ATW August 2024 : simplebooklet.com

71SPECIAL INTEREST ARTICLEBeneﬁ cial microbes and their combinations improved the growth and health performanceof female Holstein calves (Bos taurus) Randy Bergola Gaquit, Rudy Castro Flores, Pedro Victory Duque and Jonathan Manzano Salas ABSTRACT TRANSLATIONSTECH-2-TECH Ultrasound to ultrasee – the use of ultrasound to determine pregnancy in mice Caroline Karam and Kirsty KempPOSTERSHands-on reﬁ nements: establishing improved husbandry processes for aged C57BL/6 mice Aimee PatersonNon-invasive sampling methods for genotyping: improvements towards the 4r principle Sivatharsini Thasian-Sivarajah, Rebecca Mohr, Maria Walter, John Gbadegoye and Miriam HopfeIntravesical dosing in the female Sprague Dawley rat Sarah Graham Chenery and Zac TurnerPublic Engagement and Openness in animal research at The Crick Jamie Redden, Ewelina Kozlowska, Clare Brazill-Adams, Caroline Zverev, and Sarah Hart-JohnsonVol 23 No 2 August 2024Editorial Diane Hazlehurst, Chair of the Editorial BoardReport of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting Marine Barnabé, Ken Applebee, Jane Hurst, Vikki Neville, Tom Childs, Miguel Maravall, Julia Bartlett, Jane Tyson, Tina O’Mahony, Haley Daniels and Kerith LuchinsThe 3Hs Initiative – housing, handling, habituation Julia Bartlett, Justyna Hinchcliffe, Megan Jackson and Emma RobinsonEditorialJas Barley, Chair of the Editorial BoardReport of the 2019 RSPCA/UFAW RodentWelfare Group meetingChloe Stevens, Emily Finnegan, Jasmine Clarkson,Charlotte Burns, Sonia Bains, Colin Gilbert,Caroline Chadwick, Samantha Izzard, Charlotte Inman,Penny Hawkins (Secretary) and Huw GolledgeReduction of the negative effects ofmethionine on bone parameters in broilers’embryos by intra-egg injection of Vitamin B12Mohammad Naser Nazem, Shima Tasharofi,Negin Amiri and Sepideh SabzekarThe care of the Childr en’s Python(Antaresia children)Alexander Hosking and Gary MartinicFeline-assisted therapy: a promising part of animal assisted therapy (AAT)Eliska Mičková and Krityna MachovaThe care of Central and Pygmy Bearded DragonsAlexander Hosking and Gary MartinicPAPER SUMMARY TRANSLATIONSFrench, German, Italian, SpanishLOOKING BACKPhysical hazards in the laboratory animal houseR.T. CharlesThe incidence of a pathogenic strain of pseudomonas in a rabbit colonyG.R. Alpen and K. MaerzTECH-2-TECHDevelopment of a sifting cage change method for rats to improve welfareSeonagh HendersonVol 1 9 No 2 A ugust 2020CONTENTSiAugust20:Animal Technology and Welfare 4/8/20 10:48 Page i101817712191109129133137139SCHOLARSHIP AWARDLAS TECHNICIANThe LAS Technician ScholarshipAward has been developed toshow recognition to oneLaboratory Animal ScienceTechnician who has shownconstant dedication to improvingboth animal and personnelwelfare through championingbest practise procedures andinspiring loyalty,trust and teamwork.A culture of care & dedicationDEADLINE FOR APPLICATIONS: 15 JANUARY 2025www.tecniplast.it/ukscholarship@tecniplastuk.com | 0345 050 4556ANZLAA 2025TASMANIA, AUSTRALIATECNIPLASTVARESE, ITALY

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vOFFICERSPresidentDr Robin Lovell-Badge CBE FRSImmediate Past PresidentProfessor Sir Richard Gardner MA PhD FRSBFIAT (Hon) FRSVice-PresidentsSenga Allan MIAT RAnTech, David Anderson MRCVS,Stephen Barnett BA MSc FIAT (Hon) CBiol FRSBRAnTech, Miles Carroll PhD, Paul Flecknell MA Vet MBPhD DLAS DipLECVA MRCVS FIAT (Hon), PennyHawkins PhD BSc, Wendy Jarrett MA, Judy MacArthur-Clark CBE BVMS DLAS FRSB DVMS (h.c.) DipECLAMFRAgS DipACLAM MRCVS, Fiona McEwen BSc BVM&SMSc MRCVS, Tim Morris BVetMed PhD DipACLAMDipECLAM CBiol FRSB CertLAS MRCVS, Clive PageOBE PhD BSc, Jan-Bas Prins PhD MSc, Vicky RobinsonCBE BSc PhD, Paul Sanders MIAT RAnTech, DavidSpillane FIAT, Gail Thompson RLATG, RobertWeichbrod PhD RLATGLife MembersKen Applebee OBE FIAT CBiol FRSB RAnTech,Charlie Chambers MIAT RAnTech, Roger Francis MScFIAT RAnTech, Pete Gerson MSc FIAT RAnTech,Cathy Godfrey FIAT RAnTech, John Gregor y BSc (Hons)FIAT CBiol FRSB RAnTech, Patrick Hayes FIAT DipBARAnTech, Robert Kemp FIAT (Hon) RAnTech,Phil Ruddock MIAT RAnTech, Ted Wills FIAT (Hon)RAnTechHonorary MembersMark Gardiner MIAT RAnTech, Sarah Lane MSc FIAT,Sue McHugh BSc FIAT, Norman Mortell BA (Hons)MIAT RAnTech, Wendy Steel BSc (Hons) FIATMembers of CouncilMatthew Bilton, Kally Booth, Steven Cubitt,Simon Cumming, Haley Daniels, Glyn Fisher,Nicky Gent, Alan Graham, Linda Horan, Sam Jameson,Elaine Kirkum, Adele Kitching, Theresa Langford,Sylvie Mehigan, Steve Owen, Alan Palmer, AllanThornhill, John Waters, Lynda Westall, Carole Wilson,Adrian WoodhouseCouncil OfficersChair: Linda Horan BSc (Hons) MIAT RAnTechVice Chair: Glyn Fisher FIAT RAnTechHonorary Secretary:Simon Cumming BSc FIAT RAnTechHonorary Treasurer: Glyn Fisher FIAT RAnTechChair of Board of Educational Policy:Steven Cubitt MSc FIAT RAnTechChair Registration & Accreditation Board:Glyn Fisher FIAT RAnTechATW Editor: Jas Barley MSc FIAT RAnTechBulletin Editor: Carole Wilson BSc MIATATW/Bulletin Editorial Board:IAT REPRESENTATIVESAugust20:Animal Technology and Welfare 4/2/21 13:19 Page vvOFFICERSPresidentDr Robin Lovell-Badge CBE FRSImmediate Past PresidentProfessor Sir Richar d Gardner MA PhD FRSBFIAT (Hon) FRSVice-PresidentsSenga Allan MIAT RAnTech, David Anderson MRCVS,Stephen Barnett BA MSc FIAT (Hon) CBiol FRSBRAnTech, Miles Carroll PhD, Paul Flecknell MA Vet MBPhD DLAS DipLECVA MRCVS FIAT (Hon), PennyHawkins PhD BSc, Wendy Jarrett MA, Judy MacArthur-Clark CBE BVMS DLAS FRSB DVMS (h.c.) DipECLAMFRAgS DipACLAM MRCVS, Fiona McEwen BSc BVM&SMSc MRCVS, Tim Morris BVetMed PhD DipACLAMDipECLAM CBiol FRSB CertLAS MRCVS, Clive PageOBE PhD BSc, Jan-Bas Prins PhD MSc, Vicky RobinsonCBE BSc PhD, Paul Sanders MIAT RAnTech, DavidSpillane FIAT, Gail Thompson RLATG, RobertWeichbrod PhD RLATGLife MembersKen Applebee OBE FIAT CBiol FRSB RAnTech,Charlie Chambers MIAT RAnTech, Roger Francis MScFIAT RAnTech, Pete Gerson MSc FIAT RAnTech,Cathy Godfrey FIAT RAnTech, John Gregor y BSc (Hons)FIAT CBiol FRSB RAnTech, Patrick Hayes FIAT DipBARAnTech, Robert Kemp FIAT (Hon) RAnTech,Phil Ruddock MIAT RAnTech, Ted Wills FIAT (Hon)RAnTechHonorary MembersMark Gardiner MIAT RAnTech, Sarah Lane MSc FIAT,Sue McHugh BSc FIAT, Norman Mortell BA (Hons)MIAT RAnTech, Wendy Steel BSc (Hons) FIATMembers of CouncilMatthew Bilton, Kally Booth, Steven Cubitt,Simon Cumming, Haley Daniels, Glyn Fisher,Nicky Gent, Alan Graham, Linda Horan, Sam Jameson,Elaine Kirkum, Adele Kitching, Theresa Langford,Sylvie Mehigan, Steve Owen, Alan Palmer, AllanThornhill, John Waters, Lynda Westall, Carole Wilson,Adrian WoodhouseCouncil OfficersChair: Linda Horan BSc (Hons) MIAT RAnTechVice Chair: Glyn Fisher FIAT RAnTechHonorary Secretary:Simon Cumming BSc FIAT RAnTechHonorary Treasurer: Glyn Fisher FIAT RAnTechChair of Board of Educational Policy:Steven Cubitt MSc FIAT RAnTechChair Registration & Accreditation Board:Glyn Fisher FIAT RAnTechATW Editor: Jas Barley MSc FIAT RAnTechBulletin Editor: Carole Wilson BSc MIATATW/Bulletin Editorial Board:IAT REPRESENTATIVESAugust20:Animal Technology and Welfare 4/2/21 13:19 Page vRat tickling in gestation females Alice Lomax, Daren Lurkins and Helen HornseyReﬁned method of Tropicamide application in minipigs Keal GraceyRehoming of laboratory animals Neil OdeySustainability and animal research – a collaborative effort Joshua KerrThe purpose and use of singly housed animals in scientiﬁc research Mark Harrington and George ZimmermanPOSTER PRESENTATIONSAssessing pain in models of Rheumatoid ArthritisSamuel Singleton, Meriam Nefla, Ngaire Dennison, Simon Arthur and Tim HalesRefinements to health monitoringHannah Jones and Rebecca KingBiosecurity risks and the pre-implantation embryo; lessons from the mouseJean Cozzi, Mendy Verrier and Jimmy MancipEnvironmental enrichment for a small colony of ratsNick Blackburn, Gemma Cronshaw and Mike MitchellOestr us checking – increasing productivity and embracing the 3RsSamantha Hoskins and Jack BrownUsing habituation to reduce str ess for rats being transported short distancesSarah TaylorShining a light on rearing pigmentless ZebrafishJacqueline Glover, Thom Berriman, Dimitra Mantzorou, William Havelange,Sam Berry and Bruno Correia da SilvaThe jacket with pulling power – a novel approach to early stage evaluationof magnetic nanoparticlesAlison Ritchie, James Dixon, Phil Clarke and Anna GrabowskaiiCONTENTSIndex to AdvertisersABPI ..................................................................x,xi LBS ..................................................................iiAS-ET ...............................................................OBC Somni Scientific ................................................ivDatesand Ltd......................................................IFC Special Diets Services .....................................viiiInstitute of Animal Technology ...............................vii Tecniplast UK Ltd .............................................xiiIPS Product Supplies Ltd.....................................IBCAugust20:Animal Technology and Welfare 12/8/20 07:54 Page iiATW PROFILEATW aims to be the medium for Animal Technologists and all those concerned with the care and welfare of animals used for research purposes to communicate ‘good practice’.ATW especially aims to promote and develop the 3Rs particularly in respect of Reﬁnement. More importantly, ATW promotes the generally accepted 4th R, Responsibility. The responsibility that all Animal Technologists have in ensuring dissemination of ‘good practice’ to every institution using animals in research.ATW enjoys a unique position as the scientiﬁc publication for the leading organisation for the welfare of animals used in research.ATW seeks to publish peer-reviewed articles, technical notes and reviews allied to animal science and technology, management and education. Particular encouragement is given to authors submitting papers leading to improvements in environmental enrichment and the care and welfare of genetically altered animals.A commemorative plaque, a cheque for £250 and free registration at the IAT Congress awaits the winner of the Marjorie (Sandiford) Whittingham Memorial Prize awarded annually to the best original article.Editorial– Peer-reviewed papers– Translations of paper summaries into 4 European languages– Tech-2-Tech informal short articles on l new or reﬁned techniques l discussion forum reports l commercial submissions welcome (speciﬁc equipment advertorials will not be accepted)– Posters from international meetings– Book reviews– Letters to the editorMember StatesEFAT comprises of Member States of the European Union and Council of Europe https://www.efat.org/AFSTAL Association Francaise des Sciences et Techniques de L’Animal de Laboratoire DALAS Dutch Association for Laboratory Animal ScienceSECAL Sociedad Española para las Ciencias del Animal de LaboratorioATWAnimal Technology and Welfarewww.atwjournal.com 145148150152143

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73vOFFICERSPresidentDr Robin Lovell-Badge CBE FRSImmediate Past PresidentProfessor Sir Richar d Gardner MA PhD FRSBFIAT (Hon) FRSVice-PresidentsSenga Allan MIAT RAnTech, David Anderson MRCVS,Stephen Barnett BA MSc FIAT (Hon) CBiol FRSBRAnTech, Miles Carroll PhD, Paul Flecknell MA Vet MBPhD DLAS DipLECVA MRCVS FIAT (Hon), PennyHawkins PhD BSc, Wendy Jarrett MA, Judy MacArthur-Clark CBE BVMS DLAS FRSB DVMS (h.c.) DipECLAMFRAgS DipACLAM MRCVS, Fiona McEwen BSc BVM&SMSc MRCVS, Tim Morris BVetMed PhD DipACLAMDipECLAM CBiol FRSB CertLAS MRCVS, Clive PageOBE PhD BSc, Jan-Bas Prins PhD MSc, Vicky RobinsonCBE BSc PhD, Paul Sanders MIAT RAnTech, DavidSpillane FIAT, Gail Thompson RLATG, RobertWeichbrod PhD RLATGLife MembersKen Applebee OBE FIAT CBiol FRSB RAnTech,Charlie Chambers MIAT RAnTech, Roger Francis MScFIAT RAnTech, Pete Gerson MSc FIAT RAnTech,Cathy Godfrey FIAT RAnTech, John Gregor y BSc (Hons)FIAT CBiol FRSB RAnTech, Patrick Hayes FIAT DipBARAnTech, Robert Kemp FIAT (Hon) RAnTech,Phil Ruddock MIAT RAnTech, Ted Wills FIAT (Hon)RAnTechHonorary MembersMark Gardiner MIAT RAnTech, Sarah Lane MSc FIAT,Sue McHugh BSc FIAT, Norman Mortell BA (Hons)MIAT RAnTech, Wendy Steel BSc (Hons) FIATMembers of CouncilMatthew Bilton, Kally Booth, Steven Cubitt,Simon Cumming, Haley Daniels, Glyn Fisher,Nicky Gent, Alan Graham,Linda Horan, Sam Jameson,Elaine Kirkum, Adele Kitching, Theresa Langford,Sylvie Mehigan, Steve Owen, Alan Palmer, AllanThornhill, John Waters, Lynda Westall, Carole Wilson,Adrian WoodhouseCouncil OfficersChair: Linda Horan BSc (Hons) MIAT RAnTechVice Chair: Glyn Fisher FIAT RAnTechHonorary Secretary:Simon Cumming BSc FIAT RAnTechHonorary Treasurer: Glyn Fisher FIAT RAnTechChair of Board of Educational Policy:Steven Cubitt MSc FIAT RAnTechChair Registration & Accreditation Board:Glyn Fisher FIAT RAnTechATW Editor: Jas Barley MSc FIAT RAnTechBulletin Editor: Carole Wilson BSc MIATATW/Bulletin Editorial Board:IAT REPRESENTATIVESAugust20:Animal Technology and Welfare 4/2/21 13:19 Page vvOFFICERSPresidentDr Robin Lovell-Badge CBE FRSImmediate Past PresidentProfessor Sir Richar d Gardner MA PhD FRSBFIAT (Hon) FRSVice-PresidentsSenga Allan MIAT RAnTech, David Anderson MRCVS,Stephen Barnett BA MSc FIAT (Hon) CBiol FRSBRAnTech, Miles Carroll PhD, Paul Flecknell MA Vet MBPhD DLAS DipLECVA MRCVS FIAT (Hon), PennyHawkins PhD BSc, Wendy Jarrett MA, Judy MacArthur-Clark CBE BVMS DLAS FRSB DVMS (h.c.) DipECLAMFRAgS DipACLAM MRCVS, Fiona McEwen BSc BVM&SMSc MRCVS, Tim Morris BVetMed PhD DipACLAMDipECLAM CBiol FRSB CertLAS MRCVS, Clive PageOBE PhD BSc, Jan-Bas Prins PhD MSc, Vicky RobinsonCBE BSc PhD, Paul Sanders MIAT RAnTech, DavidSpillane FIAT, Gail Thompson RLATG, RobertWeichbrod PhD RLATGLife MembersKen Applebee OBE FIAT CBiol FRSB RAnTech,Charlie Chambers MIAT RAnTech, Roger Francis MScFIAT RAnTech, Pete Gerson MSc FIAT RAnTech,Cathy Godfrey FIAT RAnTech, John Gregor y BSc (Hons)FIAT CBiol FRSB RAnTech, Patrick Hayes FIAT DipBARAnTech, Robert Kemp FIAT (Hon) RAnTech,Phil Ruddock MIAT RAnTech, Ted Wills FIAT (Hon)RAnTechHonorary MembersMark Gardiner MIAT RAnTech, Sarah Lane MSc FIAT,Sue McHugh BSc FIAT, Norman Mortell BA (Hons)MIAT RAnTech, Wendy Steel BSc (Hons) FIATMembers of CouncilMatthew Bilton, Kally Booth, Steven Cubitt,Simon Cumming, Haley Daniels, Glyn Fisher,Nicky Gent, Alan Graham, Linda Horan, Sam Jameson,Elaine Kirkum, Adele Kitching, Theresa Langford,Sylvie Mehigan, Steve Owen, Alan Palmer, AllanThornhill, John Waters, Lynda Westall, Carole Wilson,Adrian WoodhouseCouncil OfficersChair: Linda Horan BSc (Hons) MIAT RAnTechVice Chair: Glyn Fisher FIAT RAnTechHonorary Secretary:Simon Cumming BSc FIAT RAnTechHonorary Treasurer: Glyn Fisher FIAT RAnTechChair of Board of Educational Policy:Steven Cubitt MSc FIAT RAnTechChair Registration & Accreditation Board:Glyn Fisher FIAT RAnTechATW Editor: Jas Barley MSc FIAT RAnTechBulletin Editor: Carole Wilson BSc MIATATW/Bulletin Editorial Board:IAT REPRESENTATIVESAugust20:Animal Technology and Welfare 4/2/21 13:19 Page vCouncil OfﬁcersChair: Glyn Fisher FIAT RAnTechVice Chair: Robin Labesse MIAT RAnTechHonorary Secretary: Haley Daniels MBA MSc CIPD RAnTechHonorary Treasurer: Sam Jameson MIAT RAnTechChair of Board of Educational Policy: Robin Labesse MIAT RAnTechChair Registration & Accreditation Board: Simon Cumming BSc FIAT RAnTech ATW Editor: Diane Hazlehurst MIAT RAnTechBulletin Editor: Carole Wilson BSc MIATATW/Bulletin Editorial Board: Diane Hazlehurst (Chair), Carole Wilson, Lynda Westall Branch Liaison Ofﬁcer: Lynda Westall BSc FIAT RAnTech DMSEFAT Representatives:Glyn Fisher, Robin Labesse, Toby SandersWebsite Coordinator: Allan Thornhill FIAT RAnTechWebsite Support: Sam JamesonAnimal Welfare Group:Carmen Abela (Chair), Kally Booth, Diane Hazlehurst, Sylvie Mehigan, Carole WilsonBoard of Educational Policy:Robin Labesse (Chair), Adele Kitching (Secretary), Richard Berks, Diane Hazlehurst, Theresa Langford, Tina O’MahonyCommunications Group:Adrian Woodhouse (Chair) Carmen Abela, Kally Booth, Sam Jameson, Theresa Langford, Sylvie Mehigan, Toby Sanders, Allan Thornhill, Lynda WestallVice-PresidentsSenga Allan MIAT RAnTech, David Anderson MRCVS, Miles Carroll PhD, Penny Hawkins PhD BSc, Wendy Jarrett MA, Judy MacArthur-Clark CBE BVMS DLAS FRSB DVMS (h.c.) DipACLAM DipELAM CBiol FRSB CertLAS MRCVS, Fiona McEwen BSc BVMOS MSc MRCVS, Tim Morris BVetMed PhD DipACLAM DipECLAM CBiol FRSB CertLAS MRCVS, Clive Page OBE PhD BSc, Jan-Bas Prins PhD MSc, Sally Robinson MA PhD, Vicky Robinson CBE BSc PhD, David Spillane FIAT, Gail Thompson RLATG, Robert Weichbrod PhD RLATGLife MembersKen Applebee OBE FIAT CBiol FRSB RAnTech, Jas Barley MSc FIAT RAnTech, Charlie Chambers MIAT RAnTech, Roger Francis MSc FIAT RAnTech, Pete Gerson MSc FIAT RAnTech, Cathy Godfrey FIAT RAnTech, John Gregory BSc (Hons) FIAT CBiol FRSB RAnTech, Patrick Hayes FIAT DipBA RAnTech, Robert Kemp FIAT (Hon), Phil Ruddock MIAT RAnTech, Ted Wills FIAT (Hon) RAnTechHonorary MembersAndy Domone MIAT, Kate Heath FIAT RAnTech, Martin Heath MSc FRSB FIAT FIScT RAnTech, Stuart Mackrell FIAT RAnTech, Steve Owen FIAT RAnTech, Sarah Reed FIAT RAnTech, John Waters MIAT RAnTech, Pete Willan DMS FInstMgt MIAT RAnTech Members of CouncilCarmen Abela, Richard Berks, Kally Booth, Simon Cumming, Haley Daniels, Glyn Fisher, Alan Graham, Diane Hazlehurst, Linda Horan, Sam Jameson, Adele Kitching, Robin Labesse, Theresa Langford, Sylvie Mehigan, Tina O’Mahony, Emma Owen, Claire Pearce, Toby Sanders, Allan Thornhill, Lynda Westall, Carole Wilson, Adrian WoodhouseFront cover credit: The Francis Crick Institute

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74BRANCH SECRETARIES 2022Cambridge: Tony Davidge cambridgebranch@iat.org.ukEdinburgh: Kery-Anne Lavin-Thomson edinburghbranch@iat.org.ukHuntingdon, Suffolk & Norfolk: Jo Martin hssbranch@iat.org.ukIreland: Lisa Watson irelandbranch@iat.org.ukLondon: Rebecca Towns londonbranch@iat.org.ukMidlands: Ian Fielding midlandsbranch@iat.org.ukNorth East England: Zoe Smith and John Bland northeastbranch@iat.org.ukNorth West: Nicky Windows cheshirebranch@iat.org.ukOxford: Adam Truby oxfordbranch@iat.org.ukSurrey, Hampshire & Sussex: Francesca Whitmore shsbranch@iat.org.ukWest Middlesex: Josefine Woodley westmiddxbranch@iat.org.ukWales & West: Rhys Perry waleswestbranch@iat.org.ukWest of Scotland: Joanne King westscotlandbranch@iat.org.ukIAT OFFICERS M AY BECONTAC TED VIA:IAT Administrator:admin@iat.org.ukOR VIA THE IAT WEBSITE AT :www.iat.org.ukOR THE REGISTERED OFFICE:5 South Parade, Summertown,Oxford OX2 7JLAdvertisement Managers:PRC Associates LtdEmail: mail@prcassoc.co.ukAlthough every effort is made to ensure that no inaccurate or misleading data, opinion or statement appear in thejournal, the Institute of Animal Technology wish to expound that the data and opinions appearing in the articles,poster presentations and advertisements in ATW are the responsibility of the contributor and advertiser concerned.Accordingly the IAT, Editor and their agents, accept no liability whatsoever for the consequences of any suchinaccurate or misleading data, opinion, statement or advertisement being published. Furthermore the opinionsexpressed in the journal do not necessarily reflect those of the Editor or the Institute of Animal Technology.© 2024 Institute of Animal TechnologyAll rights reserved. No part of this publication may be reproduced without permission from the publisher.CPD Officer: Alan Palmer MIAT RAnTechRegistration and Accreditation Board:Glyn Fisher (Chair), John Gregor y,Cathy Godfrey, Kathy Ryder (Home Office),Stuart StevensonObserver: Ngaire Dennison (LAVA)Congress Committee:Alan Graham (Chair), Haley Daniels, Adele Kitching,Allan Thornhill, John WatersDiversity Officer:Haley Daniels MBA MSc MIAT RAnTech CIPDUK Biosciences ASG Representative/Home Office:Alan Palmer MIAT RAnTechviAugust20:Animal Technology and Welfare 12/8/20 07:54 Page viRegistration and Accreditation Board:Simon Cumming (Chair), Glyn Fisher (Secretary), Linda HoranCongress Committee:Alan Graham (Chair), Haley Daniels, Adele Kitching, Claire Pearce, Allan ThornhillEquity, Diversity and Inclusion Group:Haley Daniels (Chair) MBA MSc MIAT RAnTech CIPD, Simon Cumming, Linda Horan, Emma OwenIndex to AdvertisersBRANCH SECRETARIES 2022Cambridge: Tony Davidge cambridgebranch@iat.org.ukEdinburgh: Kery-Anne Lavin-Thomson edinburghbranch@iat.org.ukHuntingdon, Suffolk & Norfolk: Jo Martin hssbranch@iat.org.ukIreland: Lisa Watson irelandbranch@iat.org.ukLondon: Rebecca Towns londonbranch@iat.org.ukMidlands: Ian Fielding midlandsbranch@iat.org.ukNorth East England: Zoe Smith and John Bland northeastbranch@iat.org.ukNorth West: Nicky Windows cheshirebranch@iat.org.ukOxford: Adam Truby oxfordbranch@iat.org.ukSurrey, Hampshire & Sussex: Francesca Whitmore shsbranch@iat.org.ukWest Middlesex: Josefine Woodley westmiddxbranch@iat.org.ukWales & West: Rhys Perry waleswestbranch@iat.org.ukWest of Scotland: Joanne King westscotlandbranch@iat.org.ukIAT OFFICERS M AY BECONTAC TED VIA:IAT Administrator:admin@iat.org.ukOR VIA THE IAT WEBSITE AT :www.iat.org.ukOR THE REGISTERED OFFICE:5 South Parade, Summertown,Oxford OX2 7JLAdvertisement Managers:PRC Associates LtdEmail: mail@prcassoc.co.ukAlthough every effort is made to ensure that no inaccurate or misleading data, opinion or statement appear in thejournal, the Institute of Animal Technology wish to expound that the data and opinions appearing in the articles,poster presentations and advertisements in ATW are the responsibility of the contributor and advertiser concerned.Accordingly the IAT, Editor and their agents, accept no liability whatsoever for the consequences of any suchinaccurate or misleading data, opinion, statement or advertisement being published. Furthermore the opinionsexpressed in the journal do not necessarily reflect those of the Editor or the Institute of Animal Technology.© 2024 Institute of Animal TechnologyAll rights reserved. No part of this publication may be reproduced without permission from the publisher.CPD Officer: Alan Palmer MIAT RAnTechRegistration and Accreditation Board:Glyn Fisher (Chair), John Gregor y,Cathy Godfrey, Kathy Ryder (Home Office),Stuart StevensonObserver: Ngaire Dennison (LAVA)Congress Committee:Alan Graham (Chair), Haley Daniels, Adele Kitching,Allan Thornhill, John WatersDiversity Officer:Haley Daniels MBA MSc MIAT RAnTech CIPDUK Biosciences ASG Representative/Home Office:Alan Palmer MIAT RAnTechviAugust20:Animal Technology and Welfare 12/8/20 07:54 Page viBRANCH SECRETARIES 2024Berkshire and Wiltshire: Cheryl Yalden berkshirewiltshirebranch@iat.org.ukCambridge: Tony Davidge cambridgebranch@iat.org.ukEdinburgh: Kerry Lavin-Thomson edinburghbranch@iat.org.ukHuntingdon, Suffolk & Norfolk (HNS): Joanna Owen hssbranch@iat.org.ukIreland: Lisa Watson irelandbranch@iat.org.ukLondon: Louise Fisher londonbranch@iat.org.ukMidlands: Alison Richie midlandsbranch@iat.org.ukNorth East England: John Bland & Zoe Smith northeastbranch@iat.org.uk North West: Emma Owen cheshirebranch@iat.org.ukOxford: Adam Truby oxfordbranch@iat.org.ukSurrey, Hampshire & Sussex (SHS): Francesca Whitmore shsbranch@iat.org.ukWest Middlesex: Hannah Easter westmiddxbranch@iat.org.ukWest of Scotland: Nicola Munroe westscotlandbranch@iat.org.ukInstitute of Animal Technology ..............75, 76, 78, 79, 100, 127, 128, 132, 136, 147, 0BCIPS Product Supplies Ltd ................................IBCLBS Serving Biotechnology Ltd .........................80Tecniplast UK Ltd ..........................................IFC

