox-congress.com/2020/

16–18 September ANZLAA, Brisbane, Australia. For further information see http://www.anzlaa.org/

16–18 September GV-SOLAS, Worzburg, Germany. For further information see http://www.gv-solas.

de/

25–29 October AAALAS National meeting, Charlotte, NC. Further information to follow.1–3 December AFSTAL Annual Conference, Marseille, France. For further information visit https://

www.colloque-afstal.com/2020/

2021

13–15 April ScandLAS 50th Symposium, Tallin, Estonia. For further information visit https://www.

scandlas2020.ee/

16–19 March IAT Congress, UK. For further information visit www.iat.org.uk

Index to Advertisers APRIL 2020

AALAS 130Altromin International OBCAnLab Ltd 195AVID plc 129

Charles River Laboratories IFC

Datesand Ltd 128

Fine Science Tools GmbH 124

Granovit AG / Kliba Nafag 123GVG Diagnostics GmbH 134

Laboratory Animals Ltd (LAL) 192, 195LBS 131

North Kent Plastics 132

Special Diets Services 193ssniff Spezialdiaten GmbH 133

Tecniplast SpA IBC

ZOONLAB 127

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https://www.nc3rs.org.uk/events/3r-symposium-%E2%80%93-alternatives-co2

https://www.nc3rs.org.uk/sites/default/files/documents/LASCourse_Registration_Juni2020_form.pdf

https://www.nc3rs.org.uk/sites/default/files/documents/LASCourse_Registration_Juni2020_form.pdf

https://www.epithelix.com/support/LIVe2020

https://www.epithelix.com/support/LIVe2020

research.animals.org.uk

http://wc11maastricht.org/

http://www.eurotox-congress.com/2020/

http://www.eurotox-congress.com/2020/

http://www.anzlaa.org/

http://www.gv-solas.de/

http://www.gv-solas.de/

https://www.colloque-afstal.com/2020/

https://www.colloque-afstal.com/2020/

https://www.scandlas2020.ee/

https://www.scandlas2020.ee/

International

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yaltrominSpezialfutter GmbH & Co. KGIm Seelenkamp 20D- 32791 Lagewww.altromin.com

Your global operating expertsin lab animal diets.

Documents

Volume 54 Number 2 April 2020 ISSN 0023-6772 Laboratory ......J-P Mocho El XV Congreso de la SECAL (Sociedad Espan˜ola para las Ciencias del Animal de Laboratorio) tuvo lugar en Sevilla