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If the answer to the above two questions is yes then listen up:The Institute of Animal Technology (IAT) is actively seeking new Council members, so whether you are interested in welfare initiatives, communications, education and training or just want to have a say, then why not get more involved in your professional body and join Council?Our Mission: Advancing and promoting excellence in the care and welfare of animals in researchAre you currently a full Member or Fellow of the Institute with two years’ consecutive membership, who wants to make a difference? Do you want to be involved in developing the future of the Institute of Animal Technology?Visit our website www.iat.org.uk for further informationInstitute of Animal TechnologyYOUR COUNCIL NEEDS YOU!Why bother?• the opportunity to shape the future• great networking and personal development opportunities• choose the area that interests you on Council and join that group• expenses are paid to attend Council meetings• discounted Congress attendance• the Council Election form is simple to completeStill unsure?Contact us and we can talk you through the process, provide more information about Council activities and groups or you can visit the IAT website members’ section.You would need to be proposed and seconded by either two Members or Fellows of the Institute or nominated by a Branch. The IAT can assist with this too, if you have any difficulties.Interested?Nomination forms should be completed online from the IAT website using this link http://iatforms.org.uk/view.php?id=16312When the form is submitted it is sent to the IAT Administrator (admin@iat.org.uk). The closing date for nominations is Friday 1st November 2024. Haley DanielsHonorary SecretaryVisit our website www.iat.org.uk for further information

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77August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareEditorialJas BarleyChair of the Editorial BoardLooking back over issues of the Journal through its various identities, one thing is apparent and that is the contribution thatoverseas authors have made to the content. Topics have varied from dealing with exotic species, lack of sophisticated equipment,different attitudes to everyday problems, staff training and education and disease outbreaks. However, the resolute thathas beenconstant throughout, despite the differences across the world, is the love and concern for the animals being cared for.Many include interesting photographs but I unfortunately am unable to use them as the quality of images is so poor whenrepr oduced, to the extent in some cases, they become worthless.Obviously, things have changed over seven decades and the technology described in contributions from overseas is less differentfrom what we use in the UK. This issue welcomes contributions from Australia, the Czech Republic and Iran as well, of coursefrom the UK. Since ATW became an Open Access publication and is being published electronically, it is enjoying a wider audienceand is attracting more contributions than usual. Not all are relevant to our profession, but knowledge is transferable so whatseems ‘off beat’ today may become useful in the future. However, as Editor I will always strive to maintain the quality of ourpublications and the usefulness to our readers.In this issue we include the RSPCA 2019 Rodent and Rabbit Welfare group meeting repor t. The 26th meeting that the RSPCA haveorganised focussed on ‘sentience, positive welfare and psychological well being’. The report contains contributions from 11presenters as well as notes on the interactive discussion session on sentience that closed the meeting.A paper from Iran, a first as far as I can see for the Journal, on reducing the negative effects of methionine on bone parametersin broilers’ embryos may seem of little relevance but it offers a better understanding of how methionine affects bone structurewhich is important to most species. Similarly, Feline Assisted Therapy as described by the team at the University of Life SciencesPrague does not appear to fall into the realms of Animal Technology but it gives us a better understanding of how animals can havea positive effect on some people, which in thecurrent situation may be of significant benefit to a wider population. Our final paperfrom the team at Western Sydney University, details the care of the Children’ Python and two species of Bearded Dragons. Notperhaps the run of the mill laboratory animals but just as important to many Animal Technologists globally as mice and rats. If youkeep reptiles at home or know of someone who is contemplating one as a pet these papers make useful reference documents. Wealso offer twopapers from previous issues of the Journal which were very different in appearance and content than today’s Journalof Animal Technology and Welfare and not only because of the change of title. Issues were printed in black and white and in the veryearly days were produced by hand. The paper from France on Physical Hazards in the laboratory animal house will bring back manymemories for some of the older technicians, myself included, but not necessarily good ones. The use of ether as an anaestheticwhich I know is still used in some countries where resources are limited, for human surgery, presented a very real danger to bothanimals and staff. Disease in laboratory animal units was often a r ecurring problem, bacterial infections such as Pseudomonas asdescribed in the reprint of the article were still presenting Animal Technologists with problems as late as the end of the 1980s. Whenimporting animals and tissues from overseas it is important to realise that they may be carrying disease not seen in the UK forseveral decades. In recent times, Ectromelia was introduced into a unit in the USA via antibodies produced overseas. Precautionsmust be taken until such time as you are sure that the animals and tissues are clear of any underlying infections.We are also able to offer in this issue an interesting Tech-2-Tech ar ticle by Seonagh Henderson of the University of Glasgow, ona novel technique of cage cleaning which hasa positive effect on the welfare of laboratory rats. Finally, we included several postersprepared for AST2020 but sadly at the moment remain unpresented.Thanks again to all of our authors, past and present, both internationally and here in the UK. There would not have been 70 yearsof the Journal without you. Here is to the next seven decades and beyond.THE INSTITUTE OF ANIMAL TECHNOLOGYETHICAL STATEMENT“In the conduct of their Professional duties, Animal Technologists have a moral and legalobligation, at all times, to promote and safeguard the welfare of animals in their care,recognising that good laboratory animal welfare is an essential component of goodlaboratory animal technology and science.The Institute recognises and supports the application of the principles of the 3Rs(Replacement, Reduction, Refinement) in all areas of animal research.”ixAugust20:Animal Technology and Welfare 12/8/20 07:54 Page ixIf you were able to attend IAT Congress this year, I am conﬁ dent that you thought it a great success with many presentations, posters and workshops to visit and participate in, and came away with new concepts on how to care for your animals. In this edition we have included articles written based on presentations and provided a selection of posters from Congress 2024.The Best Poster – Animal Welfare winner Aimee Paterson discusses the reﬁ nements introduced to improve the husbandry processes for aged C57BL/6 mice. They are comparable to humans as they develop a reduction in muscle mass, weight loss, changes of gait and development of age-related illnesses. They reviewed their husbandry processes and introduced new measures. These new improved routines help to identify health concerns sooner during their routine checking regime. Ultrasound to ultra-see is an article based around the early detection of pregnancy in time mated mice using an ultrasound machine instead of visual checking or palpating the abdomens of females. This method allows females which are not pregnant to be mated again and this supports the 3Rs.The 3Hs’ initiative – housing, handling and habituation was a presentation on an approach that considers the lifespan of laboratory animals, seeking to improve each animal’s experience. The animals are housed in highly enriched cages to promote natural behaviours and to reduce stereotypical behaviours associated with stress and negative experiences. Playpens are used for added enrichment which have shown a positive impact on Animal Welfare.This issue starts with the Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting. A variety of different topics were presented and discussed. This meeting has been held annually for the past 30 years and allows members to discuss current welfare research, welfare issues and shared experiences of the implementation of the 3Rs for Replacement, Reduction and Reﬁ nement regarding rodents. Its theme was looking back and looking forward. This report is a summary of the meeting along with take home messages and action points for readers to consider raising at their own establishments where they work.Jamie Redden’s poster on public engagement and openness in animal research is one of The Crick’s strategic priorities by looking at ways to engage and inspire members of the public with discovery and science. They are open and transparent about their research that uses animals and what the beneﬁ ts are. The Crick is 1 of over 100 signatories to the Understanding Animal Research (UAR) Concordat on Openness. They use different methods of engagement with the public such as a website, social media, public exhibits and events.Remember that our journal can be used as sources of references for both your independent learning and your IAT educational journal and to gather the great ideas that people come up with to improve the Health and Welfare of the animals in our care.We are here to provide you with the opportunity to publish your work as we are always looking for new material for ATW focussing on Animal Welfare and the 3Rs which is free of charge. The assistance the AWT editorial board provide you will turn your ideas and concepts into realities.EditorialDiane HazlehurstChair of the Editorial Board Editor’s email: atweditor@iat.org.uk April 2024 Animal Technology and Welfare

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The Andrew Blake Tribute Award commemorates the work and life of Andrew Blake, who suffered from Friedrich’s ataxia, a hereditary condition described as one of the “worst of neurological diseases”. Andrew died in May 2002 aged 39. Andrew was passionate about the need to support scientists in their work and his commitment to speaking out against animal rights activists took up much of the last ten years of his life. He died shortly before he was to collect his MBE.ANDREW BLAKETRIBUTE AWARDSPONSORED BY THE NC3RsANDREW BLAKETRIBUTE AWARDDON’T KEEP YOUR GOOD IDEA TO YOURSELF!WE WANT TO HEAR ABOUT IT FOR THE 2025 AWARDDETAILS OF THE AWARD This Award is given annually, where sponsorship allows, to the Animal Technician/Technologist judged to have made the most significant contribution to improving standards in laboratory animal welfare over the previous twelve months. All qualified Animal Technologists are guided in their work by the Institute of Animal Technology’s Ethical Statement: In the conduct of their Professional duties Animal Technologists have a moral and legal obligation, at all times, to promote and safeguard the welfare of animals in their care, recognising that good laboratory animal welfare is an essential component of good laboratory animal technology and science. The Institute recognises and supports the application of the principles of the 3Rs (Replacement, Reduction, Refinement) in all areas of animal research. The Award is made to acknowledge the professional and personal commitment of Animal Technologists to improving standards in all aspects of laboratory animal care and welfare. THE PRIZE CONSISTS - CONGRESS 2025 FREE ATTENDANCEnext March WHICH WILL INCLUDE DISPLAYING YOUR POSTER(WITH THE OPTION TO ALSO GIVE AN ORAL PRESENTATION)- AN ENGRAVED GLASS PLAQUE - AND £250 CASH AWARDCLOSING DATE FRIDAY 18TH OCTOBER 2024 Need advice – or you wish to discuss anything regarding a possible entry? Then please email the IAT Administrator admin@iat.org.uk with your contact details and one of the organisers will respond and give you all the support you need.ARE YOU AN ANIMAL TECH?HAVE YOU BEEN PART OF A TEAM OR HAVE YOU REFINED ANIMAL CARE AND WELFARE IN YOUR FACILITY?ALL ANIMAL TECHNICIANS AND TECHNOLOGISTS, QUALIFIED AT ANY LEVEL AND PRIMARILY WORKING IN THE UK CAN ENTERCRITERIA – The topic of work that you describe in your application may be undertaken as part of a project and PRESENTED AS A POSTER.YOUR POSTER SUBMISSION SHOULD CONTAIN THE FOLLOWING HEADINGS: TITLE, AIM, METHOD, RESULTS, DISCUSSION, CONCLUSION, REFERENCES and ACKNOWLEDGEMENTS The Poster should also contain the content below:- Why did you undertake this work? (what was the potential problem you were trying to improve?) - How did you undertake it? (species, numbers, sex, materials used) - Describe in a comprehensive and concise manner that allows a complete understanding facilitating reproducibility. - Explain if the work contributes to one of the 3Rs. - Explain how the welfare of the animals was improved. - Describe the results you obtained including data generated with assessment. - Were there any statistics undertaken? Please provide this information. Include a brief CV outlining your overall contribution to the work. Please also list your supervisors or PPL holder if applicable for the work. Submit your Poster online via this link https://www.iat.org.uk/abta where you will see the Submission form for completion.To allow others to be able to replicate the work, please consult the ARRIVE guidelines: https://www.nc3rs.org.uk/arrive-guidelines

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81August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2024 Animal Technology and WelfareIntroductionThe RSPCA/UFAW Rodent Welfare Group has held a one-day meeting every autumn for the last 30 years. This is to allow its members to discuss current welfare research, exchange views on welfare issues and share experiences of the implementation of the 3Rs for Replacement, Reduction, and Reﬁnement with respect to rodent use.This meeting was held in person at the Francis Crick Institute in London in October 2023 and allowed participants the opportunity to engage in face-to-face discussions throughout the day and as part of a group discussion session at the end of the day. The theme for the day was looking back and looking forward, and talks covered topics ranging from housing and husbandry to reﬁnement in procedures, education, and learning from companion rat care. This report summarises the meeting and includes some action points for readers to consider raising at their own establishments.Rodent husbandry: where have we come from – and where are we going?Ken ApplebeeKen began his career in 1972 as a Junior Animal Technician at the Medical College of St Bartholomew’s Hospital and resigned in 2020 after 10 years as the Director of Biological Services at King’s College London. During his career, Ken witnessed many advances in rodent husbandry. In this presentation, he highlighted some of the major changes that have occurred over the last 20 years and made suggestions on new improvements to rodent welfare that could be implemented in the near future. He focussed particularly on ways in which Animal Technologists could support such improvements.Caging Ken spoke of the changes in rodent cage technology that had impacted both animal cages and the overall design and structure of animal facilities. The development of Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group MeetingMARINE BARNABÉ1, KEN APPLEBEE2,3, JANE HURST4, VIKKI NEVILLE5, TOM CHILDS6, MIGUEL MARAVALL7, JULIA BARTLETT8, JANE TYSON9, TINA O’MAHONY10, HALEY DANIELS11 and KERITH LUCHINS121 Animals in Science Department, RSPCA, Horsham RH12 1GY2 Applebee Advisory, Hornchurch RM12 6RJ3 College of Laboratory Animal Science & Technology, Rushden NN10 0SH4 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool CH64 7TE5 Bristol Veterinary School, Bristol BS40 5DU6 Francis Crick Institute, London NW1 1AT7 Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton BN1 9RH8 School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol BS8 1QU9 Companion Animals Department, RSPCA, Horsham RH12 1GY10 Institute of Animal Technology, Oxford OX2 7JL11 University of York, York YO10 5DD12 University of Chicago, United States of AmericaCorrespondence: animalsinscience@rspca.org.uk

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82Animal Technology and Welfare August 2020individually ventilated cages (IVCs) in particular was important as it presented a new way of working. Ken also noted the changes in guidelines requiring more spacious cages (regarding ﬂoor area and height) for both mice and rats. Cage changing practices have also developed a great deal. Ken noted that in the 1980s, animals were housed with high density in rooms with low air changes and 2 or 3 cage changes per week. These practices have changed signiﬁcantly with increasing awareness of the effects of cage changing on rodent welfare, particularly the link with aggression in male mice. Rodents are now primarily kept at lower stocking density in rooms with high air density (if using conventional open-top cages) or in IVCs. Most importantly, cages are cleaned out much less frequently, usually every 10 days, and are spot cleaned, both of which reduce disruption and aggression. Enrichment In the 1970s and 1980s, there was much less evidence supporting the welfare and scientiﬁc beneﬁts of environmental enrichment. Household products such as shredded paper and tissues may have been provided, with treats such as sunﬂower seeds, rice grains and yoghurt. Today, there is an important industry producing specially designed enrichment items including tunnels, chew blocks, hides, and toys. The effects of providing environmental enrichment need to be scientiﬁcally evaluated to ensure they have a positive measurable impact on Animal Welfare – a useful resource for evaluating environmental enrichment was produced by the NC3Rs, RSPCA and IAT.HandlingFinally, Ken mentioned the reﬁnements made to rodent handling, including the use of tunnels or cupped hands to pick up mice. The importance of calm, non-aversive handling in all aspects of rodent husbandry has become increasingly recognised, leading to positive changes in the way mice and rats are picked up and held, including routine activities such as weighing.Room for improvementWith regards to areas of potential improvement, Ken raised the issue of chronic cold stress experienced by laboratory mice. According to the UK Home Ofﬁce guidelines, mice should be kept at a room temperature of 20-24°C, even though this is signiﬁcantly below the thermoneutral zone of 26-34°C for these animals. This may be improved by providing sufﬁcient nesting material to allow mice to control their environment and create adequate nests, which can reach temperatures of 30-32°C. This is particularly important for newborn pups who lack the ability to thermoregulate and are dependent on their mother and siblings for warmth. The Animals in Science Regulation Unit (ASRU) Change Programme and the associated increased requirement for establishment self-regulation were also discussed. In this new system, Ken believes that issues may arise in establishments with poorly functioning Animal Welfare and Animal Welfare and Ethical Review Bodies (AWERBs) and inconsistent standards of Animal Welfare. For establishments to effectively self-regulate, the AWERB and animal care staff must be competent, diligent, and innovative. Finally, an increase in external scrutiny was noted. This was exempliﬁed by the recent critical evaluation of the forced swim test by the Animals in Science Committee (ASC) and the fact that this test is no longer being performed at King’s College London as of 2015.Take-home messages and action points– Think about the use of enrichment in your own establishment – is its implementation sufﬁciently driven by evidence? You may wish to take a look at the NC3Rs/RSPCA/IAT tool for evaluating environmental enrichment.– Consider the handling methods you use with the animals in your care. If you would like to switch over to reﬁned handling methods but are unsure of how to start, you could take the free, online reﬁned mouse handling course offered by the NC3Rs. Progress on reﬁned mouse handling – has it made a difference?Jane HurstRoutine handling of animals in the laboratory is an essential but frequently ignored component of animal experiments and husbandry. It has considerable potential to inﬂuence anxiety and aversion to human approach and contact. Jane and colleagues showed that the tail method traditionally used to pick up mice consistently induces aversion and high anxiety (even with the animal’s weight supported). Conversely, use of handling tunnels, scooping mice up on the open hand (cupping), or on a cage ladder leads to voluntary approach of the handler, low stress and anxiety, and improved tolerance of physical restraint. Now there is substantial evidence that these less aversive methods provide major reﬁnements over tail handling for laboratory mice, uptake is spreading worldwide. However the time and effort required to achieve this level of engagement has been far greater than Jane ever imagined. In this presentation, she shared her experience with some of the major barriers that have slowed the uptake of reﬁned handling within animal facilities and how progress has been achieved.Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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83August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareHaving previously spent time working with wild rodents, Jane noted that laboratory mice showed very high levels of anxiety compared to their wild counterparts. This triggered an initial study to compare the use of four different methods to pick up mice.1 The study design included animals of different strains and sexes and multiple human handlers. Results revealed that tail handled mice consistently showed greater aversion to handling as well as higher levels of stress and anxiety than mice handled using a tunnel or cupped hands. These differences were most notable when comparing tail and tunnel handling. Since the publication of Jane’s paper in Nature Methods in 2010, other research groups have independently published further evidence of the beneﬁcial effects of non-aversive handling, showing strong consistency and reproducibility. Jane highlighted that the method of handling used in a study inﬂuences multiple aspects of the experiments. These effects are evidenced in multiple areas, including behavioural test results (including elevated maze and open ﬁeld tests and exposure to a novel stimulus), breeding success, litter mortality, physiological stress responses (changes in corticosterone and blood glucose), and responses to speciﬁc procedures such as injections, anaesthesia and oral gavage. All papers published showed that tail-handled mice showed greater anxiety, a ﬁnding that was shown to be consistent across research labs, handlers, mouse strains and tests.However although there is now substantial evidence to support the implementation of non-aversive handling methods, there are also signiﬁcant barriers to adopting new practices. These include fear that changing to a different handling method will require additional time and training, incur ﬁnancial costs, pose practical issues, or simply that it will make no difference to the research and so is not worth doing. Jane noted that the resources and training, including webinars and videos showing how to correctly handle the mice, provided by the NC3Rs had made a positive impact on encouraging researchers and technicians to adopt tunnel handling. Since its inception in 2016, the NC3Rs-based resource page dedicated to mouse handling has been accessed by users from 107 different countries and posters were translated into ﬁve languages and sent to over 400 institutions.Jane also noted the essential role of champions – those who were initially sceptical but have experienced the beneﬁts of non-aversive handling ﬁrst-hand and are able to speak to and convince others facing similar issues. Animal Technologists may be particularly concerned about the practicalities of implementing novel animal handling protocols and may beneﬁt from demonstrations by others who have successfully moved towards using tunnel handling and who can provide advice and support.Take-home messages and action points– Although scientiﬁc evidence is important, it is not enough. To effect change, it is essential to hear from those who have experienced the challenges and beneﬁts of implementing reﬁnements. These early adopters are invaluable in their roles as advocates for change.– For faster implementation, focus on collaborating with other research groups and stakeholders.– Enforcement through policy may be considered, although it should be positively framed as handling mice in the most appropriate, reﬁned manner, rather than banning tail handling. A mapping review of reﬁnements to laboratory rat housing and husbandryVikki NevilleReﬁning the housing and husbandry of laboratory rats is an important goal. Indeed, standard laboratory rat housing may be deemed unacceptable according to several deﬁnitions of ‘good’ welfare. However the implementation of any reﬁnement should have a strong evidence base, to ensure that the rats will genuinely beneﬁt and to avoid wasting resources. There is a large existing body of work focussing on reﬁnements to rat housing and husbandry and synthesising these data is therefore an important step before drawing conclusions. The aim of Vikki’s work was to compile, assess and summarise previous research on reﬁnements made to rat housing and husbandry and the resulting review was recently published in Lab Animal.2Vikki and her team conducted a mapping review of studies investigating potential reﬁnements of laboratory rat housing and husbandry to assess what reﬁnements have, and have not, been studied and to brieﬂy assess whether evidence supports any impact on rat welfare. The literature search was designed to cover three key focus areas: rats, welfare and housing/husbandry. The criteria for inclusion into this analysis were the publication language (English), the type of study (empirical), the species studied (Rattus norvegicus) and the experimental design (involving a change made to housing or husbandry that had the potential to be a reﬁnement, and the inclusion of both treatment and control groups). After screening and selection, a total of 1,017 studies were included in the analysis. The ﬁrst of these studies was published in 1944 and the number of publications per year increased signiﬁcantly over time. Overall, this body of work showed a strong bias towards using male rats only (60.2%) and it was noted that some studies (1.9%) did not even report the sex of the animals used. In addition, over half of studies (50.6%) did not mention the use of randomisation in their study design and very Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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84Animal Technology and Welfare August 2020few reported using blinding (14.1%) or provided sample size justiﬁcation (3.8%). Randomisation, blinding, sample size and sex are all items listed in the ARRIVE guidelines Essential 10, meaning they should be included in all manuscripts as a basic minimum.3 Without these, readers cannot effectively assess the reliability of the ﬁndings reported.There were many reﬁnements studied and a range of readouts used to assess the impact of these. Many reﬁnements related to cage contents, followed by cage type and cage mate number, although the rationale for manipulations were often unclear. Vikki questioned what impact these additional enrichment items may have other than initially providing a novelty to the animals. In addition, potential reﬁnements were often studied concurrently with others, making it difﬁcult to tease apart the speciﬁc aspects that are most beneﬁcial to rat welfare. Outcome measures were widely varied, spanning behaviour, physiology and neurophysiology. The most reported measure was body weight, followed by open ﬁeld test results and elevated plus maze results. Some of the physiological indicators reported, such as heart rate, cortisol concentrations, and blood pressure, may rise in stressful situations, but can also increase in situations that are neutral or even rewarding, which complicates their interpretation in terms of welfare. These outcome measures may not provide reliable information about the impact of the housing and husbandry reﬁnements.Results from preference studies, such as choice, consumer demand and conditioned place preference tests, may be less ambiguous. Overall, preference studies showed that rats preferred having complex environments to perform different activities. This includes having multiple items in the cage, such as natural objects, chewable objects, shelters, a running wheel and a foraging device among others. Preference for size, bedding, and temperature was dependent on several factors, including sex, the number of animals in a cage, the photoperiod and particular behaviours, among others.Take-home messages and action points– Rats prefer complex environments that provide different areas to fulﬁl different functions.– A one-size-ﬁts-all approach to reﬁnements is not appropriate because different reﬁnements impact different rats in different ways.– A complete overhaul of rat housing may be needed perhaps using a consultation process as has been done regarding pet rat housing.4 – Looking forward, future research should focus on reﬁnements to transport, investigating controllable/ predictable manipulations and using/validating reliable measures of welfare. Improving efﬁciency and accuracy of Animal Welfare reporting through automationTom ChildsDuring this talk, Tom explored how automating (making a process operate automatically without the need for manual interference) certain processes at The Crick has improved the efﬁciency and accuracy of Animal Welfare reporting. This change was driven by the industry’s high expectations surrounding swift, efﬁcient and accurate reporting of Animal Welfare to ensure legal and ethical standards are met. The use of automation presents several beneﬁts, including saving time and freeing up staff to perform other tasks, reducing opportunities for human error and increasing traceability and reliability of results. However there will always need to be a balance between efﬁcient automated systems and competent, empathetic human observers.Starting off small, Tom and his team identiﬁed processes that were repetitive, complex and time-consuming. A good candidate for automation was deﬁned as a process that: • involves data stored in an accessible format• is logical, repeatable and scalable with high impact• requires minimal human decision-making• may be prone to human error and time-sensitive• is tedious, repetitive or boring With this knowledge, the team began mapping and automating jobs that would have the highest impact. This resulted in the automation of a number of processes but the most impactful project was the automation of Home Ofﬁce returns. These reports are produced following a speciﬁc format and provide details of the number of procedures and animals used, and the nature and purpose of the procedures performed under the project licence. The Home Ofﬁce returns process fulﬁlled all the criteria outlined above. In addition, these are subject to signiﬁcant time pressure as the window for submitting returns is approximately 4 weeks, which makes this process stressful for staff members. In its ﬁrst year, this initiative signiﬁcantly reduced the workload of four staff members assisting project licence holders with their annual returns from a full month of hard, repetitive and complex work to just a couple of hours per lab. This was not achieved without some challenges, including issues with data storage, signiﬁcant training requirements, initial lack of trust and resistance to change from users and staff, niche cases that did not ﬁt the standard process and quite a few bugs! In addition to streamlining processes, this experience also allowed team members to develop IT skills, including coding and gave them the opportunity to gain experience with project management software. The outcome is a platform that users can access to Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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85August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfaregain an aggregated overview of all data relating to an animal research project, broken down and organised into collapsible categories and sub-sections that can be searched. The database also allows users to record and keep track of actual severity, which has signiﬁcantly reduced the number of errors in Home Ofﬁce returns. The data is refreshed daily and can be exported to Excel, making it very user-friendly.Since that ﬁrst year, Tom and his team have introduced the use of a fully functional report to allow each individual animal to be reviewed and have expanded this report for niche cases, tools for auditing and more. In addition, the team has automated a mailing system to allow reporting of standard condition 18 reports and breaches, as well as producing daily reports for project licence holders and colony managers showing stock levels, single-housed animals, ﬁnancial allocations and health issues, among others.Tom hopes to inspire others to look at processes in their own workﬂows and create their own systems. The following points may help to begin this process:– Identify processes that take a lot of time and effort. Which of these may be suitable for automation?– Consider the pros and cons of automation. Is the time saved worth the signiﬁcant time and resource investment? – Clearly map out your process, starting with small, focussed and achievable goals.Free exploration in a modular labyrinth: a novel open-source design for mouse experimentsMiguel MaravallBrains evolved to guide animals’ interactions with their environment. Animals in nature sense their surroundings by actively engaging with them and process the resulting signals according to their behavioural utility. Laboratory based neuroscience research has often focussed on achieving tight experimental control and high statistical power. These aims are necessary for rigorous and reproducible research but have traditionally only been attainable under restrictive conditions. These approaches can place animals in a non-natural behavioural state and induce severe stress. An opportunity for reﬁnement is provided by new developments in machine vision and microelectronics, which can enable the tracking of motion and posture with unprecedented detail and allow experiments to unfold automatically depending on the animal’s behaviour. This requires an architecture that can be easily adopted, altered and shared, and is based on cheap and readily available components. In the presentation, Miguel introduced his team’s new modular maze for mice, designed to interrogate sensing and decision-making in freely moving animals while providing precise experimental control and ﬂexible protocols. This labyrinth follows previous contributions in that it is made of plastic that is opaque under visible illumination but transparent in the infrared range, allowing animals to be tracked as they move. Mice feel safe in an enclosed environment and can express their natural curiosity to explore. Partitions can slot in between posts and be replaced by panels incorporating different textures, gratings, or 3D-printed shapes, as well as reward ports. This allows ﬂexible reconﬁguration of the maze and creates associations between stimuli, locations and rewards. Tunnel crossings from the cage into the maze and entries into regions of interest can also be tracked. With these components, animals can encounter multiple stimuli as they move from the labyrinth’s origin to any endpoint, permitting the experimenter to set up automated rules deciding whether the mouse will be rewarded. Mice display high levels of motivation to explore and quickly learn to navigate the labyrinth. Fast navigational learning is achieved without ﬂuid or food restriction or any other regulated procedure, as the animal’s natural motivation to explore is leveraged. To encourage uptake of this system, Miguel and his team have set up a Github repository site for maze designs and code. These are openly shared and distributed as testing continues to enable quick, cheap and easy replication of the maze by others.Take-home messages and action points– Visit the repository comprising images, maps, and coding relating to the mouse mazes developed by Miguel’s team.Reﬁnements of handling and dosing methods for rats and miceJulia BartlettMany scientiﬁc procedures involving animals require the administration of test substances. This almost always entails each animal to be restrained, which is known to cause stress. Julia and her colleagues have developed and implemented several techniques to reduce the stress caused by all aspects of oral dosing, including handling, restraint and drug administration. This work forms part of the ‘3Hs’ Initiative’ (housing, handling and habituation), a holistic approach to reﬁnement that considers the lifetime experience of laboratory animals to promote positive experiences and reduce cumulative suffering. Habituation is particularly important as it requires only a small-time commitment and yet has a signiﬁcant impact on the animal, who is learning from every experience with the handler. By receiving positive reinforcement (a reward), the animal learns Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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86Animal Technology and Welfare August 2020to anticipate a positive experience. This reduces the stress associated with all subsequent handling, making procedures easier and saving time in the long term.Oral dosing of rats and mice is commonly carried out using an oesophageal cannula. This procedure is distressing for the animals and requires physical restraint. There are also risks of adverse events including incorrect placement, tracheal dosing and oesophageal trauma. This method also requires the experimenter to have a personal licence, which can leave studies vulnerable to disruption associated with staff absences. An alternative approach is to use voluntary ingestion of test substances in palatable solutions. Julia’s experience has shown that rats and mice readily take palatable solutions such as peanut butter, condensed milk and apple juice from a syringe. Drugs can be accurately administered in this way with much less stress for the animal and handler.Although this approach has previously been reported by several research groups, it is still not widely used and it is not clear if this is due to a lack of awareness or whether researchers have encountered problems using this approach. Animals may initially show neophobia but this can be overcome by introducing the new palatable substance gradually. Rodents may also develop conditioned aversion, which refers to the association of the aversive effects of a drug with the palatable solution in which it was administered. To reduce the potential for this to occur, Julia’s team developed a modiﬁed protocol in which the palatable substance on its own is offered to the animal a few hours after the drug was administered. This reduces the negative association of the adverse effects of the drug with the treat substance. Using this approach, the team has been able to administer a wide range of psychiatric drugs without issue. This reﬁned method reduces the distress caused to the animal during the substance administration procedure and eliminates the potential risks associated with oral gavage. Although the approach will not be compatible with all test substances, this method offers both welfare and scientiﬁc beneﬁts. Reﬁnements have also been implemented for intraperitoneal injections, in which the animal is typically ﬁrmly restrained, causing stress for both animals and staff. Julia’s team has modiﬁed their handling techniques for rats and mice to eliminate the need for scrufﬁng in rats. By reducing physical restraint, the animal can relax abdominal muscles at the injection site, which reduces pain during the procedure. Animals are also less likely to bite when they are not restrained and show lower cortisone levels and overt behaviours (struggling, vocalising and defecation).5 There is also no evidence of intestinal damage using this method.Similarly, mouse scrufﬁng was also reﬁned to avoid pulling or touching the animal’s tail, which is known to be aversive. Reﬁned scrufﬁng is done entirely on a VetBed or on the handler’s arm using the animal’s body but without touching the tail. Analysis of objective measures of affective state and the stress response (urination, vocalisation, struggling, aversion on release and defecation) showed that this method offers signiﬁcant improvements in Animal Welfare. Overall, the reﬁnement techniques described here are easy and practical ways of reducing the stress caused by handling and dosing. Making reﬁnements to these common procedures can improve the lifetime experience of the animal and reduce cumulative severity over time. Tail handling and oral gavage are no longer used in Julia’s research lab, with beneﬁts to both animals and the staff members working with them. These techniques have also been used by researchers with a range of prior experience and all have demonstrated competency within a short period of time. Implementing habituation into animal research protocols has important beneﬁts for all aspects of animal handling and should be encouraged.Take-home messages and action points– Visit the 3Hs’ Initiative website for detailed examples of how to implement these reﬁnements in your own lab.– Complete the 3Hs’ online CPD course to test your knowledge and fulﬁl your CPD requirements.What can carers of companion rodents and laboratory rodents learn from each other?Jane TysonSeveral rodent species have been kept as companion animals for many years. They can be easily acquired from pet shops and many have a short life expectancy, often thought of as being good pets for children because they require little of the complex time, ﬁnancial and care commitments of other animals. However this perception is far from the truth, which can lead to problems for owners and compromised welfare for pets when the difference between expectations and reality is realised. In this presentation, Jane described some of the recommendations provided to carers of companion rodents in relation to housing and the types of things they should be considering in their pets’ environment to help meet their needs. Some of these may be transferable into the laboratory world to help improve the lives of rodents in these settings too.Knowledge around pet rodents’ needs, husbandry and housing is still limited and little research has been undertaken to investigate these. Much of the early housing guidance provided to pet owners was based Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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87August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfareon information gleaned from research institutions and laboratories. There are few studies looking speciﬁcally at the welfare needs of pets and very little research has investigated their spatial requirements. Housing can frequently be a welfare concern as commercially available cages are often small and inadequate to allow animals to express their full behavioural repertoire and contain all the resources they may need. The RSPCA promotes a pragmatic outcomes-based approach to pet rodent housing, aiming to empower owners to provide these highly active and intelligent animals with the space and resources they require and to also enable them to fulﬁl their natural behavioural patterns. SpaceEnclosures should be large enough to comfortably accommodate all resources whilst ensuring all animals can comfortably perform all their natural behaviours. All individuals need to be able to perform the same behaviours at the same time to help minimise competition or monopolisation of resources so multiples of each item should be included within enclosures housing multiple animals. There should still be enough space for animals to move around and this should not be constrained by the quantity of resource provision. Environments need to be kept interesting as rodents are naturally inquisitive and enjoy exploring so will easily get bored in an unstimulating enclosure. If all resources do not comfortably ﬁt in their cage and allow plenty of space for moving around, including running, jumping, climbing, the enclosure is not large enough. Housing designMany rodents enjoy using different levels. Vigilance is a natural anti-predator tactic, so rodents want a good view of their surroundings. To get the most beneﬁt, they need to stand fully upright without touching the roof of their enclosure. As rodents are prey species, an anti-predatory tactic is simply to ﬂee and hide. It is crucial all animals have access to their own safe, secure hiding place, with safe sleeping areas to rest undisturbed and plenty of nesting material. Providing various types of nesting material gives choice and helps them construct better nests. Of course, these can be bought commercially but cardboard boxes can also be used. Use hiding places with two entry points to prevent one animal trapping another inside or preventing access, and make sure there is at least one more hiding place than the number of animals.Housing provided should also be designed with the animal’s individual needs in mind. It is useful to consider the biology and natural behaviours of the species.For example, some rodents like to climb while others like to jump. However every individual has their own preferences. Whilst each species may enjoy digging, an individual animal may have a particular preference over the type of litter they dig in. It is worth taking some time to identify an individual’s preferences as this helps meet their needs in the best way.Jane concluded her presentation by highlighting that although laboratory rodents live in very different environments to pets and are cared for in a different way, the fundamental needs of a laboratory animal are the same as those of a pet. Traditional cages for pet rodents are still widely available and well used, although these animals require more than a simple cage to satisfy their needs – they need plenty of enrichment and space. Pet owners are increasingly thinking about enclosures that are not simply off the shelf and making their own improvements. Small changes to housing and husbandry can make a big difference to Animal Welfare. Increasing space allowances and the complexity of the environment means rodents are more likely to explore and engage with their surroundings which will keep them physically and mentally active and help to improve their wellbeing. Jane also noted that safe, supervised time outside of the home cage is important to provide animals with opportunities to explore somewhere new. This is something that has been introduced in many laboratory settings as playpens. Finally, Jane highlighted the importance of interacting with these animals, as they can ﬁnd positive interactions with us a rewarding experience and actively enjoy spending time with their human caretakers.Take-home messages and action points– Think about the differences in the ways lab and pet rodents are housed and cared for. Are there any practices for companion rodents that you could adapt? For more information, you can read the RSPCA Companion Animals resources on keeping pet rodents.Educated animal care staff: intrinsic for Animal WelfareTina O’Mahony and Ken ApplebeeFrom delivering endpoint assessments, to creating syllabuses and moderating coursework, IAT Education is committed to ensuring professional education standards are upheld in industries. IAT Education is the endpoint assessment organisation for multiple apprenticeships. In these programmes, the apprentice develops technical knowledge and real practical experience, along with the functional and personal skills required as an Animal Technician. These are acquired through a combination of learning in the workplace, formal off-the-job training and the opportunity to practise and embed new skills in a real-work context. An apprenticeship is a paid job where the employee learns and gains valuable workplace Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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88Animal Technology and Welfare August 2020experience. Alongside on-the-job training, apprentices spend at least 20% of their working hours off-the-job training. Apprentices must demonstrate competency in 3 areas throughout their apprenticeships: knowledge (facts and information), skills (the ability to do a task well) and behaviours (the way in which a person acts in response to a particular situation or stimulus, particularly with respect to others). Areas of competency include ensuring animals are provided with the appropriate amount and type of food; have access to clean, fresh water; are provided with species-appropriate enrichment activities for mental stimulation; and are housed in the correct environment. In addition, apprentices may be required to check an animal’s health and take the appropriate action depending on the situation, manage breeding colonies and maintain strict records as required under the Animals (Scientiﬁc Procedures) Act 1986 (ASPA). With good training, apprentices can ensure good welfare for the animals they look after.The IAT in consultation with CLAST has also developed a wide range of optional smaller higher education (HE) units that not only provide a route to ultimately attain a Level 6 qualiﬁcation or ﬁrst degree and IAT Fellowship but also lend themselves to continuing professional development (CPD). An example of this is the partnership between CLAST and the Advance Training Centre at MRC Harwell, which provides high-quality courses in their purpose built, state-of-the-art training facility. Individual training units are available on any different topics and fees will vary depending on the size, content and modes of delivery. More information can be found at https://clast.org.uk/ In this presentation, Tina noted the beneﬁts of some of these courses not just for the learner and for rodent welfare but also for the employer. Some of these include: • increased staff loyalty and sense of being valued by the company• greater staff retention• knowledge that staff are experts in Animal Welfare• maintaining a Culture of CareThe wide range of modules meet CPD needs and can be incorporated into staff appraisals. With access to these training opportunities, staff members can develop conﬁdent public speaking and presentation skills, can prioritise and manage their time effectively and develop scientiﬁc writing skills for use in the company. Take-home messages and action points– Speak to your manager about completing some additional training or CPD courses in an area where you feel you could beneﬁt from developing new skills. Visit https://clast.org.uk/ to see which courses may be of interest.The psychological contract of Animal Technicians as ‘dirty workers’ in the biomedical research industryHaley DanielsThe psychological contract is fundamental to the employment relationship. It can be argued that Animal Technicians in the biomedical research industry experience certain challenges associated with the dirty aspects of the role, which may potentially lead to a disruption or violation of the psychological contract. This disruption ultimately impacts on the employment relationship (the relationship between employee and employer). In this presentation, Haley provided an overview of her journey in this industry and her PhD research to date: an exploration of the psychological contract of Animal Technicians and how the dirtier tasks associated with the role they conduct inﬂuence the state of the psychological contract. Dirty work involves physical, moral, or emotional taint. Some examples of professions considered to involve this include abattoir workers, sex workers, street workers, criminal lawyers, animal academics and researchers. These roles may be stigmatised, or even perceived as having a low status, which can in turn lead to social isolation and workers becoming disengaged and developing feelings of shame, guilt, fear and anger.A psychological contract refers to a fair day’s work for a fair day’s pay. It is intangible and outside a written contract but it includes a mutual agreement between employer and employee of reciprocity and the expectation of loyalty and being treated well. This agreement governs the relationship between the employee/employer and may be transactional and/or relational in nature. The psychological contract begins during recruitment and can be disrupted, repaired, or renegotiated during its lifetime. Haley’s research aims to explore how inputs, processes, and policies in the workplace can impact attitudes and behaviours. These can have signiﬁcant consequences in the context of an animal facility in which the Animal Technician’s role is already associated with critical pressures. These themes were explored by conducting interviews with 60 animal research professionals (technicians and managers).The 5 obligations most important to individuals at both employee (Animal Technician) and employer (manager) level were trust, job security, personal safety, salary and fairness. Interviews further delved into whether these obligations were being met. Job security was viewed as the most important expectation or promise and was high (during COVID). Trust and honesty were good but could be improved. However fairness, loyalty and consultation were thought to be poor and obligations to provide emotional support and physical safety were not met at all. Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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89August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareParticipants were asked about the way they viewed their psychological contract: whether it was relational (i.e. based on loyalty, trust and long-term goals) or transactional (i.e. based on short-term returns and beneﬁts, such as earning a salary and training before moving on to another post). For the majority of participants (77%), the psychological contract was relational and they viewed animal research as their long-term career, with some participants stating: “I am still here, and I am still passionate about working with animals.” “This is my life; I am committed to my team and the animals.” “I cannot imagine doing anything else now.”However others had mixed feelings associated with their work, which they viewed through the lens of a transactional psychological contract. Some participants are quoted here: “I could not stay in the role as an Animal Technician long term, I need more than this, it is crap work for a crap salary.” “I am leaving as I can not handle killing animals and I wanted to leave for 13 years. I do like looking after the animals and I have worked here now for a long time and it is hard. I recently worked 19 days in a row without a day off, we have to work 365 days a year including Christmas and bank holidays and it is too much.”Finally, interviewees were asked about potential stigma associated with the work of an Animal Technician and whether they considered this profession to be “dirty work”. The overwhelming majority of participants (95%) felt that stigma still existed around the role and 80% viewed their job as dirty work. Furthermore, 46 of 60 interviewees felt that the psychological contract had been breached in their workplace, although 74% of them wanted to remain in the industry. The reasons for this included being committed to the animals in their care, perceived beneﬁts to science, and commitment to their team and colleagues.Haley’s work has highlighted some of the issues facing staff working with animals in research institutions regarding the psychological contract. It has also shown that many Animal Technicians exist in a liminal state in which the psychological contract has been broken but is not being repaired, leaving people stuck in their institutions with no clear path to renegotiation or exit. For positive changes to occur, changes are needed at the individual, organisational and societal level to increase the visibility of Animal Technicians and acceptance of their work.Take-home messages and action points– Speak to your colleagues and/or manager if you ever feel that you are struggling with the emotional labour associated with your work.– Visit the North American 3Rs Collaborative website for resources, webinars and tools to cope with compassion fatigue and the emotional burden of working as an Animal Technologist.Replacing sentinel rodents with environmental health monitoring: why and howKerith LuchinsResearch institutions are increasingly replacing their soiled bedding sentinel rodents with environmental health monitoring (EHM). This switch can facilitate full animal replacement, more accurate results and even reduce programme costs. However concerns remain about the strength of the scientiﬁc evidence behind this practice and options for racks that have cage-level ﬁltration. In this presentation, Kerith discussed results of a systematic literature review, common challenges and solutions and resources for practical implementation of this replacement at their facility.In 2016, a systematic review6 evaluating the efﬁcacy of soiled bedding sentinels was published. This review highlighted that, as of 2016, only 15 articles, conference presentations and posters had investigated the efﬁcacy of soiled bedding sentinels and data available concluded that this practice was only effective in detecting 5 pathogens. Another systematic review was undertaken in 2022 by Kerith and the North American 3Rs Collaborative, in which 33 papers were included. EHM was shown to be superior to soiled bedding sentinels. It detected pathogens more often, regardless of the sampling method or pathogen type, and was highly effective in detecting 22 pathogens, including viruses, bacteria, fungus and parasites. There is a strong evidence base supporting the superiority of EHM over sentinels and it is therefore a scientiﬁc and ethical priority to replace the use of sentinels with EHM.Next, Kerith and colleagues ran a survey7 between 2021 and 2023 to explore the prevalence of EHM, factors inﬂuencing its use and possible barriers. Results showed that although many respondents still used a combination of EHM and sentinels, the exclusive use of EHM had increased from 7% in 2021 to 48% in 2023. The main barrier to implementing EHM reported was the type of caging and rack design used. This is because one EHM system (exhaust dust testing) can only be used in conjunction with an individually ventilated cage (IVC) system that has rack level ﬁltration. However sentinel-free soiled bedding is another form of EHM Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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90Animal Technology and Welfare August 2020that can be used with any type of caging, providing a suitable alternative for all facilities. Another important factor was the cost associated with the implementation of EHM. However as EHM reduces the numbers of animals needed, costs associated with ordering, shipping, and keeping sentinel animals are avoided completely. Kerith and her team conducted a study8 at the University of Chicago showing that the total annual cost of using EHM was 26% lower than that of using sentinels. The third most signiﬁcant perceived barrier (16%) was the accuracy of EHM in detecting pathogens and participants mentioned concern over a lack of published data on EHM accuracy and reliability. Given the rapid increase in data and novel testing methods in the past few years, respondents listing accuracy as a barrier could be operating on an outdated view of the methods that encompass EHM. It is clear that recent publications have not become mainstream knowledge and Kerith hopes that the team’s latest systematic literature review, due to be published soon, will change that. Accuracy was actually perceived as an advantage of EHM by 37% of respondents. Overall, the number of participants reporting that there are no barriers had increased from 18% in 2021 to 34% in 2023, indicating that many barriers were gradually being removed. It is also worth noting that the use of sentinels involves an additional emotional burden associated with the need to euthanise animals purely for health testing, which is unnecessary and can be removed when switching to EHM.Take-home messages and action pointsFor those who are interested in switching to EHM, Kerith has included some tips here:1. Determine your caging type, which dictates your options.– If you have IVC racks that exhaust at rack level (i.e. Allentown Inc, TecniplastTM) you can use exhaust dust testing.– Regardless of your caging/rack type, you can use sentinel-free soiled bedding.2. Reach out to your diagnostic vendors to determine their EHM preferences.3. Gather an internal advocacy group and get buy-in.– Analyse cost savings via cost analysis tool on our resource hub.– Use our editable slide deck to convince stakeholders.– Evaluate if current sanitation methods are acceptable.4. Decide how to make the transition.– Consider a hybrid approach or change 100% to EHM.– See case studies on how two institutions converted on resource hub.5. Develop materials and timeline for implementing EHM.– Our SOPs can help you start.There are more resources available on the North American 3Rs Collaborative website, including steps to follow when making the switch and successful case studies. In conclusion, EHM presents many advantages: it is 3Rs compliant, increases accuracy, reduces labour and costs, and reduces emotional fatigue.References1 Hurst, J.L. & West, R.S. (2010). Taming anxiety in laboratory mice. Nat. Methods 7, 825–826.2 Neville, V. et al. (2023). A mapping review of reﬁnements to laboratory rat housing and husbandry. Lab Anim. 52, 63–74.3 Percie du Sert, N. et al. (2020). Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. 18, e3000411.4 Neville, V., Hunter, K., Benato, L., Mendl, M. & Paul, E.S. (2022). Developing guidelines for pet rat housing through expert consultation. Vet. Rec. 192, e1839.5 Stuart, S.A. & Robinson, E.S.J. (2015). Reducing the stress of drug administration: implications for the 3Rs. Sci. Rep. 5, 14288.6 de Bruin, W.C.C., van de Ven, E.M.E. & Hooijmans, C.R. (2016). Efﬁcacy of Soiled Bedding Transfer for Transmission of Mouse and Rat Infections to Sentinels: A Systematic Review. PLoS One 11, e0158410.7 Luchins, K.R. et al. (2023). A Cross-sectional Survey on Rodent Environmental Health Monitoring Practices: Benchmarking, Associations, and Barriers. J. Am. Assoc. Lab. Anim. Sci. 62, 64–73.8 Luchins, K.R., Bowers, C.J., Mailhiot, D., Theriault, B.R. & Langan, G.P. (2020). Cost Comparison of Rodent Soiled Bedding Sentinel and Exhaust Air Dust Health-Monitoring Programs. J. Am. Assoc. Lab. Anim. Sci. 59, 508–511.Report of the 2023 RSPCA/UFAW/IAT Rodent Welfare Group Meeting

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91August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAbstractThe 3Hs Initiative is a concept that arose from the research and management of laboratory mice and rats of Professor Emma Robinson’s laboratory at the University of Bristol. Our research is predominantly concerned with the treatment of psychiatric disorders and particularly symptoms of depression and apathy. Due to the nature of our work, it is important that we are conﬁdent that the animals we are working with are not being put into a negative affective state by their environment or as an unintended consequence of our interactions with them. Our approach focussed on methods which provide the optimal control populations and ensure we can quantify speciﬁc and relevant behavioural changes arising for experimental manipulations. As our research programme evolved, we have become more aware of the impact of stress caused to our animals by routine management approaches and physical restraint during procedures. We have looked at our housing, handling and habituation approaches to identify areas to make improvements. This is critical for our research outputs and has the beneﬁt of reducing the cumulative suffering the animals experience by reducing stress and negative affective experiences associated with their day-to-day lives. We identiﬁed some key changes which can be made which beneﬁt both Animal Welfare and our scientiﬁc objectives and experimental outcomes. IntroductionOne of the principal areas of interest for our research is emotional disorders and investigating the mechanisms of action of antidepressants.1,2 Until recently, objective methods to quantify the affective state of non-human animals has been limited. Methods such as measuring stress hormones or overt signs of distress lack both sensitivity and speciﬁcity.3 Our approach builds from human experimental medicine and uses tasks designed to quantify affective state using cognitive tasks to quantify affective biases. Affective biases are observed when the emotional state of the subject biases their behaviour and affective biases have been shown to inﬂuence a number of different cognitive domains including attention, learning and memory and decision-making.4 Just as is seen in humans, studies in non-human animals have found that similar affective biases can be quantiﬁed in rats and mice.5 Using these methods we have been able to assess the reﬁnements we have put in place and show that they are reducing the stress caused to the animals and having a positive impact on their affective state. At least some aspects of the techniques and protocols that we use can be easily implemented by other researchers and animal care staff throughout the industry. To disseminate our work more widely, we have launched a website: www.3Hs-initiative.co.uk . Here we provide examples of our work, explain the rationale behind our methods and the research we have undertaken for validation. We provide guidance documents and videos so that others can replicate our methods at their facilities. The initiative is an ongoing project, and we plan to continue to develop our understanding of how to manage laboratory animals for scientiﬁc procedures while also reducing their cumulative suffering.HousingWhen considering how to house laboratory animals there is a need to balance scientiﬁc and husbandry requirements with Animal Welfare. Ideal housing from a welfare perspective would allow the animals to express their full range of natural behaviours, meet their needs The 3Hs Initiative – housing, handling, habituation JULIA BARTLETT, JUSTYNA HINCHCLIFFE, MEGAN JACKSON and EMMA ROBINSONUniversity of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TDCorrespondence: 3hsinitiative@gmail.com August 2024 Animal Technology and Welfare

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92Animal Technology and Welfare August 2020in terms of shelter and provide optimal social interaction opportunities. However we also must consider that all animals need to be welfare checked daily, soiled cages need changing on a regular basis and we need to be able to easily access animals for experimental procedures.Basic shoebox cages and independently ventilated cages (IVCs) have beneﬁ ts in terms of space requirements, reducing cross-contamination and making Animal Welfarechecks and cage changing simpler but they are not idealfor allowing animals to express their natural behaviours. Adding cage furniture and enrichment items increases the available space in the cage as well as providing areas to hide or create nests. The right types of enrichment can lead to signiﬁ cantly lower stress levels in animals.6,7However the addition of too many items can make it difﬁ cult to perform Animal Welfare checks and may leadto competition for resources. When deciding on the bestenrichment to provide, the principal factor is identifyingitems that provide the most opportunity for enhancingspace and supporting the expression of naturalbehaviours. For example, increasing the surface areawithin the cage and incorporating nesting materials can enhance the home cage environment. We also largelyoverlook the fact that mice and rats explore their surroundings using touch and olfaction and so providing greater variety in terms of textures and scents can providegreater value.There are other factors that we must consider when selecting enrichment materials. In group-housed, malemice, aggression can be a problem and can be exacerbated by high value resources and additional monitoring after the introduction of novel enrichment items may be useful.Also, aged animals, post-surgery animals and animals with reduced mobility may require modiﬁ ed enrichment to enable interaction without risking injury.Using playpens (Figure 1) can be a good compromise between the needs of the animals and the welfare and scientiﬁ c requirements as the animals have access to a large area with lots of enrichment where they can run and explore for a set amount of time and they can be monitored throughout if required. Playpens have been additional monitoring after the introduction of novel enrichment items may be useful. Also, aged animals, post-surgery animals and animals with reduced mobility may require modified enrichment to enable interaction without risking injury. Using playpens (figure 1) can be a good compromise between the needs of the animals and the welfare and scientific requirements as the animals have access to a large area with lots of enrichment where they can run and explore for a set amount of time, and they can be monitored throughout if required. Playpens have been shown to be rewarding for rats and mice and they can also be used to provide supervised social interaction for rats which may have limited opportunity in their home cage.8,9. Figure 1 rat and mouse play pens A study conducted by our laboratory showed that when placed in a ball pit or a playpen, rats emitted a high number of 50kHz calls (figure 2) which are associated with positive events.8,10,11 These high numbers of calls were sustained over consecutive days which suggests that the Figure 1. Rat and mouse play pens.shown to be rewarding for rats and mice and they can also be used to provide supervised social interaction for rats which may have limited opportunity in their homecage.8,9A study conducted by our laboratory showed that when placed in a ball pit or a playpen, rats emitted a high number of 50kHz calls (Figure 2) which are associated with positive events.8,10,11 These high numbers of calls were sustained over consecutive days which suggests that the animals are not just reacting to the novelty of the playpen and that the positive effect does not decline with repeated exposure.animals are not just reacting to the novelty of the playpen and that the positive effect does not decline with repeated exposure. Figure 1 50kHz calls emitted by rats exposed to ball pit or playpen over four consecutive days.8 Figure 2. 50kHz calls emitted by rats exposed to ball pit or playpen over four consecutive days.In a follow-up study we found that when the animals wereinjected with a drug that induces a strong anxiogenic effect, if they were subsequently given access to a playpen, the effect of the drug was attenuated. This suggests that, The 3Hs Initiative – housing, handling, habituation

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93August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe 3Hs Initiative – housing, handling, habituationif the animals must undergo an aversive intervention, allowing them playpen time after the negative event could mitigate against the negative affective experience caused by the procedure.HabituationWhen we talk about habituation, our aim is to gradually introduce animals to human interaction, experimental equipment and procedures in a way that promotes positive rather than negative associations. This is especially important for handling as this is something that the animals will encounter often throughout their lives from husbandry to regulated procedures and can result in high levels of stress.12,13If we can reduce or remove the stress from these situations, we can have a signiﬁ cant impact on the cumulative stress that the animal experiences over its lifetime.14,15,16 Put simply, if an animal forms a positive rather than negative association with human contact, the cumulative impacts of routine care and procedures can beneﬁ t their welfare rather than add to their suffering. The beneﬁ ts of habituation may also be felt by the animal care staff and researchers as positive human-animal interactions may be linked to decreased compassion fatigue in laboratory animal workers and increased satisfaction in their work.17Examples are shown in Figure 3 of the protocols we use for mouse and rat handling habituation.The key things to note are that the ﬁ rst interactions are kept short. This reduces the chance that anything negative could happen during the interaction and reduces any stress the animal might experience from the novel situation. It is immediately followed by a reward to build the positive association. When progressing through the habituation protocol you should not move to a new stage before the animal is comfortable with the current one. Some animals and strains are more anxious than others, so it is important that you do not undo all your work building a positive association by jumping ahead before the animal is ready. It is much easier to build a positive association from scratch than it is to overcome a negative one. Keeping a record of overt measures of stress e.g. vocalisation, urination and faecal pellets may be useful to ensure the animal is not ﬁ nding the current stage aversive. A habituated animal should be showing very few, if any, of the overt signs listed above.Keep in mind that every time we interact with our animals, they are learning something from us and the ﬁ rst interaction they have might be the most important. If they have a negative experience the ﬁ rst time they meet you, then the next time you approach them they will be anticipating another negative experience and they will avoid you. This leads to them trying to evade handling which will increase their stress and increase Figure 3. Habituation protocols.Mouse Habituation Rat HabituationDay 1 – Open cage and remove all furnishings accept a familiar tube. Keeping the tube within thecage, lift the mouse in the tube and gently encourage to step over your hand back into the cage. Repeat a few times and for each animal in the cage and then place reward into the cage and replace all furniture and lid.Day 1 – Cage lid off, furniture removed, remaining within the conﬁ nes of the cage, pick up the rat around the shoulder then immediately put back in cage. Repeat with each cage mate then give reward in home cage .Day 2 – Repeat day one but try to have the mouse pause on the hand with gentle cupping each time. With cup handling, the mouse should not feel restrained and should be released if it starts trying to evade handling. For animals which do not require physical handling for the procedures repeat tube handling habituation days 3 to 5.Day 2 – Cage lid off, furniture removed, pick up and transfer to a travel box containing rewards, move all rats from the cage to the travel box, give additional rewards then once consumed, pick up and return to cage, give reward in cage .Day 3 – For animals requiring physical restraintfor procedures progress to cup handling. Repeat day 2 but cup restrain the mouse for a brief period before release. Gradually increase the time restrained as the animal’s tolerance increases.Day 3 – Open cage lid and wait for rat to approach then pick up, transfer to travel box containing reward, move all rats from the cage to the travel box, give additional rewards then once consumed, pick up and return to cage, give reward in cage .Day 4-5 – repeat day 3 increasing time animal is cup restrained .Individual and strain speciﬁ c differences will impact on the number of sessions required for the animals to accept each stage and it is important not to progress to a new stage before the animal is comfortable with the current one.Day 4-5 – Repeat day 3 and start introducing dosing positions and taking palatable solutions from a syringe. If using palatable dose training, this can also serve as the reward.Habituation to sitting on a piece of Vetbed can also be useful for certain procedures e.g. subcutaneous injections and administration or withdrawal of substances through surgically implanted devices.

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94Animal Technology and Welfare August 2020their negative association with you. By starting with a positive experience paired with a reward you will make every subsequent interaction easier as they are anticipating the reward.Once the animal is habituated to being handled you can start to introduce holding them in dosing positions and even sham injections with an empty syringe without a needle to get them used to the whole process before beginning a dosing study. If you wish to use a Vetbed as part of your handling or dosing procedures, then providing rewards whilst the animal is sitting on the Vetbed will help the animals form a positive association Figure 4. Rewards.Reward OptionsReward Primary Macronutrient GroupFeaturesSunﬂower seeds (whole or crushed)Fat Can be provided irradiated, autoclavable, contaminant screenedYogurt Drops Carbohydrate/FatCan be provided irradiated, autoclavable, contaminant screenedForage Mixes Carbohydrate Can be provided irradiated, autoclavable, contaminant screenedCereal Grains Carbohydrate Can be provided irradiated, autoclavable, contaminant screened, low calorieDried Fruits/VegetablesCarbohydrate Can be provided irradiated, autoclavable, contaminant screenedDried MealwormsProtein Can be provided irradiated, autoclavable, contaminant screenedPrecision Reward Pellets20% Sucrose enriched food pelletCan be provided irradiated, autoclavable, contaminant screenedwith the context which can mitigate the effects of subsequent mildly aversive procedures such as subcutaneous injection or procedures in animals with head mounted devices.Even if time and management systems limit your ability to use the full handling habituation protocol, providing animals with a food reward after each human interaction can mitigate their negative experiences and lead them to anticipate human contact with a positive outcome.There is a wide variety of options for food reward available from commercial suppliers. All can be provided irradiated or certiﬁed contaminant free and they can be autoclaved for further biosecurity. Depending on the dietary requirements of your animal and study, there will be a suitable reward to meet your needs.HandlingOnce you have built up positive associations with the handler, the animals should be quite easy to handle for basic husbandry and health checks but there are still going to be some occasions when we need to physically restrain the animals for procedures. The instinct a lot of the time is to restrain the animal as securely as possible as this is perceived to be better for the safety of the animal and the handler and the accuracy of the procedure but physical restraint is highly aversive for animals and is widely used to model depression.18,19,20 The negative impacts of restraint do not seem to decrease with repeated exposure although animals may exhibit passive coping strategies such as learned helplessness when overt signs of distress decline but the affective experience does not.21 It has even been shown that, when comparing animals that were restrained and underwent an intraperitoneal (I.P) injection with animals that were just restrained, the stress response was the same across both groups.22 This suggests that the primary source of stress during this procedure was the restraint and not the injection. The stress caused to the animal will have an impact on the results of your study as well as the welfare of the animal handler.23 Instead, maybe we should be asking, what is the least amount of restraint we can use to achieve the outcome we need, or can we even remove the need for restraint altogether?Oral dosing Oral dosing of rats and mice is commonly conducted using an oesophageal cannula. This procedure is distressing for the animals requiring physical restraint.24 There are also risks of adverse events including incorrect placement and tracheal dosing and oesophageal The 3Hs Initiative – housing, handling, habituation

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95August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe 3Hs Initiative – housing, handling, habituationtrauma.25,26 This method also requires the experimenter to have a high level of skill and may result in bite or scratch injuries and stress. An alternative approach is to use voluntary ingestion of test substances in palatable solutions. Although this approach has previously been reported by several research groups it is still not widely used and it is not clear if this is due to a lack of awareness or whether researchers have encountered problems using this approach.27,28,29,30 Our own experience has been that rats and mice readily take palatable solutions from a syringe but can quickly form negative associations with ingestion of some drug solutions which we hypothesise arises if drugs have a bitter taste or from conditioned aversion i.e. where any aversive effects of the drug become associated with the palatable solution in which it was administered. To reduce the potential for this to develop, we have developed a protocol to increase the reliability of this dosing method. The animals are introduced to the palatable substance a week before the start of the dosing study so that they can become familiar with the novel taste and drinking from a syringe. The palatable solution can even be used as a reward during their initial habituation.31 The animals develop a positive association with the syringe and will quickly approach when it is presented. Once the animals are reliably drinking the palatable solution from the syringe, the required drug can be added and the animals should readily consume the drug without the need for gavage. By forming the strong positive association with the palatable solution in the syringe before combining it with a drug we reduce the likelihood that the animals will make a connection between any aversive effects of the drug and the palatable solution thus decreasing the risk of conditioned aversion. We can further mitigate against this by providing a second syringe of palatable solution without drug on dosing days at a later timepoint. By doing this, the animals will have drunk the palatable solution twice that day but only felt the effects of the drug on one occasion so again, we are reducing the likelihood of an association forming between the aversive effects and the solution.Using this approach, we have administered a wide range of psychiatric drugs without issue31 (Figure 5). This reﬁ nedmethod takes what would previously have been a stressful and risky procedure and replaces it with a positive experience for both the animal and the person carrying out the procedure. Although the approach will not be compatible with all test substances, this method offers both welfare and scientiﬁ c beneﬁ ts and could reduce variability between subjects and improve the quality and reproducibility of scientiﬁ c studies. We are continuing to reﬁ ne this dosing method by exploring methods of masking the bitter taste of certain drugs to increase the range of drugs that can be reliably dosed by voluntary ingestion.Dosing by injectionThere will be occasions where we still need to inject our animals and in these cases we try to use very low restraint methods. We have modiﬁ ed our handling techniques for rats and mice to eliminate the need for scrufﬁ ng in rats or tail handling in mice prior to injecting the animal. We have been able to use objective measures of affective state and the stress response to show that these methods offer welfare beneﬁ ts.32,33 Importantly, these techniques have been taught to and used by researchers with a range of prior experience levels and all have demonstrated competency within a short period of time. These methods offer both welfare and scientiﬁ c beneﬁ ts and could reduce variability between subjects and improve the quality and reproducibility of scientiﬁ c studies.IP injection in ratsThe conventional handling methods for I.P. dosing all involve the rat being tightly held with its abdomen stretched out.34reduce the likelihood that the animals will make a connection between any aversive effects of the drug and the palatable solution, thus decreasing the risk of conditioned aversion. We can further mitigate against this by providing a second syringe of palatable solution without drug on dosing days at a later timepoint. By doing this, the animals willhave drunk the palatable solution twice that day but only felt the effects of the drug on one occasion so, again, we are reducing the likelihood of an association forming between the aversive effects and the solution.Using this approach, we have administered a wide range of psychiatric drugs without issue (figure 5). This refined method takes what would previously have been a stressful and risky procedure and replaces it with a positive experience for both the animal and the person carrying out the procedure. Although the approach will not be compatible with all test substances, this method offers both welfare and scientific benefits and could reduce variability between subjects and improve the quality and reproducibility of scientific studies. We are continuing to refine this dosing method by exploring methods of masking the bitter taste of certain drugs to increase the range of drugs that can be reliably dosed by voluntary ingestion.Figure 5 mice and rats voluntarily drinking palatable solutions from a syringe31.Figure 5. Mice and rats voluntarily drinking palatable solutions from a syringe.

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96Animal Technology and Welfare August 2020To avoid the stress caused by this ﬁ rm restraint we use a method where the animal is held lightly, in a relaxed position, with a rounded abdomen to decrease the pain of the injection.31 The rats are habituated to being held in this position and sham injected with an empty syringe with no needle attached. By getting them used to the hold and pairing it with reward, the rats have a positive association with being held in this way and this leads to signiﬁ cantly lower overt signs of stress as well as physiological and behavioural indicators of stress and aversion35 (Figure 7). Figure 6 conventional restraint methods for IP injections in rats34 To avoid the stress caused by this firm restraint we use a method where the animal is held lightly, in a relaxed position, with a rounded abdomen to decrease the pain of the injection. The rats are habituated to being held in this position and sham injected with an empty syringe with no needle attached. By getting them used to the hold and pairing it with reward, the rats have a positive association with being held in this way and this leads to significantly lower overt signs of stress as well as physiological and behavioural indicators of stress and aversion.35 (figure 7). Figure 6. Conventional restraint methods for IP injections in rats. Figure 7 refined handling method for IP injection in rats31 Subcutaneous dosing in rats Once a rat is habituated to being handled, subcutaneous (S.C.) dosing can be carried out with almost no restraint at all. The rat can be habituated to being held against the handler’s body, on the handler’s lap or on a piece of Vetbed. Once the animal is comfortable sitting in the required position you can introduce sham injections with an empty syringe with no needle attached and go through the motions of tenting the skin and injecting in the appropriate place. By pairing this scenario with positive rewards, when the actual injection occurs, the rat will easily tolerate the transitory pain of the needle insertion without struggling or attempting to evade the needle. This results in a virtually zero restraint procedure. Figure 7. Reﬁ ned handling method for IP injection in rats.Subcutaneous dosing in ratsOnce a rat is habituated to being handled, subcutaneous (S.C.) dosing can be carried out with almost no restraint at all. The rat can be habituated to being held against the handler’s body, on the handler’s lap or on a piece of Vetbed.31 (Figure 8) Once the animal is comfortable sitting in the required position you can introduce shaminjections with an empty syringe with no needle attachedand go through the motions of tenting the skin and injecting in the appropriate place. By pairing this scenariowith positive rewards, when the actual injection occurs, The 3Hs Initiative – housing, handling, habituation

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97August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe 3Hs Initiative – housing, handling, habituationthe rat will easily tolerate the transitory pain of the needle insertion without struggling or attempting to evade theneedle. This results in a virtually zero restraint procedure.Alternative scrufﬁ ng methods in miceIt has been very well established that tail handling is aversive for mice and that experimental outcomes can be improved by using cup or tunnel handling instead of tail handling.36 The majority of United Kingdom (UK) establishments have moved away from tail handling for their husbandry and basic handling but when it comes to procedures, common practice still involves using the base of the tail to facilitate scrufﬁ ng the mouse. This can be replaced by placing the mouse onto your arm or a piece of Vetbed31 (Figure 9), cupping a hand on top of them, letting them squeeze their head out between your thumb and foreﬁ nger and then scrufﬁ ng them from there. This technique produces the same level of control as the conventional method but causes signiﬁ cantly lower overt signs of stress such as struggling, vocalisation and aversion on release.33 (Figure 10) Figure 8 SC dosing of rats.31 Alternative scruffing methods in mice It has been very well established that tail handling is aversive for mice and that experimental outcomes can be improved by using cup or tunnel handling instead of tail handling.36 The majority of United Kingdom (U.K.) establishments have moved away from tail handling for their husbandry and basic handling but, when it comes to procedures, common practice still involves using the base of the tail to facilitate scruffing the mouse. This can be replaced by placing the mouse onto your arm or a piece of Vetbed (figure 9), cupping a hand on top of them, letting them squeeze their head out between your thumb and forefinger, and then scruffing them from there. This technique produces the same level of control as the conventional Figure 8. SC dosing of rats.method but causes significantly lower overt signs of stress such as, struggling, vocalisation and aversion on release.33 Figure 9 refined scruffing methods in mice31 Figure 9. Reﬁ ned scrufﬁ ng methods in mice.method but causes significantly lower overt signs of stress such as, struggling, vocalisation and aversion on release.33 Figure 9 refined scruffing methods in mice31 Figure 10. Overt measures of stress showing signiﬁ cantly decreased struggling (p<0.0001), vocalisation (p<0.0121),and aversion on release (p<0.0001).ConclusionBy implementing the 3Hs you do need to put in a little bit more work at the beginning of your study but it will make everything that you do throughout the study easier and possibly save time overall. This will prevent stress to you and your animals. This is so important when we think about the cumulative suffering that an animal will experience throughout its lifetime. Moreover, the effects stress to the animals and to you will have on the outcomes of your experiments.

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98Animal Technology and Welfare August 2020Whilst the examples shown in this article and on our website are taken speciﬁcally from the laboratory environment that we work in; the principles of the 3Hs can be translated into any other laboratory environment. Animals housed in higher biosecurity containment such as IVCs and isolators will still beneﬁt from positive reinforcement after human interaction and all animals will beneﬁt from habituation protocols that introduce new interactions with objects or personnel in a gradual way that establishes positive associations.If you do not have the capability to provide a large playpen due to space or biosecurity concerns then you could provide a smaller space with enhanced enrichment that would not be practical within the home cage but could provide greater opportunities for natural behaviour such as digging, foraging, novel smells and textures. If biosecurity or cross contamination are a concern, you could consider using items that can be easily cleaned between animals or have separate labelled items for different groups to minimise these risks. If there are methods already in place in your facilities for decontamination e.g. fogging chambers, autoclaves, ethylene oxide, it is likely that you will have a suitable method already available for any novel enrichment or positive reinforcement aid you wish to use. By creating the 3Hs Initiative website, we hope to provide technicians and researchers throughout the industry with evidence-based protocols that can be easily implemented in their own facilities. We have videos and images of all the techniques mentioned as well as downloadable guidance documents. We can improve our animals’ lives and our experimental outcomes by enhancing housing, reﬁning handling and introducing habituation protocols.References1 Robinson, E.S.J. Translational new approaches for investigating mood disorders in rodents and what they may reveal about the underlying neurobiology of major depressive disorder. Philosophical Transactions of the Royal Society B: Biological Sciences [Internet]. 2018 Mar 19 [cited 2021 May 18]; 373(1742). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790833/ 2 Kamenish, K., Emma. Neuropsychological Effects of Antidepressants: Translational Studies. Current topics in behavioral neurosciences. 2023 Jan 1;3 Berton, O., Hahn, C.G., Thase, M.E. Are We Getting Closer to Valid Translational Models for Major Depression? Science. 2012 Oct 4;338(6103):75–9.4 Rockm, P.L., Roiser, J.P., Riedel, W.J., Blackwell, A.D. Cognitive impairment in depression: a systematic review and meta-analysis. Psychological Medicine [Internet]. 2013 Oct 29;44(10):2029–40. Available from: https://www.cambridge.org/core/journals/psychological-medicine/article/cognitive-impairment-in-depression-a-systematic-review-and-metaanalysis/ 0EE176727AC50D44326A3D8DF2AB88A7 5 Robinson, E.S., Roiser, J.P. Robinson ES, Roiser JP. Affective Biases in Humans and Animals. Current Topics in Behavioral Neurosciences. 2016;28:263-86. doi: 10.1007/7854_20105_5011. PMID: 27660073.6 Belz, E.E., Kennell, J.S., Czambel, R. Kenneth., Rubin, R.T., Rhodes, M.E. Environmental enrichment lowers stress-responsive hormones in singly housed male and female rats. Pharmacology Biochemistry and Behavior. 2003 Dec;76(3-4):481–6.7 Gurfein, B.T., Stamm, A.W., Bacchetti, P., Dallman, M.F., Nadkarni, N.A., Milush, J.M., et al. The Calm Mouse: An Animal Model of Stress Reduction. Molecular Medicine. 2012 Feb 29;18(4):606–17.8 Hinchcliffe, J.K., Jackson, M.G., Robinson, E.S. The use of ball pits and playpens in laboratory Lister Hooded male rats induces ultrasonic vocalisations indicating a more positive affective state and can reduce the welfare impacts of aversive procedures. Laboratory Animals. 2022 Aug;56(4):370-379. doi: 10.1177/00236772211065920. Epub 2022 Jan 13. PMID: 35023405; PMCID: PMC9388951.9 Ratuski, A.S., Makowska, I.J., Dvorack, K.R., Weary, D.M. Using approach latency and anticipatory behaviour to assess whether voluntary playpen access is rewarding to laboratory mice. Scientiﬁc Reports. 2021 Sep 21;11(1).10 Burgdorf, J., Kroes, R.A., Moskal, J.R., Pfaus, J.G., Brudzynski, S.M., Panksepp, J. Ultrasonic vocalizations of rats (Rattus norvegicus) during mating, play, and aggression: Behavioral concomitants, relationship to reward, and self-administration of playback. Journal of Comparative Psychology. 2008; 122(4):357–67.11 Rygula, R., Pluta, H., Popik, P. Laughing Rats Are Optimistic. Chapouthier G, editor. PLoS ONE. 2012 Dec 26;7(12):e51959.12 Balcombe, J.P., Barnard, N.D., Sandusky, C. Laboratory routines cause animal stress. Contemparory Topics in Laboratory Animal Science. 2004;43(6):42-51.13 Kylie, J., Cooper, D.M., Kurpinski, J.K., Chase, F.T., Muzyka, M.D., Plachta, T.C. Evaluation of Potential Low-stress Handling Methods in Crl:CDSD Rats (Rattus norvegicus). Journal of the American Association for Laboratory Animal Science. 2024 Jan 1;63(1): 10–9.14 Bigelow, L.J., Pope, E.K., MacDonald, D.S., Rock, J.E., Bernard, P.B. Getting a handle on rat familiarization: The impact of handling protocols on classic tests of stress in Rattus norvegicus. Laboratory Animals. 2023 Jun;57(3):259-269. doi: 10.1177/00236772221142687. Epub 2023 Jan 5. PMID: 36604974; PMCID: PMC10288167.15 Costa, R., Tamascia, M.L., Nogueira, M.D., Casarini, D.E., Marcondes, F.K. Handling of adolescent rats improves learning and memory and decreases anxiety. PubMed. 2012 Jan 1;16 Marcotte, M., Bernardo, A., Linga, N., Pérez-Romero, C.A., Guillou, J.L., Sibille, E., et al. Handling Techniques to Reduce Stress in Mice. Journal of Visualized Experiments. 2021 Sep 25;(175).The 3Hs Initiative – housing, handling, habituation

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99August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe 3Hs Initiative – housing, handling, habituation17 LaFollette, M.R., Riley, M.C., Cloutier, S., Brady, C.M., O’Haire, M.E., Gaskill, B.N. Laboratory Animal Welfare Meets Human Welfare: A Cross-Sectional Study of Professional Quality of Life, Including Compassion Fatigue in Laboratory Animal Personnel. Frontiers in Veterinary Science. 2020 Mar 5;7.18 Armario, A., Vallès, A., Dal-Zotto, S., Márquez, C., Belda, X. A Single Exposure to Severe Stressors Causes Long-term Desensitisation of the Physiological Response to the Homotypic Stressor. Stress. 2004 Sep;7(3):157–72.19 Thaddeus, W.W., Pace, Spencer, R.L. Disruption of mineralocorticoid receptor function increases corticosterone responding to a mild, but not moderate, psychological stressor. American Journal of Physiology-endocrinology and Metabolism. 2005 Jun 1;288(6):E1082–8.20 Płaźnik, A., Stefański, R., Kostowski, W. Restraint stress-induced changes in saccharin preference: The effect of antideprressive treatment and diazepam. Pharmacology Biochemistry and Behavior. 1989 Aug; 33(4):755–9.21 Sikora, M., Konopelski, P., Pham, K., Wyczalkowska- Tomasik, A., Ufnal, M. Repeated restraint stress produces acute and chronic changes in hemodynamic parameters in rats. Stress. 2016 Oct 20; 19(6): 621–9.22 Davis, J.N., Courtney, C.L., Superak, H., Taylor, D.K. Behavioral, clinical and pathological effects of multiple daily intraperitoneal injections on female mice. Lab Animal. 2014 Mar 20;43(4):131–9.23 Kaźmierowska, A.M., Kostecki, M., Szczepanik, M., Tomasz Nikolaev, Hamed, A., Michałowski, J.M., et al. Rats respond to aversive emotional arousal of human handlers with the activation of the basolateral and central amygdala. Proceedings of the National Academy of Sciences of the United States of America. 2023 Nov 7;120(46).24 Administration of Substances Archives – Research Animal Training [Internet]. researchanimaltraining.com. [cited 2024 Apr 15]. Available from: https://research animaltraining.com/article-categories/administration- of-substances 25 Arantes-Rodrigues, R., Henriques, A., Pinto-Leite, R., Faustino-Rocha, A., Pinho-Oliveira, J., Teixeira-Guedes, C., et al. The effects of repeated oral gavage on the health of male CD-1 mice. Lab Animal. 2012 May; 41(5):129–34.26 Germann, P.G., Ockert, D. Granulomatous inﬂammation of the oropharyngeal cavity as a possible cause for unexpected high mortality in a Fischer 344 rat carcinogenicity study. PubMed. 1994 Aug 1;44(4): 338–43.27 Abelson, K.S.P., Katja Kemp Jacobsen, Renée Sundbom, Kalliokoski, O., Hau, J. Voluntary ingestion of nut paste for administration of buprenorphine in rats and mice. Laboratory Animals. 2012 Oct 1;46(4): 349–51.28 Diogo, Ln., Faustino, Ra A., Sa P., Monteiro, Ec, Ai, S. Voluntary Oral Administration of Losartan in Rats. PubMed. 2015 Sep 1;54(5):549–56.29 Hocking, A.J., Elliot, D., Hua, J., Klebe, S. Administering Fixed Oral Doses of Curcumin to Rats through Voluntary Consumption. Journal of the American Association for Laboratory Animal Science. 2018 Sep 1;57(5):508–12.30 Kaźmierowska, A.M., Kostecki, M., Szczepanik, M., Tomasz Nikolaev, Hamed, A., Michałowski, J.M., et al. Rats respond to aversive emotional arousal of human handlers with the activation of the basolateral and central amygdala. Proceedings of the National Academy of Sciences of the United States of America. 2023 Nov 7;120(46).31 The 3Hs Initiative [Internet]. www.3hs-initiative.co.uk. [cited 2024 Apr 15]. Available from: https://www. 3hs-initiative.co.uk/the-3hs 32 Davies, J.R., Purawijaya, D.A., Bartlett, J.M., Robinson, E.S.J. Impact of Reﬁnements to Handling and Restraint Methods in Mice. Animals. 2022 Aug 24;12(17): 2173.33 Stuart, S.A., Butler, P., Munafò, M.R., Nutt, D.J., Robinson, E.S. A Translational Rodent Assay of Affective Biases in Depression and Antidepressant Therapy. Neuropsychopharmacology [Internet]. 2013 Aug 1 [cited 2021 Jan 27];38(9):1625–35. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717539/ 34 Turner, P.V., Brabb, T., Pekow, C., Vasbinder, M.A. Administration of substances to laboratory animals: routes of administration and factors to consider. Journal of the American Association for Laboratory Animal Science. 2011 Sep;50(5):600-13. 35 Stuart, S.A., Robinson, E.S.J. Reducing the stress of drug administration: implications for the 3Rs. Scientiﬁc Reports. 2015 Sep 23;5(1).36 Hurst, J.L., West, R.S. Taming anxiety in laboratory mice. Nature Methods. 2010 Sep 12;7(10):825–6.

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101August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2024 Animal Technology and WelfareAbstractThis study was conducted to evaluate the growth and health performance of Holstein Friesian calves (Bos taurus) supplemented with beneﬁcial microbes and their combinations at varying levels for 30 days from 1st September 2021 to 1st October 2021 in Poaka Farm, Tokoroa, New Zealand. A total of 48 female calves were randomly distributed in 8 treatments, having 3 replications with 2 calves per replication, following a factorial experiment in a completely randomised design.Factor A was the types of beneﬁcial microbes (BM): BM1- Lactobacillus subtilis, BM2- Biﬁdobacterium animalis, BM3- Lactic Acid Bacteria Serum, and BM4- Lactobacillus + Biﬁdus + LABS. Factor B was the levels of supplementation (LS): LS1- 10ml per calf/day and LS2- 20ml per calf/day. The parameters were: feed consumption, milk consumption, weight gained, average daily gain (ADG), feed conversion ratio (FCR), feed and milk cost, income, morbidity, survival, faecal colour, faecal odour, faecal consistency, degree of scouring and general appearance scores.Results of the study revealed a highly signiﬁcant difference among means on the average feed consumption which indicate a positive effect caused by the types of beneﬁcial microbes and the levels of supplementation. The signiﬁcant improvement in feed consumption was the beneﬁcial effect of the Lactobacillus subtilis, Biﬁdobacterium animalis, and Lactic Acid Bacteria Serum due to enhanced intestinal health and an increased digestive capacity of the experimental calves. While the remaining parameters assessed were not signiﬁcantly affected by the types of beneﬁcial microbes and levels of supplementation. The diverse types of beneﬁcial microbes and their combinations at varying levels can be used as supplement for the calves to enhance intestinal health is important for increasing their digestive capacity in order that they will consume more feeds and therefore increase their growth rate. Beneﬁcial microbes and their combinations improved the growth and health performance of female Holstein calves (Bos taurus) RANDY BERGOLA GAQUIT1, RUDY CASTRO FLORES2*, PEDRO VICTORY DUQUE1 and JONATHAN MANZANO SALAS21 Poaka Farm, New Zealand Faculty of Animal Science, College of Agriculture 2 Bataan Peninsula State University- Abucay Campus, PhilippinesCorrespondence: rcﬂores@bpsu.edu.phSPECIAL INTEREST SECTION

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102Animal Technology and Welfare August 2020Introduction Importance of the study Between June 2011 and June 2020, 4.19 million dairy calves on average were born in New Zealand.1 Approximately ¼ are kept as replacement heifers.2 New Zealand’s dairy system is primarily pasture-based. Calves are raised on pasture grass from infancy; and thus potential risk factors for mortality and morbidity. The management of calves in the ﬁrst 3 weeks of life is critical for their health and longevity.3,4,5 During this time neonatal calves are particularly susceptible to infectious gastrointestinal and respiratory diseases.3,6 Diarrhoea can have both infectious and non-infectious aetiologies and co-infection is common in scouring calves.7,8,9Probiotic microorganisms beneﬁt from a natural image and can expect a promising future in animal nutrition. Controlled research studies demonstrate that they can positively balance gastrointestinal microbiota and improve animal production and health.10 Probiotics may play a role in suppressing the injurious effect derived from the instability of microﬂora colonisation, encouraging immunisation and inhibiting epithelial and mucosal adherence and epithelial invasion by harmful pathogens such as entero-toxigenic Escherichia coli.11 The combination of probiotics and phytobiotics as a feed additive to the diet of dairy calves during rumen fermentation and biochemical blood indices, including the inclusion of phytobiotic with rosmarinic acid as the main bioactive components or probiotics does not affect growth performance and physiology indices.12 But probiotics may be alternative to the antibiotics commonly used as growth promoters in calves.13 Probiotic administration before weaning could improve calf health and decrease mortality of 0.04% and medication.14 Lactic Acid Bacteria Serum (LABS) is an anaerobic microorganism that decomposes sugar without oxygen which are separated and cultured with rice-washed water and milk.15 These should maintain their viability during animal feed processing and storage.16 Beneﬁcial microbes or probiotic feed additives are attracting increased attention as a cost-effective alternative to controlling animal disease.17This study attempted to improve the growth and health performance of new-born calves by using Lactobacillus subtilis, Biﬁdobacterium animalis and homemade Lactic Acid Bacteria Serum (LABS) and their combinations as a supplement to be drenched once or twice daily. The general objective of this study was to evaluate the growth and health performance of the calves supplemented with different beneﬁcial microbes and their combinations at varying levels. Speciﬁcally, this study aimed to assess their growth and health performance in terms of feed consumption, milk consumption, gain in body weight, average daily gain, feedconversion ratio, feed and milk cost per kilogram (kg) of gain in weight, income over feed, milk, beneﬁcial microbes and calf cost, morbidity rate, survival rate, faecal colour score, faecal odour score, degree of scouring score, faecal consistency score and general appearance score.Materials and Methods AnimalsThis study used a total of 48 Holstein Friesian female calves raised in Poaka Farm, Tokoroa, New Zealand that were selected from the 600 newborn calves at the dairy farm. Only healthy female calves that were ﬁve days old with an average of 29.41 kg bodyweight at the start of the experiment were selected to avoid discrepancies and to minimise bias. Those chosen were randomly distributed in a factorial experiment following a completely randomised design (CRD). The experimental house was prepared by thorough cleaning and disinfection one week before the arrival of the calves. The shed had sufﬁcient ventilation to ensure a regular circulation of clean air through it to ensure the calves remained warm and dry. Each calf was provided with 2 square meters (2m2 to allow the calves to move around freely, explore and play. An all-in all-out method which is the practice of keeping animals together in groups, avoiding animals from different groups to mix during their stay on the farm, which are closely matched by age, weight, production stage and condition. The group is moved into a phase of production together, such as into an empty nursery and is moved out of that phase as a group according to a production schedule. When a group moves forward, the facility is completely emptied. This was done to minimise scours and animal health issues. Bedding was changed every 10 days to ensure that calves remained dry. They had access to fresh water and feed and their environmental temperature range was between 15 to 25°C as it was Spring time in New Zealand at the time of the experiment.A strict biosecurity measure was observed during the experimental period to ensure the calves’ health protection. The entry of an unauthorised personnel in the area was prohibited which helped to reduce spreading dust and dirt inside the pens. Cleanliness was maintained in the experimental area by regular disinfection and a clean water supply. Removal of manure and replacement of litter materials was performed weekly to prevent the accumulation of ammonia and to prevent the entry of pathogens that could cause diseases to the calves. The calves were placed in pens according to the proposed treatments after taking their initial bodyweight. Body- weights were performed every 7 days to monitor their growth and development. Their ﬁnal bodyweight was taken when they reached 35 days old.Preparation of supplementsTo prepare the Lactic Acid Bacteria Serum (LABS), the following materials are needed: • 1kg uncooked rice, molasses or brown sugar• 1L fresh or pasteurised milkBeneﬁcial microbes and their combinations improved the growth and health performance of female Holstein calves

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103August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareBeneﬁcial microbes and their combinations improved the growth and health performance of female Holstein calves• unchlorinated clean water• plastic or glass clear jar or container• a plastic basin• clean cloth as a strainer• digital weighing scale• funnel• paper• rubber band The steps to follow are:1. Make ‘rice washing’ by adding 700ml of clean water to 1kg of uncooked rice.2. Stir, swirl and crush by hand until milky-coloured water was attained from the mixture. 3. Obtain the rice washing by ﬁltering the mixture using a clean cloth.4. Place 500ml rice washing in a clear jar and cover with paper and a rubber band. 5. Leave the jar in a cool dark place for 5 to 7 days.6. When the bran has risen, it will smell a little sour and form 3 layers, this has indicated the rice washing is infected with various microbes.7. 100ml of clear water was taken by straining the mixture again.8. 1L of fresh milk was added to 100ml clear water after straining to obtain a 1:10 water and milk ratio.9. The clear jar was covered with paper again and left for 5 to 7 days in a cool dark place.10. Curds appeared after 7 days (made of carbohydrates, protein and fat) or the white part and yellow liquid or whey, enriched with lactic acid bacteria seen from the fermentation of the milk.11. The whey was extracted by pouring it through a strainer to create serum.12. Molasses/brown sugar in equal amounts were weighed and added to the collected whey (serum) – This acts as a stabilising agent for the bacteria, keeping the bacteria alive in a stable dormant stage at room temperature.13. The produced LABS was preserved at room temperature by placing it in a clean container with a loose cap to avoid air pressure increase and labelled accordingly.15 Experimental procedureThe experimental calves were fed with the required amount of 1kg per calf per day starter feeds for 30 days, followed by restricting their food the restricted system of feeding. Fresh drinking water was always provided in buckets and available. A commercially available preparation of Lactobacillus subtilis containing 10x108 microbes per L was used as a source of beneﬁcial microbes. 1ml of Lacto bacillus subtilis was mixed with 1L of water, containing an estimated 106 microbes, before drenching to each calf assigned for the treatment. Another source of beneﬁcial microbes available commercially is Biﬁdobacterium animalis. This preparation contains 10x109 microbes per kg. One gram of Biﬁdobacterium animalis was mixed with 1L of drinking water to obtain an estimated 108 microbes before drenching it to each calf assigned for the treatment.Factor A beneﬁcial microbes (BM)• BM1- Lactobacillus subtilis, • BM2- Biﬁdobacterium animalis • BM3- Lactic Acid Bacteria Serum (LABS) • BM4- Lactobacillus subtilis + Biﬁdobacterium animalis + LABS Factor B varying levels of supplementation (LS):• LS1- 10ml per calf/day • LS2- 20ml per calf/dayThe experimental treatments were as follows: • BM1LS1- 10ml Lactobacillus subtilis per calf/day• BM1LS2- 20ml Lactobacillus subtilis per calf/day• BM2LS1- 10ml Biﬁdobacterium animalis per calf/day• BM2LS2- 20ml Biﬁdobacterium animalis per calf/day• BM3LS1- 10ml LABS per calf/day• BM3LS2- 20ml LABS per calf/day• BM4LS1- 10ml Lactobacillus subtilis + Biﬁdobacterium animalis + LABS per calf/day• BM4LS2- 20ml Lactobacillus subtilis + Biﬁdobacterium animalis + LABS per calf/dayDrenching of 10ml and 20ml of the prepared Lactobacillus subtilis, Biﬁdobacterium animalis, and LABS was performed every morning for 30 days. A plastic drencher was used to administer the preparation to avoid spoilage. Each calf was correctly restrained to avoid injury to both the researcher and calves. It was performed in a gentle manner of handling by approaching the head from the side (not the front), running the hand from the neck under the ear and along the jawbone, then cup the jaw in one hand from the animals head in case it jerks up, making sure that personal protective equipment were worn and safe work standards followed.The following data was gathered and analysed using the analysis of variance for a CRD: average initial weight (AIW), average feed consumption (AFC), average milk consumption (AMC), average gain in weight (AGW), average daily gain (ADG), feed conversion ratio (FCR), average feed and milk cost per kilogram of calf produced (FCKCP), average income over feed, milk, beneﬁcial microbes and calf cost (IOFMBC), average morbidity rate, average survival rate. Meanwhile, to determine the inﬂuence of different beneﬁcial microbes at varying levels on the health performance of the experimental calves, faecal samples were collected and evaluated daily using a manure scoring guide.22,23

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104Animal Technology and Welfare August 2020Manure score guideAll the data gathered was analysed following the analysis of variance (ANOVA) for a factorial experiment in a Completely Randomised Design (CRD). Comparison among means was obtained using the Least Signiﬁ cant Difference (LSD). Texture ScoreManure with normal (ﬁ rm to soft) consistency; brown to light brown colour; normal odour1Manure with soft to loose consistency; yellow, brown, or green; mucus; slight odour2Manure with Loose to Watery consistency; yellow or green colour; mucus; strong odour3Manure with Watery consistency; yellow, green, or clear colour; mucus; slight blood; strong odour4Manure with Watery consistency; clear colour,mucus; bloody5ResultsGrowth performance parametersThe parameters on the growth performance of the calves included the average feed consumption, milk consumption, weight gain, ADG, feed conversion ratio, feed and milk cost to produce a kg weight gain, income over feed, milk and calf cost. The results of the experiment revealed the following:1. A highly signiﬁ cant difference in the average feedconsumption (Figure 1) of the experimental calves was obtained (P<0.05). This indicates that the feedconsumption was highly affected by the types of beneﬁ cial microbes and the level of supplementation due to their ability to enhance intestinal health resulting in increased digestive capacity. The average feed consumption of the experimental calves presented in Figure 1 shows that those given with Lactobacillus + Biﬁ dus + LABS (BM4) supplement consumed the highest number of feeds with an average of 1.80kg/day followed by Biﬁ dobacterium animalis (BM2) and Lactobacillus subtilis (BM1) with 1.79kg and 1.56kg, respectively. The lowest number of feeds consumed was observed in calves assigned in BM3 (Lactic AcidBacteria Serum or LABS) with a mean value of 1.52kg.Levels of supplementation, as drenched, showed that the calves in LS2 (20ml per calf/day) had a higher average feed consumption of 1.72kg compared to the 1.62kg average feed consumption of calves inLS1 (10ml per calf/day). For the interaction between the types of beneﬁ cial microbes and levels of supplementation (BM x LS), as reﬂ ected in Figure 1,calves supplemented with combinations of Lactobacillussubtilis, Biﬁ dobacterium animalis, and LABS (BM4LS2- 20mL L+B+LABS per calf/day) with a mean value of 1.92kg obtained the highest mean on average feed consumption, followed by BM2LS1 (10mL Biﬁ dobacterium animalis per calf/day) with 1.86kg, while calves in BM1LS2 (20ml Lactobacillus subtilis per calf/day), BM2LS2 (20mL Biﬁ do bacterium animalisper calf/day), BM4LS1 (10ml L+B+LABS per calf/day), BM3LS1 (10mL LABS per calf/day), and BM3LS2(20ml LABS per calf/day) consumed an average of 1.75kg, 1.71kg, 1.69kg, 1.55kg, and 1.49kg feeds, respectively. The lowest mean was observed in BM1LS1(10mL Lactobacillus subtilis per calf/day) with 1.38kgaverage feed consumption. Figure 1. Average feed consumption per kg.2. The analysis of variance revealed no signiﬁ cant difference among treatment means among the different types of beneﬁ cial microbes, including their combinations and the varying levels of supplementation on the average milk consumption (Figure 2), average gain in weight (Figure 3), average daily gain (Figure 4), feed conversion ratio (Figure 5), feed and milk cost per kg of gain in weight (Figure 6), income over feed, milk, beneﬁ cial microbes and calf costs (Figure 7). Figure 1 average feed consumption per kg 2. The analysis of variance revealed no significant difference among treatment means among the different types of beneficial microbes, including their combinations, and the varying levels of supplementation on the average milk consumption (Figure 2), average gain in weight (Figure 3), average daily gain (Figure 4), feed conversion ratio (Figure 5), feed and milk cost per kg of gain in weight (Figure 6), income over feed, milk, beneficial microbes and calf costs (Figure 7). Figure 2 average milk consumption per L Average feed consumptionFigure 2. Average milk consumption per L. Figure 1 average feed consumption per kg 2. The analysis of variance revealed no significant difference among treatment means among the different types of beneficial microbes, including their combinations, and the varying levels of supplementation on the average milk consumption (Figure 2), average gain in weight (Figure 3), average daily gain (Figure 4), feed conversion ratio (Figure 5), feed and milk cost per kg of gain in weight (Figure 6), income over feed, milk, beneficial microbes and calf costs (Figure 7). Figure 2 average milk consumption per L Average milk consumptionBeneﬁ cial microbes and their combinations improved the growth and health performance of female Holstein calves

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105August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareBeneﬁ cial microbes and their combinations improved the growth and health performance of female Holstein calvesHealth performanceStatistically, there were no signiﬁ cant differences amongmeans on the morbidity rate (Figure 8), survival rate (Figure 9), faecal colour (Figure 10), faecal odour (Figure 11), faecal consistency (Figure 12), degree of scouring (Figure 13), and average general appearance (Figure 14)scores of the calves used in this study (P>0.05).Figure 3. Average gain in weight per kg. Figure 3 average gain in weight per kg Figure 4 average daily gain per kg Figure 5 feed conversion ratio Average gain in weightFigure 4. Average daily gain per kg. Figure 3 average gain in weight per kg Figure 4 average daily gain per kg Figure 5 feed conversion ratio Average daily gain per kgFigure 5. Feed conversion ratio. Figure 3 average gain in weight per kg Figure 4 average daily gain per kg Figure 5 feed conversion ratio FCRFigure 6. Average feed and milk cost/kg gain in weight, NZ$ Figure 6. Average feed and milk cost/kg gain in weight, NZ$ Figure 7. Average income over calf, feeds, beneficial microbes, and milk cost in NZ$ Health performance Statistically, there were no significant differences among means on the morbidity rate (Figure 8), survival rate (Figure 9), faecal colour (Figure 10), faecal odour (Figure 11), faecal consistency (Figure 12), degree of scouring (Figure 13), and average general appearance (Figure 14) scores of the calves used in this study (P>0.05). Figure 8 % morbidity rate Average feed and milk cost/kg gain in weightFigure 7. Average income over calf, feeds, beneﬁ cial microbes, and milk cost in NZ$. Figure 6. Average feed and milk cost/kg gain in weight, NZ$ Figure 7. Average income over calf, feeds, beneficial microbes, and milk cost in NZ$ Health performance Statistically, there were no significant differences among means on the morbidity rate (Figure 8), survival rate (Figure 9), faecal colour (Figure 10), faecal odour (Figure 11), faecal consistency (Figure 12), degree of scouring (Figure 13), and average general appearance (Figure 14) scores of the calves used in this study (P>0.05). Figure 8 % morbidity rate Average income over calf, feeds, beneﬁ cial microbes, and milk costFigure 8. Morbidity rate. Figure 6. Average feed and milk cost/kg gain in weight, NZ$ Figure 7. Average income over calf, feeds, beneficial microbes, and milk cost in NZ$ Health performance Statistically, there were no significant differences among means on the morbidity rate (Figure 8), survival rate (Figure 9), faecal colour (Figure 10), faecal odour (Figure 11), faecal consistency (Figure 12), degree of scouring (Figure 13), and average general appearance (Figure 14) scores of the calves used in this study (P>0.05). Figure 8 % morbidity rate Morbidity rate

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107August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareBeneﬁcial microbes and their combinations improved the growth and health performance of female Holstein calvesDiscussionGrowth performanceThere is a highly signiﬁcant difference among treatment means on the average feed consumption of the experimental calves used in this study (P<0.01). This indicated that the types of beneﬁcial microbes including their combination and the varying levels of supplementation signiﬁcantly affected the average feed consumption. Comparison among means on the type of beneﬁcial microbes revealed that BM2 (Biﬁdobacterium animalis) and BM4 (Lactobacillus subtilis + Biﬁdobacterium animalis + LABS) were comparable with each other but signiﬁcantly higher over BM1 (Lactobacillus subtilis) and BM3 (LABS) at (P<0.01). This means that the average feed consumption was signiﬁcantly affected by the types of beneﬁcial microbes used as supplements in this experiment. This signiﬁcant improvement in feed consumption supports what was mentioned in a review that lactic acid bacteria (LAB) strains, species belonging to the genera Lactobacillus, Biﬁdobacterium, and Enterococcus, are considered beneﬁcial due to their ability to enhance intestinal health by stimulating the development of a healthy microbiota (predominated by beneﬁcial bacteria), preventing enteric pathogens from colonising the intestine, increasing digestive capacity, lowering the pH and improving mucosal immunity.18 Mean comparison on the levels of supplementation shows that LS1 (10ml per calf/day) is signiﬁcantly higher than LS2 (20ml per calf/day). This means that the average feed consumption of the calves was signiﬁcantly affected by the levels of supplementation. These ﬁndings coincide with what was noted19,20 as cited that the use of direct-fed microbes (DFM) in young calves causes the rapid establishment to adapt to solid feed by stabilising the rumen and intestinal microbes,18 resulting in increased digestive capacity.18Meanwhile, the milk consumption, gain in weight, ADG, FCR, feed and milk cost per kilogram gain in weight, income over feed, milk, beneﬁcial microbes and calf cost revealed no signiﬁcant difference among treatment means on the different types of beneﬁcial microbes and the levels of supplementation based on the analysis of variance (P>0.05). This indicates that the types of beneﬁcial microbes including their combination and the varying levels of supplementation had not signiﬁcantly affected these parameters.Health performance The analysis of variance revealed no signiﬁcant difference among treatment means on the morbidity rate, survival rate, faecal colour score, faecal odour score, faecal consistency score, degree of scouring score and the general appearance score of the calves (P>0.05). These indicate that the different beneﬁcial microbes and their combinations at varying levels have not signiﬁcantly affected the health parameters evaluated in this study.This study proved that the different beneﬁcial microbes could be used as supplements due to improved feed consumption. The signiﬁcant improvement in feed consumption was due to the beneﬁcial effects of the Lactobacillus subtilis, Biﬁdobacterium animalis and Lactic Acid Bacteria Serum and their ability to enhance intestinal health, therefore increasing their digestive capacity. Since no signiﬁcant differences existed among means on all other parameters evaluated in this study, the use of either Lactobacillus subtilis, Biﬁdobacterium animalis, Lactic Acid Bacteria Serum (LABS) or their combinations at 10 ml or 20 mL per calf/day was found to be beneﬁcial.The distinct types of beneﬁcial microbes and their combinations at varying levels can be used as supplement to enhance intestinal health, which is important for increasing the digestive capacity of young calves in order that they will consume more feeds and, therefore, increase their growth rate.A follow-up study on using these beneﬁcial microbes at varying levels for a longer period to further evaluate their beneﬁts on the growth and health of young animals is also recommended.Acknowledgements The authors express their sincerest thanks to Mr. Gerard van der Mark and Ms. Marcelle van der Mark of Poaka Farm, Tokoroa, New Zealand for allowing them to conduct this experiment on their dairy farm.References1 New Zealand Statistics. (2020). Livestock Numbers by Regional Council. http://nzdotstat.stats.govt.nz/wbos/Index.aspx?DataSetCode=TABLECODE7423 2 Hickson, R.E., Zhang, I.L., & McNaughton, L.R. (2015). Brief communication: Birth weight of calves born to dairy cows in New Zealand. In Proceedings of the New Zealand Society of Animal Production (Vol. 75, pp. 257-259).3 Waltner-Toews, D., Martin, S.W., & Meek, A.H. (1986). The effect of early calf hood health status on survivorship and age at ﬁrst calving. Canadian Journal of Veterinar y Research, 50(3), 314.4 Bach, A. (2012). Ruminant Nutrition Symposium: Optimizing performance of the offspring: Nourishing and managing the dam and postnatal calf for optimal lactation, reproduction and immunity. Journal of Animal Science, 90(6), 1835-1845.5 de Passillé, A.M., & Rushen, J. (2016). Using automated feeders to wean calves fed large amounts of milk according to their ability to eat solid feed. Journal of Dair y Science, 99(5), 3578-3583.

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108Animal Technology and Welfare August 20206 Trotz-Williams, L.A., Martin, S.W., Leslie, K.E., Dufﬁeld, T., Nydam, D.V., & Peregrine, A.S. (2007). Calf-level risk factors for neonatal diarrhea and shedding of Cryptosporidium parvum in Ontario dairy calves. Preventive veterinary medicine, 82(1-2), 12-28.7 Hall, G.A., Reynolds, D.J., Parsons, K.R., Bland, A.P., & Morgan, J.H. (1988). Pathology of calves with diarrhoea in southern Britain. Research in veterinary science, 45(2), 240-250.8 Dela Fuente, R., Luzon, M., Ruiz-Santa-Quiteria, J.A., et al. (1999). Cryptosporidium and concurrent infections with other major enteropathogens in 1 to 30-day-old diarrheic dairy calves in central Spain. Veterinar y parasitology, 80(3), 179-185.9 Al Mawly, J., Grinberg, A., Prattley, D., Moffat, J., Marshall, J., & French, N. (2015). Risk factors for neonatal calf diarrhea and enteropathogen shedding in New Zealand dairy farms. The Veterinar y Journal, 203(2), 155-160. 10 Chaucheyras-Durand, F., Walker, N.D., & Bach, A. (2008). Effects of active dry yeasts on the rumen microbial ecosystem: Past, present and future.Animal Feed Science and Technology, 145(1-4), 5-26.11 Nagashima, K., Daisure, Y., Kentaru, Ryoji, N., Tooru, K., Toshiharu, M., & Shu, K. (2010). Effect of Lactobacillus species on the incidence of diarrhea in calves and chase microﬂora associated with growth. Bioscience Microﬂora, Vol. 29 (2), 97-11012 Barbara, S., Sroka, J., Katzer, F., Gdnksi, P., & Nowak, W. (2021) The effect of probiotics, phytobiotics and their combination as feed additives in the diet of dairy calves on the performance, rumen fermentation, and blood metabolites during the pre-weaning period. Animal Feed Science and Technology, Vol.27213 Frizzo L.S., Brun, M.V.S., Soto, L.P., & Signorini, M.L. (2011). Effects of probiotics on growth performance in young calves: Meta-analysis of randomized controlled trials. Animal Feed Science and Technology, Vol.169 issues 3-4, pp 147-156.14 Gorgulu, M., Slutaa, A., Yurtseven, S., Ongel, E., & Kutlu, H.R. (2003). Effect of Probiotics on growing performance and health of calves. Cuban Journal of Agricultural Science, pp 125-129.15 Gamboa, J.C. (2009). Korean Natural Farming Handbook. (2009). Korean Natural Farming Lactic Acid Bacteria (LAB) pp 126. https://rooftopecology.wordpress.com/2009/09/29/korean-natural-farming-lactic-acid-bacteria-lab/16 Lin, W-H, Yu, B., Sheng-Hon, J., & Hau-Yang, T. (2007). Different probiotic properties for Lactobacillus fermentum strains isolated from swine and poultry. Anaerobe 13: 107-113.17 Melegy, T., Khaled, N. F., El-Bana, R., & Abdellatif, H. (2011). Effect of dietary supplementation of Bacillus subtilis PB6 (CLOSTATTM) on performance, immunity, gut health, and carcass traits in broilers. Journal of American Science, 7(12), 891-898.18 Uyeno, Y., Shigemori, S., & Shimosato, T. (2015). Effect of Probiotics/Prebiotics on Cattle Health and Productivity. Microbes and Environments, 30(2), 126–132. https://doi.org/10.1264/jsme2.ME1417619 Dhama, K., Mahendran, M., Tomar, S., & Chauhan, R.S. (2008). Beneﬁcial effects of probiotics and prebiotics in livestock and poultry: The current perspectives. Intas Polivet, 9: 1-12.20 Puniya, A.K., Salem, A.Z.M., Kumar, S., Dagar, S.S., & Grifﬁth, G.W. (2015). Role of live microbial feed supplements with reference to anaerobic fungi in ruminant productivity: A review. J. Integr. Agric., 14: 550-560.21 Rifat U.K., Shabana N., Kuldeep D., et al. (2016). Direct-Fed Microbial: Beneﬁcial Applications, Modes of Action and Prospects as a Safe Tool for Enhancing Ruminant Production and Safeguarding Health. Inter. J. Pharmacol. 12:220-231.22 Hutjens, M.F. (2018). Manureology101. University of Illinois, USA (Hutsjensm@illinois.edu). Available at https://nagonline.net/wp-content/uploads/2018/ 04/d10181-manureology-101.pdf 23 Dzaaijer, W.D.J., Kremer, J.P., & Noordhuizen, T.M. (2001). Manure Score Card: Consistency. Available at https://nagonline.net/wp-content/uploads/2018/ 04/Manure-score-card.pdf Beneﬁcial microbes and their combinations improved the growth and health performance of female Holstein calves

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109August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2024 Animal Technology and WelfarePAPER SUMMARY TRANSLATIONSCONTENU DE LA REVUERapport de la réunion 2023 du Groupe de protection des rongeurs de la RSPCA/UFAW/IAT MARINE BARNABÉ1, KEN APPLEBEE2,3,JANE HURST4, VIKKI NEVILLE5, TOM CHILDS6, MIGUEL MARAVALL7, JULIA BARTLETT8, JANE TYSON9, TINA O’MAHONY10, HALEY DANIELS11 ET KERITH LUCHINS12 1 Animals in Science Department, RSPCA, Horsham RH12 1GY2 Applebee Advisory, Hornchurch RM12 6RJ3 College of Laboratory Animal Science & Technology, Rushden NN10 0SH4 Institute of Infection, Veterinary and Ecological Sciences, Université de Liverpool, Liverpool CH64 7TE5 Bristol Veterinary School, Bristol BS40 5DU6 Francis Crick Institute, London NW1 1AT7 Sussex Neuroscience, School of Life Sciences, Université de Sussex, Brighton BN1 9RH8 School of Physiology, Pharmacology and Neuroscience, Université de Bristol, Bristol BS8 1QU9 Companion Animals Department, RSPCA, Horsham RH12 1GY10 Institute of Animal Technology, Oxford OX2 7JL11 Université de York, York YO10 5DD12 Université de Chicago, Les États-Unis d’AmériqueCorrespondance: animalsinscience@rspca.org.ukIntroduction Le groupe de protection des rongeurs de la RSPCA/UFAW organise depuis 30 ans une réunion d’une journée chaque automne pour permettre à ses membres de discuter de la recherche actuelle sur le bien-être, d’échanger des points de vue sur les questions de bien-être et de partager des expériences sur la mise en œuvre des 3R pour le remplacement, la réduction et le rafﬁnement en ce qui concerne l’utilisation des rongeurs.Cette réunion, qui s’est tenue en personne au Francis Crick Institute de Londres en octobre 2023, a permis aux participants de prendre part à des discussions en face à face tout au long de la journée et dans le cadre d’une séance de discussion de groupe à la ﬁn de la journée. La journée avait pour objet d’aborder le passé et l’avenir, avec des conférences couvrant des sujets allant du logement et de l’élevage au rafﬁnement des procédures, en passant par l’éducation et les leçons tirées des soins prodigués aux rats compagnons. Ce rapport résume la réunion et comprend une liste de points d’action que les lecteurs peuvent envisager de soulever dans leurs propres établissements. ★ ★ ★

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110Animal Technology and Welfare August 2020Perfectionnements pratiques: mise en place de procédés d’élevage améliorés pour les souris C57BL/6 âgées AIMÉE PATERSONCorrespondance: aimee-paterson@hotmail.com Introduction Le vieillissement est déﬁni comme une détérioration des fonctions physiologiques qui entraîne par la suite des maladies liées à l’âge et une fragilité clinique. Pour étudier le processus de vieillissement, il est important d’envisager ses effets cumulatifs sur le corps.La recherche sur le vieillissement est essentielle pour mieux comprendre les processus biologiques et offrir d’éventuelles avancées4.Les souris sont un bon modèle de vieillissement car elles sont physiologiquement similaires et partagent environ 95 % de leur génome avec l’humain1.En termes de fragilité clinique, les souris vieillissent également de manière comparable. Elles présentent de nombreux signes cliniques tels qu’une réduction de la masse musculaire, une perte de poids, des changements de la démarche et le développement de maladies liées à l’âge2.L’objectif principal de ce projet était d’examiner les processus d’élevage en place pour la colonie de souris C57BL/6 âgées à l’Institut Babraham, d’établir un processus d’élevage afﬁné et d’améliorer les orientations locales. Deuxièmement, en analysant les données de devenir et d’observation clinique entre 2021 et 2023, ce projet visait à évaluer l’impact de ces routines améliorées sur l’identiﬁcation des problèmes de santé et le nombre de souris retrouvées mortes lors de contrôles sanitaires de routine. ★ ★ ★Initiative 3H – logement, manipulation, accoutumanceJULIA BARTLETT, JUSTYNA HINCHCLIFFE, MEGAN JACKSON ET EMMA ROBINSONUniversité d’Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TDCorrespondance: 3hsinitiative@gmail.comRésumé L’initiative 3H est un concept issu de la recherche et de la gestion des souris et rats de laboratoire du laboratoire du Professeur Emma Robinson à l’Université de Bristol. Nos recherches portent principalement sur le traitement des troubles psychiatriques et en particulier des symptômes de dépression et d’apathie. En raison de la nature de notre travail, il est important d’assurer que les animaux avec lesquels nous travaillons ne soient pas affectés de façon négative par leur environnement ou involontairement par nos interactions avec eux. Notre approche s’est concentrée sur des méthodes fournissant des populations de contrôle optimales en nous permettant de quantiﬁer les changements comportementaux spéciﬁques et pertinents survenant lors des manipulations expérimentales. Au ﬁl de l’évolution de notre programme de recherche, nous avons pris de plus en plus conscience de l’impact du stress causé à nos animaux par les approches de gestion de routine et la contrainte physique pendant les procédures. ★ ★ ★ Paper Summary Translations

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111August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePaper Summary TranslationsL’échographie pour déterminer la gestation chez la souris CAROLINE KARAM ET KIRSTY KEMPUKRI MRC Laboratory of Molecular Biology CambridgeCorrespondance: cperimnt@mrc-lmb.cam.ac.uk, ktrevenn@mrc-lmb.cam.ac.uk Résumé Plusieurs groupes du laboratoire de biologie moléculaire (LMB) du MRC ont besoin d’embryons de souris à un stade précoce pour leurs programmes de recherche scientiﬁque. Les techniciens animaliers s’appuyaient généralement sur des contrôles visuels ou la palpation de l’abdomen pour déterminer la gestation des souris. L’achat d’un échographe en 2017 permettrait de scanner des souris fécondées présentant un bouchon pour conﬁrmer la gestation avant le sacriﬁce, offrant ainsi une précision accrue pour surmonter les limites des méthodes existantes. Cela permet ainsi aux femelles qui ne semblent pas gestantes d’être accouplées à nouveau ; un perfectionnement de la pratique actuelle réduisant également le nombre d’animaux utilisés pour des expériences. Initialement, l’utilisation de l’équipement d’échographie a permis une augmentation de 36 % dans la détermination de la gestation par rapport aux méthodes traditionnelles entre E.7.5 et E12.5. Compte tenu du temps et de la pratique, la procédure a été afﬁnée pour permettre de détecter avec succès la gestation aux premières périodes de E6.5 et E5.5.Un programme de formation à l’échographie en interne a été organisé avec succès et il existe désormais plusieurs techniciens compétents. ★ ★ ★Engagement public et ouverture en recherche animale au CrickJAMIE REDDEN, EWELINA KOZLOWSKA, CLARE BRAZILL-ADAMS, CAROLINE ZVEREV ET SARAH HART-JOHNSONThe Francis Crick InstituteCorrespondance: Jamie.redden@crick.ac.ukIntroduction L’engagement du public constitue l’une des priorités stratégiques du Crick, en cherchant à engager et à inspirer le public par la découverte et la science. Une grande partie de la science qui change la vie impliquant l’utilisation d’animaux, nous cherchons à rester ouverts et transparents sur la façon dont nous utilisons ces animaux dans la science, et sur les avantages qu’ils apportent. Pour soutenir cet engagement, le Crick est l’un des plus de 100 signataires du Concordat sur l’ouverture d’Understanding Animal Research (UAR). De plus en plus d’organisations s’engagent désormais à faire preuve d’ouverture concernant leur travail avec les animaux. Les différentes méthodes par lesquelles nous pouvons communiquer avec le public et nous engager auprès de ce dernier deviennent ainsi plus largement connues et les institutions sont en mesure d’apprendre les unes des autres et de tester ce qui fonctionne le plus efﬁcacement pour leur modèle opérationnel, notamment ce qui est pratique et réalisable, tout en ayant un impact.Nous soulignons ici quelques-unes des différentes façons dont le Crick s’est engagé auprès du public pour promouvoir l’ouverture dans la recherche animale, y compris les différents médias et formats utilisés, et les différents publics que nous avons atteints. Nous visons à montrer la variété des méthodes qui sont à la disposition des organisations (comment le personnel, à différents niveaux d’ancienneté, peut être impliqué dans la sensibilisation, les possibilités de collaboration inter-service) et à démontrer comment une approche diversiﬁée et variée de l’ouverture peut s’avérer plus efﬁcace. ★ ★ ★

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112Animal Technology and Welfare August 2020INHALTVERZEICHNISBericht über die Tagung der RSPCA/UFAW/IAT-Tierschutzgruppe für Nager 2023MARINE BARNABÉ1, KEN APPLEBEE2,3,JANE HURST4, VIKKI NEVILLE5, TOM CHILDS6, MIGUEL MARAVALL7, JULIA BARTLETT8, JANE TYSON9, TINA O’MAHONY10, HALEY DANIELS11 UND KERITH LUCHINS12 1 Animals in Science Department, RSPCA, Horsham RH12 1GY2 Applebee Advisory, Hornchurch RM12 6RJ3 College of Laboratory Animal Science & Technology, Rushden NN10 0SH4 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool CH64 7TE5 Bristol Veterinary School, Bristol BS40 5DU6 Francis Crick Institute, London NW1 1AT7 Sussex Neuroscience, School of Life Sciences, University of Sussex, Brighton BN1 9RH8 School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol BS8 1QU9 Companion Animals Department, RSPCA, Horsham RH12 1GY10 Institute of Animal Technology, Oxford OX2 7JL11 University of York, York YO10 5DD12 University of Chicago, United states of AmericaKorrespondenz: animalsinscience@rspca.org.ukEinleitung Die RSPCA/UFAW-Tierschutzgruppe für Nager veranstaltet seit 30 Jahren jeden Herbst ein eintägiges Treffen, bei dem die Mitglieder über Themen aktueller Tierschutzforschung diskutieren und Erfahrungen und Meinungen über Tierschutzfragen sowie über die Umsetzung des 3R-Prinzips (Vermeidung, Reduktion und Verbesserung) beim Einsatz von Nagern austauschen können. Die Tagung fand als Präsenz-Veranstaltung im Oktober 2023 am Francis Crick Institute in London statt. Sie bot den Teilnehmern im Rahmen von Einzelgesprächen über den Tag hinweg und einer Gruppendiskussion am Ende des Tages die Möglichkeit zum persönlichen Austausch. In den Vorträgen wurden rückblickend und vorausschauend Themen wie Unterbringung, Haltung, Verfahrensverbesserung, Ausbildung und Anwendung von Erfahrungen bei der Pﬂege von Heimratten behandelt. Dieser Bericht fasst das Treffen zusammen und enthält einige Aktionspunkte, die Leser für ihre eigenen Einrichtungen in Betracht ziehen können. ★ ★ ★Paper Summary Translations

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113August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePaper Summary TranslationsPraktische Verbesserungen: Einführung optimierter Haltungsverfahren für ältere C57BL/6-MäuseAIMEE PATERSONKorrespondenz: aimee-paterson@hotmail.com EinleitungAltern ist deﬁniert als ein Abbau der physiologischen Funktionen, der in der Folge zu altersbedingten Krankheiten und klinischer Gebrechlichkeit führt. Zur Untersuchung des Alterungsprozesses ist es wichtig, die kumulativen Auswirkungen auf den Körper zu betrachten.Altersforschung ist grundlegend für ein besseres Verständnis der biologischen Prozesse und zur Erzielung möglicher Fortschritte4.Mäuse sind ein gutes Modell für den Alterungsprozess, da sie dem Menschen physiologisch ähnlich sind und etwa 95 % ihres Genoms mit dem menschlichen teilen1.Bezüglich klinischer Gebrechlichkeit altern Mäuse auf vergleichbare Weise und zeigen viele der gleichen klinischen Anzeichen wie verringerte Muskelmasse, Gewichtsverlust, Veränderungen des Gangbildes und die Entwicklung altersbedingter Krankheiten2.Das Hauptziel dieses Projekts bestand darin, die bestehenden Haltungsverfahren für die Kolonie gealterter C57BL/6-Mäuse am Babraham Institute zu überprüfen, ein verbessertes Haltungsverfahren zu etablieren und die örtliche Pﬂege zu verbessern. Mit der Analyse von zwischen 2021 und 2023 erhobenen Daten zum Verbleib und zur klinischen Beobachtung bezweckte das Projekt des Weiteren, die Auswirkungen dieser verbesserten Abläufe im Hinblick auf die Erkennung von Gesundheitsproblemen und die Zahl der bei Routineuntersuchungen tot aufgefundenen Mäuse zu bewerten.★ ★ ★Die 3H-Initiative – Housing, Handling, Habituation (Unterbringung, Handling, Gewöhnung)JULIA BARTLETT, JUSTYNA HINCHCLIFFE, MEGAN JACKSON UND EMMA ROBINSONUniversity of Bristol, Biomedical Sciences Building, University Walk, Bristol, BS8 1TDKorrespondenz: 3hsinitiative@gmail.comAbstract Die 3H-Initiative ist ein Konzept, das aus der Forschung und Haltung von Labormäusen und -ratten im Labor von Professorin Emma Robinson an der Universität Bristol hervorgegangen ist. Unsere Untersuchung befasst sich vor allem mit der Behandlung psychischer Störungen und insbesondere mit Symptomen von Depression und Apathie. Die Art unserer Tätigkeit erfordert es sicherzustellen, dass die Tiere, mit denen wir arbeiten, nicht durch ihre Umgebung oder als unbeabsichtigte Folge unserer Interaktionen mit ihnen in einen negativen affektiven Zustand versetzt werden. Unser Ansatz konzentrierte sich auf Methoden, die optimale Kontrollpopulationen liefern und gewährleisten, dass wir speziﬁsche und relevante Verhaltensänderungen, die bei Experimenten auftreten, quantiﬁzieren können.Im Laufe unseres Forschungsprogramms haben wir sukzessive die Folgen des Stresses erkannt, der unseren Tieren durch routinemäßige Haltungsmethoden und körperliche Ruhigstellung während der Verfahren zugefügt wird. ★ ★ ★

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114Animal Technology and Welfare August 2020Die Verwendung von Ultraschall zur Bestimmung der Trächtigkeit bei Mäusen CAROLINE KARAM UND KIRSTY KEMPUKRI MRC Laboratory of Molecular Biology CambridgeKorrespondenz:: cperimnt@mrc-lmb.cam.ac.uk, ktrevenn@mrc-lmb.cam.ac.uk AbstractMehrere MRC-Laboratorien für Molekularbiologie (LMB) benötigen für ihre wissenschaftlichen Forschungsprogramme Mausembr yonen im Frühstadium. Traditionell verlassen sich Tiertechniker auf Sichtkontrollen oder das Abtasten des Bauches, um eine Trächtigkeit bei Mäusen festzustellen. Im Jahr 2017 wurde ein Ultraschallgerät angeschafft, mit dem Mäuse vor der Tötung gescannt werden können, um eine Trächtigkeit zu bestätigen und dank der erhöhten Genauigkeit die Grenzen bisheriger Methoden zu durchbrechen. So können Weibchen, die nicht trächtig zu sein scheinen, erneut gepaart werden – eine Verbesserung der derzeitigen Praxis, mit der auch die Anzahl der für die Experimente verwendeten Tiere reduziert werden kann.Anfangs ermöglichte der Einsatz des Ultraschallgeräts im Vergleich zu herkömmlichen Methoden eine um 36 % höhere Wahrscheinlichkeit, eine Trächtigkeit zwischen E7,5 und E12,5 festzustellen. Im Laufe der Zeit und weiterer praktischer Anwendung wurde das Verfahren verbessert, so dass auch Trächtigkeiten in den früheren Gestationsstadien E6,5 und E5,5 erfolgreich festgestellt werden konnten.Im Rahmen eines internen Ultraschall-Schulungsprogramms wurden mehrere Techniker mit entsprechender Kompetenz erfolgreich ausgebildet. ★ ★ ★Öffentlichkeitsarbeit und Transparenz in der Tierforschung am Francis Crick InstituteJAMIE REDDEN, EWELINA KOZLOWSKA, CLARE BRAZILL-ADAMS, CAROLINE ZVEREV UND SARAH HART-JOHNSONFrancis Crick InstituteKorrespondenz: Jamie.redden@crick.ac.ukEinleitung Öffentlichkeitsarbeit ist eine der strategischen Prioritäten des Francis Crick Institute, mit der wir versuchen, das Interesse der Öffentlichkeit an Forschung und Wissenschaft zu fördern. Da ein Großteil unserer lebensverändernden Wissenschaft mit dem Einsatz von Tieren verbunden ist, ist es uns ein Anliegen, die Art und Weise unserer Verwendung von Tieren in der Forschung sowie den Nutzen, den sie uns bringen, offen und transparent zu kommunizieren. Zur Erfüllung dieser Aufgabe hat das Francis Crick Institute gemeinsam mit über 100 anderen Einrichtungen das Konkordat über Offenheit in der Tierforschung (Understanding Animal Research – UAR) unterzeichnet. Je mehr Organisationen sich verpﬂichten, offen über ihre Arbeit mit Tieren zu sprechen, desto besser können wir unsere verschiedenen Methoden der Kommunikation und Öffentlichkeitsarbeit verbreiten und desto mehr können Einrichtungen voneinander lernen und testen, was für ihr Betriebsmodell am effektivsten ist – und was für sie praktikabel und umsetzbar ist und gleichzeitig Wirkung zeigt.Hier stellen wir einige der verschiedenen Möglichkeiten vor, die das Francis Crick Institute für den Kontakt mit der Öffentlichkeit nutzt, um Offenheit in der Tierforschung zu fördern, einschließlich der verschiedenen Medien und Formate zur Ansprache der unterschiedlichen Zielgruppen. Wir möchten die vielfältigen Methoden aufzeigen, die Organisationen zur Verfügung stehen, wie Mitarbeiter verschiedener Leitungsebenen in die Öffentlichkeitsarbeit einbezogen werden können, welche Möglichkeiten abteilungsübergreifender Zusammenarbeit es gibt und wie ein diversiﬁzierter und differenzierter Offenheitsansatz eine größere Wirkung erzielen kann. ★ ★ ★ Paper Summary Translations

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115August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePaper Summary TranslationsINDICE DELLA REVISTAResoconto dell’incontro del RSPCA/UFAW/IAT Rodent Welfare Group del 2023MARINE BARNABÉ1, KEN APPLEBEE2,3,JANE HURST4, VIKKI NEVILLE5, TOM CHILDS6, MIGUEL MARAVALL7, JULIA BARTLETT8, JANE TYSON9, TINA O’MAHONY10, HALEY DANIELS11 E KERITH LUCHINS12 1 Animals in Science Department, RSPCA, Horsham RH12 1GY2 Applebee Advisory, Hornchurch RM12 6RJ3 College of Laboratory Animal Science & Technology, Rushden NN10 0SH4 Institute of Infection, Veterinary and Ecological Sciences, Università di Liverpool, Liverpool CH64 7TE5 Bristol Veterinary School, Bristol BS40 5DU6 Francis Crick Institute, London NW1 1AT7 Sussex Neuroscience, School of Life Sciences, Università del Sussex, Brighton BN1 9RH8 School of Physiology, Pharmacology and Neuroscience, Università di Bristol, Bristol BS8 1QU9 Companion Animals Department, RSPCA, Horsham RH12 1GY10 Institute of Animal Technology, Oxford OX2 7JL11 Università di York, York YO10 5DD12 Università di Chicago, Stati Uniti d’AmericaCorrispondenza: animalsinscience@rspca.org.ukIntroduzione Ogni autunno, da ormai 30 anni, il RSPCA/UFAW Rodent Welfare Group organizza un incontro della durata di un giorno per consentire ai suoi membri di discutere degli studi attuali di ricerca sul benessere, di scambiarsi opinioni sulle questioni legate al benessere e di condividere esperienze di applicazione del principio delle 3 R, ovvero sostituzione (Replacement), riduzione (Reduction) e perfezionamento (Reﬁnement), in relazione all’uso di roditori.L’incontro, tenutosi in presenza a ottobre 2023 presso il Francis Crick Institute di Londra, ha permesso ai partecipanti di interagire di persona nel corso della giornata e di intavolare discussioni nell’ambito di una sessione di gruppo tenutasi a conclusione dell’incontro. Il tema affrontato si è concentrato sulla valorizzazione del passato, con lo sguardo volto anche verso il futuro, puntando i riﬂettori su argomenti quali la stabulazione, la zootecnia, il rafﬁnamento delle procedure, l’educazione e le lezioni apprese dalla cura dei ratti da compagnia. Il presente resoconto riassume l’incontro e include alcuni punti di intervento che i lettori possono portare all’attenzione delle loro strutture. ★ ★ ★

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116Animal Technology and Welfare August 2020Perfezionamenti pratici: istituzione di processi zootecnici ottimizzati per topi C57BL/6 anziani AIMEE PATERSONCorrispondenza: aimee-paterson@hotmail.com IntroduzioneL’invecchiamento è deﬁnito come il deterioramento delle funzioni ﬁsiologiche, che sfocia a sua volta in patologie legate all’età e nella fragilità clinica. Lo studio del processo di invecchiamento richiede la valutazione degli effetti cumulativi sul corpo.Le ricerche condotte in campo di invecchiamento sono essenziali per favorire una migliore comprensione dei processi biologici e per promuovere potenziali avanzamenti4.I topi sono un buon modello di invecchiamento, in quanto sono ﬁsiologicamente simili e condividono circa il 95% del loro genoma con gli esseri umani1.In termini di fragilità clinica, i topi invecchiano in maniera comparabile e mostrano gran parte dei medesimi segni clinici, quali riduzione della massa muscolare, perdita di peso, cambiamenti nell’andatura e sviluppo di malattie correlate all’età2.Questo progetto si è proposto principalmente di esaminare i processi zootecnici in essere per la colonia di topi C57BL/6 anziani del Babraham Institute, di stabilire un processo potenziato e di migliorare le linee guida locali. In secondo luogo, basandosi sull’analisi dei dati sul destino e sulle osservazioni cliniche effettuata tra il 2021 e il 2023, ha mirato a valutare l’impatto di queste procedure migliorate sull’identiﬁcazione di problemi di salute e il numero di topi trovati morti durante check-up di routine.★ ★ ★L’Iniziativa delle 3 H: stabulazione, gestione, assuefazioneJULIA BARTLETT, JUSTYNA HINCHCLIFFE, MEGAN JACKSON E EMMA ROBINSONUniversità di Bristol, Biomedical Sciences Building, University Walk, Bristol, BS8 1TDCorrispondenza: 3hsinitiative@gmail.comAbstract L’Iniziativa delle 3 H (Housing, Handling, Habituation) è un concetto emerso dalla ricerca e dalla gestione di topi e ratti da laboratorio utilizzati presso il laboratorio del Professor Emma Robinson nell’Università di Bristol. Il nostro studio di ricerca riguarda prevalentemente il trattamento di disturbi psichiatrici e, in particolare, i sintomi della depressione e dell’apatia. La natura del nostro lavoro ci impone di accertarci che l’ambiente circostante non attivi uno stato affettivo negativo negli animali utilizzati o che tale stato derivi involontariamente dalle nostre interazioni con essi. Il nostro approccio si è incentrato su metodi che forniscono popolazioni di controllo ottimali e consentono di quantiﬁcare cambiamenti comportamentali speciﬁci e rilevanti emergenti per le manipolazioni sperimentali. L’evoluzione del nostro programma di ricerca ci ha resi più consapevoli dell’impatto dello stress causato ai nostri animali da approcci gestionali di routine e dalle restrizioni ﬁsiche imposte nel corso delle procedure. ★ ★ ★ Paper Summary Translations

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117August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePaper Summary TranslationsUna visione chiara: l’uso dell’ultrasuono per determinare la gravidanza nei topi CAROLINE KARAM E KIRSTY KEMPUKRI MRC Laboratory of Molecular Biology CambridgeCorrispondenza: cperimnt@mrc-lmb.cam.ac.uk, ktrevenn@mrc-lmb.cam.ac.uk AbstractSvariati gruppi dell’MRC Laboratory of Molecular Biology (LMB) richiedono embrioni di topo allo stadio iniziale per i loro programmi di ricerca scientiﬁca. Tradizionalmente, gli stabularisti hanno fatto afﬁdamento su controlli visivi o sulla palpazione addominale per determinare la gravidanza nei topi. Nel 2017, è stata acquistata una macchina ad ultrasuoni, che avrebbe permesso di effettuare una scansione dei topi esaminati per confermare la loro gravidanza prima di sacriﬁcarli, promuovendo una maggiore accuratezza volta a superare i limiti dei metodi esistenti. In tal modo, si rendeva di nuovo possibile l’accoppiamento delle femmine che non risultavano gravide, migliorando la pratica attuale e riducendo inoltre il numero di animali utilizzati a ﬁni sperimentali. Inizialmente, l’uso del macchinario ad ultrasuoni ha generato un incremento del 36% nella determinazione della gravidanza dei topi E.7.5-E12.5 rispetto ai metodi tradizionali. Con il passare del tempo e ulteriore pratica, la procedura è stata perfezionata, consentendo di rilevare le gravidanze dei topi E6.5 ed E5.5 nei primi periodi di gestazione.Grazie a un efﬁcace programma di formazione interno sulle tecniche ecograﬁche, adesso si dispone di svariati stabularisti competenti. ★ ★ ★Istituto Crick: coinvolgimento del pubblico e trasparenza nella sperimentazione animaleJAMIE REDDEN, EWELINA KOZLOWSKA, CLARE BRAZILL-ADAMS, CAROLINE ZVEREV E SARAH HART-JOHNSONFrancis Crick InstituteCorrispondenza: Jamie.redden@crick.ac.ukIntroduzione Il coinvolgimento del pubblico è una delle priorità strategiche dell’Istituto Crick, che si propone di agganciare e ispirare il pubblico attraverso le scoperte e la scienza. Gran parte della scienza rivoluzionaria prevede l’uso di animali e, pertanto, ci impegniamo a dichiarare apertamente e con trasparenza le modalità di utilizzo degli animali in campo scientiﬁco, evidenziando i beneﬁci da essi apportati. A supporto di tale impegno, l’Istituto Crick è uno di oltre 100 ﬁrmatari del Concordat on Openness dell’organizzazione Understanding Animal Research (UAR). Grazie a un numero sempre crescente di organizzazioni dedite a essere trasparenti in merito alle loro sperimentazioni con animali, i vari metodi di comunicazione e interazione con il pubblico da noi promossi assumono maggiore notorietà, consentendo alle istituzioni di apprendere reciprocamente e di provare le metodologie più idonee al loro modello operativo, comprese opzioni concrete e realizzabili che abbiano, comunque, un certo impatto.Questo articolo mette in evidenza i diversi modi con cui l’Istituto Crick ha interagito con il pubblico per promuovere la trasparenza nella sperimentazione animale, inclusi i vari mezzi di informazione e formati usati, e le varie utenze raggiunte. Il nostro obiettivo è mostrare la varietà di metodi disponibili alle organizzazioni, le strategie di sensibilizzazione che possono applicare dipendenti con anzianità di servizio diversa e le opportunità di collaborazione interdipartimentale, oltre a dimostrare la maggiore efﬁcacia di un approccio alla trasparenza multiforme e variegato. ★ ★ ★

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118Animal Technology and Welfare August 2020INDICE DE LA REVISTAInforme sobre la reunión del Grupo para el bienestar de roedores RSPCA/UFAW/IAT 2023MARINE BARNABÉ1, KEN APPLEBEE2,3, JANE HURST4, VIKKI NEVILLE5, TOM CHILDS6, MIGUEL MARAVALL7,JULIA BARTLETT8, JANE TYSON9, TINA O’MAHONY10, HALEY DANIELS11 Y KERITH LUCHINS12 1 Departamento de Animales para la Ciencia, RSPCA, Horsham, RH12 1GY2 Applebee Advisory, Hornchurch RM12 6RJ3 Instituto superior de Ciencia y Tecnología de Animales de Laboratorio, Rushden, NN10 0SH4 Instituto de Infecciones, Ciencias Veterinarias y Ecológicas, Universidad de Liverpool, Liverpool, CH64 7TE5 Facultad de Veterinaria de Bristol, Bristol, BS40 5DU6 Francis Crick Institute, Londres, NW1 1AT7 Neurociencia de Sussex, Facultad de Ciencias de la Vida, Universidad de Sussex, Brighton, BN1 9RH8 Facultad de Fisiología, Farmacología y Neurociencia, Universidad de Bristol, Bristol, BS8 1QU9 Departamento de Animales de Compañía, RSPCA, Horsham, RH12 1GY10 Instituto de Tecnología Animal, Oxford OX2 7JL11 Universidad de York, York YO10 5DD12 Universidad de Chicago, Estados Unidos de AméricaCorrespondencia: animalsinscience@rspca.org.ukIntroducción El Grupo para el bienestar de roedores de RSPCA/UFAW lleva celebrando una reunión de un día cada otoño durante los últimos 30 años. El objetivo es que sus miembros puedan conversar sobre investigaciones de bienestar actuales, intercambiar opiniones sobre temas relacionados con el bienestar y compartir su experiencia respecto a la implementación de las 3 R (Reemplazo, Reducción y Reﬁnamiento) en relación al uso de roedores.Esta reunión se celebró de forma presencial en el Francis Crick Institute de Londres en octubre de 2023 y ofreció a los participantes la oportunidad de participar en conversaciones presenciales a lo largo del día y también como parte de una sesión de debate grupal al ﬁnal de la jornada. El tema del día buscaba mirar hacia atrás y hacia delante, y las charlas abarcaron cuestiones que iban desde el alojamiento y la cría hasta el perfeccionamiento de los procedimientos, la educación y el aprendizaje del cuidado de las ratas de compañía. Este informe resume la reunión e incluye algunos puntos de acción para que los lectores consideren presentar en sus propios centros. ★ ★ ★Paper Summary Translations

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119August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePaper Summary TranslationsPerfeccionamientos prácticos: establecimiento de procesos de cría mejorados para ratones C57BL/6 envejecidos AIMEE PATERSONCorrespondencia: aimee-paterson@hotmail.com IntroducciónEl envejecimiento se deﬁne como un deterioro de las funciones ﬁsiológicas que posteriormente conduce a enfermedades relacionadas con la edad y a la fragilidad clínica. Para estudiar el proceso de envejecimiento, es importante observar los efectos acumulativos en el organismo.La investigación sobre el envejecimiento es esencial para comprender mejor los procesos biológicos y ofrecer posibles avances4.Los ratones sirven como un buen modelo de envejecimiento, ya que son ﬁsiológicamente similares y comparten aproximadamente el 95 % de su genoma con los humanos1.En términos de fragilidad clínica, los ratones también envejecen de forma comparable y muestran muchos de los mismos signos clínicos, como la reducción de la masa muscular, la pérdida de peso, los cambios en la marcha y el desarrollo de enfermedades relacionadas con la edad2.El objetivo principal de este proyecto era revisar los procesos de cría vigentes para la colonia de ratones C57BL/6 envejecidos en el Babraham Institute, establecer un proceso de cría perfeccionado y mejorar la orientación local. En segundo lugar, mediante el análisis de los datos sobre el destino y la observación clínica entre 2021 y 2023, este proyecto trataba de evaluar el impacto de estas rutinas mejoradas en la identiﬁcación de problemas sanitarios y el número de ratones encontrados muertos en los controles sanitarios rutinarios.★ ★ ★La Iniciativa de las 3 H: Hábitat, Habilidad, HabitualizaciónJULIA BARTLETT, JUSTYNA HINCHCLIFFE, MEGAN JACKSON Y EMMA ROBINSONUniversidad de Bristol, Biomedical Sciences Building, University Walk, Bristol, BS8 1TDCorrespondencia: 3hsinitiative@gmail.comResumen La Iniciativa de las 3 H es un concepto que surgió de la investigación y la gestión de ratones y ratas de laboratorio del laboratorio de la profesora Emma Robinson, en la Universidad de Bristol. Nuestra investigación se centra particularmente en el tratamiento de los trastornos psiquiátricos y, en particular, de los síntomas de la depresión y la apatía. Debido a la naturaleza de nuestro trabajo, es importante que estemos seguros de que los animales con los que trabajamos no se vean sometidos a un estado afectivo negativo debido a su entorno o como consecuencia no deseada de nuestras interacciones con ellos. Nuestro enfoque se centró en métodos que proporcionan poblaciones de control óptimas y aseguran que podamos cuantiﬁcar los cambios conductuales especíﬁcos y relevantes que surgen de las manipulaciones experimentales. A medida que nuestro programa de investigación ha ido evolucionando, hemos ido tomando conciencia del impacto del estrés causado a nuestros animales por los métodos de gestión rutinarios y la contención física durante los procedimientos. ★ ★ ★

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120Animal Technology and Welfare August 2020El uso de ultrasonidos para determinar gestaciones en ratones CAROLINE KARAM Y KIRSTY KEMPUKRI MRC Laboratorio de Biología Molecular de CambridgeCorrespondencia: cperimnt@mrc-lmb.cam.ac.uk, ktrevenn@mrc-lmb.cam.ac.uk ResumenVarios grupos del Laboratorio de Biología Molecular (LMB) del MRC requieren embriones de ratón en etapa temprana para sus programas de investigación cientíﬁca. Tradicionalmente, los zootécnicos se han basado en comprobaciones visuales o en la palpación del abdomen para determinar la gestación en ratones. En 2017, se adquirió una máquina de ultrasonido, ya que el equipo permitiría escanear a los ratones conectados para conﬁrmar el embarazo antes de su sacriﬁcio, proporcionando así una mayor precisión para superar las limitaciones de los métodos existentes. Por tanto, esto permite que las hembras que no parezcan estar embarazadas sean apareadas de nuevo; una mejora de la práctica actual que también reduce el número de animales utilizados para los experimentos. Inicialmente, el uso del equipo de ultrasonidos permitió un aumento del 36 % en la determinación del embarazo en comparación con los métodos tradicionales entre E.7.5 y E12.5. Con el tiempo y más práctica, el procedimiento se fue perfeccionando, lo que permitió detectar con éxito embarazos en los periodos de gestación más tempranos de E6,5 y E5,5.Se ha organizado con éxito un programa interno de formación en ecografía, por lo que ahora se cuenta con varios técnicos competentes. ★ ★ ★Compromiso público y transparencia en la investigación con animales en el CrickJAMIE REDDEN, EWELINA KOZLOWSKA, CLARE BRAZILL-ADAMS, CAROLINE ZVEREV Y SARAH HART-JOHNSONFrancis Crick InstituteCorrespondencia: Jamie.redden@crick.ac.ukIntroducción La implicación del público es una de las prioridades estratégicas del Crick, al tratar de implicar e inspirar al público con el descubrimiento y la ciencia. Dado que gran parte de nuestra ciencia transformadora implica el uso de animales, nuestro objetivo es asegurar que seamos abiertos y transparentes sobre la manera en que utilizamos animales en la ciencia y los beneﬁcios que aportan. Para respaldar este compromiso, el Crick es uno de los más de 100 signatarios del Concordato de Transparencia de la Investigación con Animales (UAR). A medida que más organizaciones se comprometen a ser transparentes sobre el trabajo que realizan con animales, cada vez hay más concienciación sobre los diversos métodos mediante los cuales podemos comunicarnos y comprometernos con el público. Ello permite a las instituciones aprender unas de otras y probar qué métodos son los más efectivos para su modelo operativo, considerando lo que es práctico y alcanzable, al mismo tiempo que logran un impacto signiﬁcativo.A continuación destacamos algunas de las diferentes formas en las que el Crick se ha comprometido con el público para fomentar la transparencia en la investigación con animales, incluidos los diferentes medios y formatos utilizados y los diferentes públicos a los que hemos llegado. Nuestro objetivo es mostrar la variedad de métodos disponibles para las organizaciones, cómo el personal de diversos niveles de antigüedad puede participar en la divulgación, las oportunidades para la colaboración entre departamentos y demostrar cómo un enfoque diverso y variado en la transparencia puede ser más efectivo. ★ ★ ★ Paper Summary Translations49Haven’t the time to write a paper but want to have something published? Then read on!This section offers readers the opportunity to submit informal contributions about anyaspects of Animal Technology. Comments, observations, descriptions of new or refinedtechniques, new products or equipment, old products or equipment adapted to new use,any subject that may be useful to technicians in other institutions. Submissions can bepresented as technical notes and do not need to be structured and can be as short or aslong as is necessary. Accompanying illustrations and/or photos should be high resolution.NB. Descriptions of new products or equipment submitted by manufacturers are welcomebut should be a factual account of the product. However, the Editorial Board gives nowarranty as to the accuracy or fitness for purpose of the product.What 3Rs idea have you developed?EMMA FILBYMira Building, University of Cambridge, University Biomedical Services,Charles Babbage Road, Cambridge CB3 0FSCorrespondence: emma.filby@admin.cam.ac.ukBased on an ar ticle written for the National Centre for the 3RsApril 2020 Animal Technology and We lfareTECH-2-TECHBackgroundEmma was invited to write an article as a 3Rschampion in NC3Rs ‘Tech 3Rs’ Issue 5, November2019.Here is her response describing how she has used anautomated system to reduce how frequently mousecage bedding is changed without compromisingcleanliness.IntroductionOur unit opened in 2017, during the procurement ofnew equipment we had the opportunity to purchase adigital ventilated rack system from Tecniplast UK. Thecages are referred to as the Digitally Ventilated Cage orDVC. This system uses the data collected by sensorsbelow the cage to flag when to clean out based on thechange in an electr omagnetic signal. To have thisfunctionality we first needed to create an algorithmduring a learning phase.The learning phase: devising analgorithmWe held a meeting to agree what warranted a cage basechange based on pictures to avoid being subjective. Wereferred to the Home Office Codes of Practice for thehousing and care of animals bred, supplied or used forscientific purposes (HOCoP) for advice on husbandr ypractices to set our criteria, balancinghygiene and theimportance of olfactory cues to rodents and their needfor control over their environment.1We started the trial, noting when the cage reached thepoint it required a base change. We assessed airquality, what proportion of the cage base was wet andwhether the animals still had choice over theirenvironment and their ability to show spatial separationof different behaviours such as nesting and excretion,for example their nest was free of faeces. During the‘learning phase’ we asked our Named VeterinarySurgeon (NVS) and Home Office inspector (HOI) tocheck that they agreed with our assessment.APRIL_1-628207435_4-628196990.e$S:Animal Technology and Welfare 24/9/20 06:51 Page 49

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121August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAbstractSeveral MRC Laboratory of Molecular Biology (LMB) groups require early-stage mouse embryos for their scientiﬁc research programmes. Traditionally, Animal Technicians have relied on visual checks or palpation of the abdomen to determine pregnancy in mice. In 2017 an ultrasound machine was purchased as the equipment would enable plugged mice to be scanned to conﬁrm pregnancy prior to sacriﬁcing, therefore, providing an increased accuracy to overcome the limitations of the existing methods. Therefore allowing females which do not appear pregnant to be mated again; a reﬁnement to the current practice also reducing the number of animals used for experiments. Initially, use of the ultrasound equipment allowed a 36% increase in determining pregnancy when compared with traditional methods between E.7.5 to E12.5. Given time and further practice the procedure was reﬁned allowing successful detection of pregnancies at the earlier gestation periods of E6.5 and E5.5.A successful in-house ultrasound training programme has been organised and there are now several competent technicians. IntroductionMice used by LMB researchers are housed in the MRC Ares breeding and experimental mouse barrier facility. All mice are bred in-house at the Ares animal facility under speciﬁc pathogen free conditions, at a temperature range of 20 to 24°C, humidity of between 45 to 65% and a 12 hour light-dark cycle. Animals are placed on Rettenmaier SDS RM1 diet with access to an automated watering system and are housed in 49Haven’t the time to write a paper but want to have something published? Then read on!This section offers readers the opportunity to submit informal contributions about anyaspects of Animal Technology. Comments, observations, descriptions of new or refinedtechniques, new products or equipment, old products or equipment adapted to new use,any subject that may be useful to technicians in other institutions. Submissions can bepresented as technical notes and do not need to be structured and can be as short or aslong as is necessary. Accompanying illustrations and/or photos should be high resolution.NB. Descriptions of new products or equipment submitted by manufacturers are welcomebut should be a factual account of the product. However, the Editorial Board gives nowarranty as to the accuracy or fitness for purpose of the product.What 3Rs idea have you developed?EMMA FILBYMira Building, University of Cambridge, University Biomedical Services,Charles Babbage Road, Cambridge CB3 0FSCorrespondence: emma.filby@admin.cam.ac.ukBased on an ar ticle written for the National Centre for the 3RsApril 2020 Animal Technology and We lfareTECH-2-TECHBackgroundEmma was invited to write an article as a 3Rschampion in NC3Rs ‘Tech 3Rs’ Issue 5, November2019.Here is her response describing how she has used anautomated system to reduce how frequently mousecage bedding is changed without compromisingcleanliness.IntroductionOur unit opened in 2017, during the procurement ofnew equipment we had the opportunity to purchase adigital ventilated rack system from Tecniplast UK. Thecages are referred to as the Digitally Ventilated Cage orDVC. This system uses the data collected by sensorsbelow the cage to flag when to clean out based on thechange in an electr omagnetic signal. To have thisfunctionality we first needed to create an algorithmduring a learning phase.The learning phase: devising analgorithmWe held a meeting to agree what warranted a cage basechange based on pictures to avoid being subjective. Wereferred to the Home Office Codes of Practice for thehousing and care of animals bred, supplied or used forscientific purposes (HOCoP) for advice on husbandr ypractices to set our criteria, balancinghygiene and theimportance of olfactory cues to rodents and their needfor control over their environment.1We started the trial, noting when the cage reached thepoint it required a base change. We assessed airquality, what proportion of the cage base was wet andwhether the animals still had choice over theirenvironment and their ability to show spatial separationof different behaviours such as nesting and excretion,for example their nest was free of faeces. During the‘learning phase’ we asked our Named VeterinarySurgeon (NVS) and Home Office inspector (HOI) tocheck that they agreed with our assessment.APRIL_1-628207435_4-628196990.e$S:Animal Technology and Welfare 24/9/20 06:51 Page 49TECH-2-TECHUltrasound to ultrasee – the use of ultrasound to determine pregnancy in miceCAROLINE KARAM and KIRSTY KEMP UKRI MRC Laboratory of Molecular Biology CambridgeCorrespondence: cperimnt@mrc-lmb.cam.ac.uk, ktrevenn@mrc-lmb.cam.ac.uk August 2024 Animal Technology and Welfare

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122Animal Technology and Welfare August 2020Tecniplast GM500 Green Line individually ventilated cages (IVCs)1 with woodchip bedding, sizzlenest nesting, wood blocks, diamond twist and fun tunnel for enrichment.2One of the research groups needed early time point embryos at E8.5. The females required for these experiments could have rare multi-allele genotypes i.e. only 1 in 25 pups could be the desired genotype. For these time points the traditional methods of pregnancy checking could not be applied, as visual checks are not possible until E12.5 and palpation until E9.5. Both methods are not accurate and can be subjective depending on who performs them. Palpating can also be quite stressful for females and can lead to further complications if not carried out correctly. Due to these limitations any timed mated mice for experiments requiring embryos at earlier time points were being sacriﬁ ced without the certainty of a successful pregnancy. This is undesirable as it led to unnecessary animal wastage. In 2017, an ultrasound scanner was purchased to reﬁ ne the detection of pregnancies. Following a period of training and practice, successful pregnancy scanning was being routinely achieved down to E7.5 with great success. In 2022, new research needs required lower time points of E5.5 and retraining was undertaken to cover these time points. The ultrasound device uses high frequency sound waves to create images of the inside of the body. The ultrasound travels through soft tissue and ﬂ uids then bounces backoff denser surfaces. The denser the object the ultrasoundhits, the more of the ultrasound bounces back. The bouncing back, or echo, is what gives the ultrasound imageits features. Varying shades of grey reﬂ ect different densities i.e. bone appears whiter where tissues are greyer.Some of the uses for the ultrasound in other facilities include blood ﬂ ow measurements3, tumour checks4, organ imaging and embryo injections5 however at Ares, ultrasound scanning is applied to conﬁ rm pregnancy or not. Mice are then either sent to the researcher for samples or experiments if pregnant and if not, they are reused for another mating as well as for checking the success of pregnancy using rotating males to speed up production.MethodOften animals are required at a speciﬁ c time point ofgestation to meet the scientiﬁ c needs. The most accurate way to do this is to arrange a timed mating. When a time point is established, the mating can then be arranged placing the two animals together until a copulation or vaginal plug (Figure 1) is found in the female and then the female will be separated from the male.6 The time pointwhen the plug is found is referred to as E0.5 (half a day) due to the nature of mice generally mating overnight. Despite the presence of a plug this does not conﬁ rm pregnancy.Ultrasound to ultrasee – the use of ultrasound to determine pregnancy in miceThere are ways of increasing the chance of a mating occurring over a required time frame and this can be doneby either seeding the cage or oestrus checking the female. Seeding the cage requires taking a small amount of malebedding and placing it in the female’s cage a day or two before the mating. Smelling the scent of the male will trigger the female to come into oestrus.Oestrus checking involves visually checking the female todetermine at what point she will be in oestrus (Figure 2),or this can be determined by producing vaginal smears of cells under a microscope.7 Although in this case visualchecks are used as they are less invasive and this is the preferred method used within Ares.confirm pregnancy or not. Mice are then either sent to the researcher for samples or experiments if pregnant and if not, they are reused for another mating as well as for checking the success of pregnancy using rotating males to speed up production. Method Often animals are required at a specific time point of gestation to meet the scientific needs. The most accurate way to do this is to arrange a timed mating. When a time point is established, the mating can then be arranged placing the two animals together until a copulation or vaginal plug (figure 1) is found in the female and then the female will be separated from the male. The time point when the plug is found is referred to as E0.5 (half a day) due to the nature of mice generally mating overnight. Despite the presence of a plug this doesn’t confirm pregnancy. Figure 1. An albino mouse with copulation plug.Figure 2. The four stages of oestrus. Figure 1 An albino mouse with copulation plug6 There are ways of increasing the chance of a mating occurring over a required time frame and this can be done by either seeding the cage or oestrus checking the female. Seeding the cage requires taking a small amount of male bedding and placing it in the female’s cage a day or two before the mating. Smelling the scent of the male will trigger the female to come into oestrus. Oestrus checking involves visually checking the female to determine at what point she will be in oestrus (figure 2), or this can be determined by producing vaginal smears of cells under a microscope. Although, in this case visual checks are used as they are less invasive and this is the preferred method used within Ares. Figure 1 An albino mouse with copulation plug6 There are ways of increasing the chance of a mating occurring over a required time frame and this can be done by either seeding the cage or oestrus checking the female. Seeding the cage requires taking a small amount of male bedding and placing it in the female’s cage a day or two before the mating. Smelling the scent of the male will trigger the female to come into oestrus. Oestrus checking involves visually checking the female to determine at what point she will be in oestrus (figure 2), or this can be determined by producing vaginal smears of cells under a microscope. Although, in this case visual checks are used as they are less invasive and this is the preferred method used within Ares. A – Proestrus B – OestrusC – Metoestrus D – Dioestrus

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123August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareOnce a female at the correct stage of oestrus is identiﬁ ed she can be placed with the male where mating will usually occur overnight.Oestrus checking is normally the quickest form of timemating but usually is most suited to wildtype lines. In most cases, a pool of wildtype females can be assessed to collect the animals required. When this involves transgenic animals, where a particular genotype is required, this can be harder to do making it a longer process.The equipment used to carry out the scans is as follows: (Figure 3 and 4)• mice – transgenic and wildtype (Hsd:ICR-CD1) lines • 70% ethanol• ultrasound gel• disinfecting hand spray• scanning probe suitable to species• LOGIQ e ultrasound monitor8• GE L8-18i-D hockey stick probe9Ultrasound to ultrasee – the use of ultrasound to determine pregnancy in miceTwo main types of scans are carried out which are thepelvis and spinal scans. The pelvis scan involves placingthe probe horizontally to give a clear view of the pelvis and then the probe is turned vertically to give a spinal view. Once these are done, the female is cleaned as much as possible before being placed gently back into the cage. Due to the mice not being anaesthetised and the short time the process takes, the mice show no signs of stress or exhibit behavioural changes.On average the whole process from start to ﬁ nish takes around 50 seconds. This can vary depending on the sizeof the mouse and gestational time point.The pelvic scan (Figure 5) is a scan showing the method used to produce a pelvic image.9 Gentle pressure is applied across the top of the hips.• mice – transgenic and wildtype (Hsd:ICR-CD1) lines • 70% ethanol • ultrasound gel • disinfecting hand spray • scanning probe suitable to species • LOGIQ e ultrasound monitor8 • GE L8-18i-D hockey stick probe9 Figure 3 LOGIO e device8 Ares is a non-tail handling unit and using this method of handling further reduces the stress that could be caused to the female during the scanning process. The aim is to produce a clear scan that will confirm the presence of pups without keeping the female away from Figure 3. LOGIO e device.Ares is a non-tail handling unit and using this method of handling further reduces the stress that could be caused to the female during the scanning process. The aim is to produce a clear scan that will conﬁ rm the presence of pups without keeping the female away from their cage too long. A researcher at the LMB with previous experience of scanning mice said that it was necessary to anaesthetise and shave the mouse abdomen. We have reﬁ ned this by carrying out a normal scruff to restrain the mouse and then applying 70% ethanol to the stomach and smoothing down the fur to remove any air bubbles that may distort the image. Other solutions were tested to see if the percentage of ethanol could be reduced or eliminated, however it was found these were not as effective. Ultrasound gel (the same type as used on humans) is then applied to the abdomen and this is what provides the connection between the body and probe to allow the sound waves to pass through. their cage too long. A researcher at the LMB with previous experience of scanning mice said that it was necessary to anaesthetise and shave the mouse abdomen. We have refined this by carrying out a normal scruff to restrain the mouse and then applying 70% ethanol to the stomach and smoothing down the fur to remove any air bubbles that may distort the image. Other solutions were tested to see if the percentage of ethanol could be reduced or eliminated, however, it was found these were not as effective. Ultrasound gel (the same as the one used on humans) is then applied to the abdomen and this is what provides the connection between the body and probe to allow the sound waves to pass through. Figure 4 set up required to perform a scan Figure 4. Set up required to perform a scan. Two main types of scans are carried out which are the pelvis and spinal scans. The pelvis scan involves placing the probe horizontally to give a clear view of the pelvis and then the probe is turned vertically to give a spinal view. Once these are done, the female is cleaned as much as possible before being placed gently back into the cage. Due to the mice not being anaesthetised and the short time the process takes the mice show no signs of stress or exhibit behavioural changes. On average the whole process from start to finish takes around 50 seconds. This can vary depending on the size of the mouse and gestational time point. The pelvic scan (figure 5) is a scan showing the method used to produce a pelvic image. Gentle pressure is applied across the top of the hips. Figure 5. Pelvic scan using hockey stick probe.

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124Animal Technology and Welfare August 2020The image in Figure 6 is produced when using this method.In the centre of the screen, the pelvis can be seen withleft and right femurs branching off at each side. Whensearching for pregnancy, the areas above the pelvis arelooked at usually to the left and right.When completing a scan, both of these angles are used as this allows for conﬁ rmation of what has been seen withno further restraint to the animal.We have completed scans over a range of different timepoints. Below is a time line of different gestational stagesfor a mouse, and shows the difference a day can make (Figures 8- 15). Figure 5 pelvic scan using hockey stick probe9 The image in figure 6 is produced when using this method. In the centre of the screen, the pelvis can be seen with left and right femurs branching off at each side. When searching for pregnancy, the areas above the pelvis are looked at usually to the left and right. Figure 6 ultrasound image of pelvic scan Figure 6. Ultrasound image of pelvic scan.The next angle is the spinal view, and to achieve this, the probe is held vertically which allows the side profile of the spinal column to be seen (Figure 7). Figure 7 spinal scan position and ultrasound image of a spinal scan When completing a scan, both of these angles are used as this allows for confirmation of what has been seen with no further restraint to the animal. We have completed scans over a range of different time points. Below is a time line of different gestational stages for a mouse, and shows the difference a day can make (Figures 8- 15). Figure 7. Spinal scan position and ultrasound image of aspinal scan.The next angle is the spinal view and to achieve this theprobe is held vertically which allows the side proﬁ le of the spinal column to be seen (Figure 7).The next angle is the spinal view, and to achieve this, the probe is held vertically which allows the side profile of the spinal column to be seen (Figure 7). Figure 7 spinal scan position and ultrasound image of a spinal scan When completing a scan, both of these angles are used as this allows for confirmation of what has been seen with no further restraint to the animal. We have completed scans over a range of different time points. Below is a time line of different gestational stages for a mouse, and shows the difference a day can make (Figures 8- 15). Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 8. Day 5.5 – The earliest time point successfully scanned.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 9. Day 6.5 – Cavities appear empty but located high.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 10. Day 7.5 – Multiple round empty cavities can be seen closerto the pelvis.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 11. Day 8.5 – Cavities increase in size but are still empty.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 12. Day 9.5 –Cavities larger with some greying mass starting to appear.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 13. Day 10.5 – Bone mass has started todevelop and can be seen in pale grey – heartbeats can be seen as a slight ﬂ icker.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 14. Day 11.5 – Thepups’ bones become denser and can be seen more clearly.Figure 10 Day 7.5-Multiple round empty cavities can be seen closer to the pelvis.ResultsFigure 8 day 5.5 – the earliest time point successfully scannedFigure 9 Day 6.5 –cavities appear empty but located high.Figure 11 day 8.5-cavities increase in size but are still empty.Figure 12 day 9.5- cavities larger with some greying mass starting to appear.Figure 13 day 10.5- bone mass has started to develop and can be seen in pale grey- heartbeats can be seen as a slight flicker.Figure 14 day 11.5-the pups bones become denser and can be seen more clearly.Figure 15 Day 15.5- Pups appears fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Figure 15. Day 15.5 – Pups appear fully formed. Eye sockets can be seen along with the rib cage and limbs. Pups can be seen moving at this point.Ultrasound to ultrasee – the use of ultrasound to determine pregnancy in mice

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125August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareResultsDuring 2018, data was collected (Figure 16) and scans carried out between and including E7.5 to E12.5. From these scans 91% were correctly identiﬁ ed as pregnant or not pregnant. The animals that were not pregnant could be reused for future time matings, 2% were incorrectly scanned and kept as not pregnant but went on to have litters which were used for colony expansion with the females being reused for time matings when cleared. 7% were scanned incorrectly and determined as pregnant. Based on the time points of these animals before the ultrasound 45% would have been used regardless as there would have been no way to conﬁ rm pregnancy and only 55% could have been visually checked or palpated but as already mentioned these methods are not ideal and this means by scanning the animals, accuracy has increased by 36%.Moving on to 2023 when new research required embryos at E5.5 and E6.5, data was collected (ﬁ gures 17 and 18) from 244 scans at these time points. 85% were scanned correctly as pregnant and not pregnant, again allowing the reuse of animals that were not pregnant. 12% were scanned incorrectly as pregnant and used unnecessarily. However before the use of ultrasound scanning was introduced all these early time point animals would have been used regardless with 41% of these were not pregnant. Ultrasound scanning has decreased unnecessary sacriﬁ cing of valuable animals by 29%. During this period scanning at these earlier time points was still being perfected, therefore moving into 2024 the aim is to see accuracy increasing further.DiscussionWhen the ultrasound was ﬁ rst purchased, the basics ofhow to use the machine and how to ﬁ nd and use the pelvis as a focal point were learnt. After this there wasno external training available, self-learning was requiredto identify what the embryos looked like on an ultrasound,starting with visibly pregnant time mated animals. Once each embryo time point was perfected and conﬁ dence gained, earlier days were worked on by decreasing one day at a time. This continued until E7.5 was reached and this initial training took around 6 months. When the need for earlier time points arose, the same process was followed until pups could be identiﬁ ed consistently at E5.5.All training is held in-house and the same pathway that was originally used is followed with new trainees, starting at E11.5 and as conﬁ dence and competence is achieved, the time point is lowered. Once trainees can complete a scan at E8.5 competence is assessed as this is the lowest commonly used time point. Training plansneed to be tailored to the individual as some trainees can analyse the screen image well whereas others are stronger at scanning and some take that bit longer to connect the two parts of the process. One thing that is the same for all trainees is that consistency is the key. Regular practice must take place to build up skill and conﬁ dence. Trainees could be ready for assessment afteraround two months.When carrying out scanning, a mixture of transgenic andwildtype mice were used. More commonly in the wildtype CD1 line, it has been found that embryos from these Figure 16. Data of scans completed between E7.5 and E11.5 gathered in 2018.Figure 17. Percentage of animals pregnant and not pregnant that were used for experiments needing embryos at E5.5 and E6.5.Figure 18. Data of scans completed between E5.5 andE6.5 gathered in 2023.Ultrasound to ultrasee – the use of ultrasound to determine pregnancy in miceE7.5 – E11.5 ResultsCorrect – Pregnant and Not PregnantIncorrect – Not Pregnant Kept and ReusedIncorrect – Not Pregnant and Sent Out91%7%2%2023 Ultrasound Data E5.5 – E6.5Correct – Pregnant and Not PregnantIncorrect – Not Pregnant Kept and ReusedIncorrect – Not Pregnant and Sent Out85%12%3%Before Ultrasound E5.5 – E6.5PregnantNot Pregnant 59%41%

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126Animal Technology and Welfare August 2020animals appear larger at the point of scan with more cavities present than those of the transgenic animals, although larger litters are more common for this line. As a consequence of these larger embryos, this can sometimes lead to incorrect assumptions that the embryos are at different time points from expected. Conﬁrmation of the correct time points is made at the time of dissection when embryo development is assessed.When handling animals for scanning, if often appears that those which are pregnant display behavioural differences from those that are not. Although all animals are handled using non-tail handling methods to reduce stress, these animals can appear more aggressive. Considering this behaviour combined with the observational evidence from the scan can help in pregnancy diagnosis. Behaviour varies from strain to strain but aggression does seem to be more prominent in transgenic lines compared to wildtypes.The use of ultrasound scanning for pregnancy has also allowed the animals to be viewed for health checks. On occasions animals have been observed to show signs of a blocked bladder, observed as a very large dark cavity in the centre of the screen. Another heath concern which has been observed has been ﬂuid ﬁlled kidneys. This again appears as a very large cavity but on one side of the animal. These animals have not presented any obvious signs of ill health at the time but have been able to provide an early warning to the research group before they make use of the animal.Ares have been very involved with the development of this idea and rate its use but also understand the need to be mindful of the caveats. – It is not possible to accurately count numbers of pups.– It is not possible to accurately determine gestation without a timed mating but can estimate.– Overweight animals are more difﬁcult to scan.– Cost of equipment.But despite this, the 3Rs beneﬁts far outweigh the limitations.Pregnancy can now be conﬁrmed at earlier time points and prevent animals from being unnecessarily used. This process itself is far less invasive and it has been possible with time and practice to make further reﬁnements and it is no longer necessary to use anaesthetics or shave the animals.In regard to pup numbers an estimation can be given which allows the opportunity to hold animals back if a litter appears small with the hope of a larger litter with the second pregnancy. This together with holding back non-pregnant females has enabled the reduction of the number of females unnecessarily used which in turn reduces numbers bred for use in these experiments. Sometimes the use of animals of the correct genotype can be limited and selection of age-appropriate animals can be difﬁcult. In these cases, animals are more commonly overweight causing the visibility of the pups to be difﬁcult to see. However in these cases the settings can be adjusted on the ultrasound machine. The depth of the sound waves can be increased, also known as the gain and this can aid bringing pups into focus through the fat tissue of the mouse. Where possible the use of younger animals is more appropriate and steps have been taken to advise better colony management such as assisting with the selection of younger females where success rates are greatly improved.This project has been expanded beyond the original expectations and is now used across the whole facility. The technique is a far more accurate way of highlighting pregnancy and a successful in-house training plan has been put together expanding our number of competent staff. We have been extremely happy with the progress of our work using ultrasound scanning to increase the rate of successful pregnancy detection in timed mated mice and will strive to continue to develop this further and promote its use, especially as it is a great 3Rs success, incorporating a fantastic reﬁnement of a traditional procedure, increasing our success rate of pregnancy determination and therefore enabling a considerable reduction in the numbers of mice sacriﬁced unnecessarily. In the future, further work is planned to explore if the numbers within a litter impacts the visibility of embryos visualised using the ultrasound scanner. Data will be gathered of numbers of embryos collected for research studies and then compared to see how numbers impact visibility.AcknowledgementsAll LMB researchers who have supplied data.Lesley Drynan – LMB Head of Biological Services Group.Ares staff.This work was supported by the Medical Research Council, as part of United Kingdom Research and Innovation (also known as UK Research and Innovation) [MC_U105184326]. For open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising.Ultrasound to ultrasee – the use of ultrasound to determine pregnancy in mice

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127August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareReferences1 Tecniplast Green Line GM500 IVC, https://www.tecniplast.it/en/product/dgm-digital-ready-ivc.html2 Datesand enrichment used in Ares mouse cages, https://www.datesand.com/subcategory/disposable-enrichmenthttps://www.datesand.com/product_page/sizzlenest3Moran, C.M., and Thomson, A.J.W. Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom. Preclinical Ultrasound Imaging – A Review of Techniques and Imaging Applications, https://www.frontiersin.org/journals/physics/articles/10.3389/fphy.2020.00124/full4Chambers, L.M., Esakov, E., Braley, C., AlHilli, M., Michener, C., and Reizes, O. Use of Transabdominal Ultrasound for the detection of intra-peritoneal tumor engraftment and growth in mouse xenografts of epithelial ovarian cancer, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190129/5Coatney, R.W. Ultrasound imaging: principles and applications in rodent research, https://academic.oup.com/ilarjournal/article/42/3/233/7814316Byers, S.L., Wiles, M.V., Dunn, S.L., and Taft, R.A. Mouse Estrous Cycles Identiﬁ cation Tool andImages, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325956/7 Reproductive Engineering Techniques in Mice, http://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/transfer.html8 GE HealthCare LOGIQ e Ultrasound Machine, https://gehealthcare-ultrasound.com/en/logiq-family/logiq-e/9 GE L8-18i-D Hockey Stick Probe, https://ameultrasounds.com/products/ge-l8-18i-d-hockey-stick-probeUltrasound to ultrasee – the use of ultrasound to determine pregnancy in miceTechnicians and Vets: a partnership for animal welfareCALL FOR PAPERSl take an active part in the UK’s leading annual meeting for our industryl present a paper and qualify for free attendance at Congressl make this your debut presentation year – ﬁrst time presenter papers are only 20 minutes long and as well as a free congress there is a prize for the one judged to be the bestl send your ideas today on the Submission form available from www.iat.org.ukl ﬁnal date for submissions: Friday 29th November 2024Contact: congress@iat.org.ukCongress2025CONGRESS Invitation to Participate4th March – 7th MarchANNIVERSARY1950 � 2025

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128151IntroductionA hallmark symptom of rheumatoid arthritis in humansis painful swollen joints. Pain can manifest before anyinflammation is noticeable1,2as well as persist longafter inflammation has resolved.3In rodent models of arthritis, ankle or footpad width isa commonly used surrogate marker of pain (seeFigure 1).Measur ing footpad width assumes that increasedswelling is proportional to enhanced pain. A mildarthritis phenotype in which there is minimal swellingmay therefore inaccurately reflect the extent of painand discomfort.POSTER PRESENTATIONSOriginally presented at:IAT Congress 2019Assessing pain in models ofRheumatoid ArthritisSAMUEL SINGLETON,1MERIAM NEFLA,1NGAIRE DENNISON,1SIMON ARTHUR2and TIM HALES1School of Life Sciences, Division of Cell Signalling and Immunology, University of Dundee,Dundee, DD1 5EH, UK2MRSU and Institute of Academic Anaesthesia, Division of Systems Medicine, NinewellsHospital, University of Dundee, DD1 9SY, UKCorrespondence: s.z.singleton@dundee.ac.ukAugust 2020 Animal Technology and WelfareFigure 1. Footpad width as a surrogate measure of pain in arthritis models. Commonly used methods to assess painare footpad width (A), ankle width (B) or footpad ankle length (C).BCAAim: We aimed to determine how well pain correlated to footpad widths using the collagen antibody arthritismodel.August20:Animal Technology and Welfare 12/8/20 07:54 Page 151WE NEED YOU!Have you made a difference in your work place?The IAT Branch Liaison Committee are looking for 100 greatimprovements that have been made in the industry. This couldbe anything.Follow the link or the QR code to submit your ideahttps://forms.office.com/e/E6A46nKd6KWE NEED YOU!Have you made a difference in your work place?The IAT Branch Liaison Committee are looking for 100 greatimprovements that have been made in the industry. This couldbe anything.Follow the link or the QR code to submit your ideahttps://forms.office.com/e/E6A46nKd6K

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129August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePOSTER PRESENTATIONS151IntroductionA hallmark symptom of rheumatoid arthritis in humansis painful swollen joints. Pain can manifest before anyinflammation is noticeable1,2as well as persist longafter inflammation has resolved.3In rodent models of arthritis, ankle or footpad width isa commonly used surrogat e marker of pain (seeFigure 1).Measur ing footpad width assumes that increasedswelling is proportional to enhanced pain. A mildarthritis phenotype in which there is minimal swellingmay therefore inaccurately reflect the extent of painand discomfort.POSTER PRESENTATIONSOriginally presented at:IAT Congress 2019Assessing pain in models ofRheumatoid ArthritisSAMUEL SINGLETON,1MERIAM NEFLA,1NGAIRE DENNISON,1SIMON ARTHUR2and TIM HALES1School of Life Sciences, Division of Cell Signalling and Immunology, University of Dundee,Dundee, DD1 5EH, UK2MRSU and Institute of Academic Anaesthesia, Division of Systems Medicine, NinewellsHospital, University of Dundee, DD1 9SY, UKCorrespondence: s.z.singleton@dundee.ac.ukAugust 2020 Animal Technology and WelfareFigure 1. Footpad width as a surrogate measure of pain in arthritis models. Commonly used methods to assess painare footpad width (A), ankle width (B) or footpad ankle length (C).BCAAim: We aimed to determine how well pain correlated to footpad widths using the collagen antibody arthritismodel.August20:Animal Technology and Welfare 12/8/20 07:54 Page 151IntroductionAgeing is deﬁned as a deterioration of physiological functions which subsequently leads to age-related illnesses and clinical frailty. To study the ageing process, it is important to look at the cumulative effects on the body.Research into ageing is essential to provide a better understanding of the biological processes and offer possible advancements.4Mice are a good model of ageing as they are physiologically similar and share approximately 95% of their genome to humans.1In terms of clinical frailty, mice also age in a comparable way and show many of the same clinical signs such as reduced muscle mass, weight loss, changes of gait and the development of age-related illnesses.2The main aim of this project was to review the husbandry processes in place for the colony of aged C57BL/6 mice at the Babraham Institute, establish a reﬁned husbandry process and improve local guidance. Secondly, by analysing fate and clinical observation data between 2021 and 2023 this project aimed to assess the impact of these improved routines on the identiﬁcation of health concerns and the number of mice found dead at routine health checks.Establishing a processAs humans age, they rely more on doctors, community nurses and other healthcare professionals to monitor their health and assist them where necessary.4 Those that provide this care are often considered experts in their ﬁeld and have a high level of understanding regarding clinical frailty in humans. When considering the use of animal models in ageing research it is therefore important to ensure that those caring for the animals also use the appropriate welfare indicators to assess the signs of frailty and have a good understanding of the difference between a sign of ageing and a sign of ill health.5 A healthy aged mouse will often begin to show subtle signs of frailty from around 17 months old.3 Some key signs of frailty in mice are changes in coat colour, stiffening of the tail, changes in gait and curvature Following posters originally presented at: IAT Congress 2024Hands-on reﬁnements: establishing improved husbandry processes for aged C57BL/6 mice AIMEE PATERSONBabraham InstituteCorrespondence: aimee-paterson@hotmail.com Sponsored byAugust 2024 Animal Technology and Welfare

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131August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe cohorts were grouped by the year in which the ageing procedure was added. The data shows the ﬁnal fate recorded for all aged mice in the 2021 and 2022 cohort. The 2023 cohort shows that there are a number of mice still alive at the time of data collection. However it is still consistent with the 2022 data showing a reduced number of mice found dead.Results– All data was collected from the Mouse Colony Management System (MCMS) in January 2024.– The fate data was grouped by the year in which the ageing procedure was recorded between 2021, 2022 and 2023.– The health observation data was grouped by the year in which the observation was recorded.Fate dataA comparison of the fate data revealed a signiﬁcant change in the number of mice found dead and culled sick between the aged cohorts of 2021, 2022 and 2023. In the 2021 cohort, 14 mice were found dead. This decreased to 10 mice found dead in the 2022 cohort. Further reduction was observed in the 2023 cohort where only 4 mice were found dead.The number of mice culled sick showed an increase over the cohorts. In the 2021 cohort, 63 mice were culled sick. This increased to 77 mice were culled sick in the 2022 cohort. Finally, in the 2023 cohort 84 mice were culled sick. A Chi-squared test was conducted to examine the association between the cohort year and the number of mice found dead and culled sick. The results indicated a statistically signiﬁcant association (χ² = 7.752, df = 2, p = 0.0207), suggesting that the probability of ﬁnding mice dead or culled sick varied signiﬁcantly across the different cohorts.Health DataA comparison of the health data revealed a signiﬁcant change in the number and type of health concerns identiﬁed in 2022 and 2023. The data demonstrate a considerable increase in the incidence of several health observations from 2022 to 2023. Speciﬁcally, there was a signiﬁcant increase in the observation of swollen ankles (0 in 2022 to 17 in 2023), palpable masses (1 in 2022 to 9 in 2023), swollen abdomens (5 in 2022 to 12 in 2023), and weight loss (16 in 2022 to 42 in 2023). However there was no difference in the number of abnormal movements recorded (1 in both 2022 and 2023).A Fisher’s Exact Test was conducted to examine the association between the health observation categories and the years 2022 and 2023. The results of the test indicated a statistically signiﬁcant association (p = 0.0387, at a statistically signiﬁcant level of p < 0.05), suggesting that the probability of observing these health concerns varied signiﬁcantly between the two years.A Fisher’s exact test was conducted to examine the association between the health observation categories and the years 2022 and 2023. The results of the test indicated a statistically significant association (p = 0.0387, at a statistically significant level of p < 0.05), suggesting that the probability of observing these health concerns varied significantly between the two years. DiscussionThe ﬁndings of this project show that there have been signiﬁcant changes in the fates recorded of aged C57BL/6 mice between 2021 and 2023. Alongside this, there was a notable increase in the number of health concerns recorded. These ﬁndings underscore the importance of continually reﬁning husbandry processes and highlight the potential for even small changes to have a signiﬁcant impact on mouse colony management.The observed reduction in the number of mice found dead over the cohorts suggests that by reﬁning the in-hand checking process, we have been able to increase early identiﬁcation of health observations leading to quicker intervention. This claim is also supported by the overall trend in health observations. In 2023, the number of health observations recorded in speciﬁc categories increased dramatically following the introduction of weekly in-hand checks.Future planned projects in this area will continue to assess the impact of daily routines on aged mice. Factors such as noise levels in the room, equipment e.g. change stations, enrichment and chemicals used to disinfect hands will be considered. By conducting comprehensive evaluations of these factors, Animal Technicians can identify additional opportunities for improvement in husbandry practices and further enhance the welfare of aged mouse colonies.Poster Presentations

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132Animal Technology and Welfare August 2020Poster PresentationsConclusionThese ﬁ ndings highlight the importance of continually reviewing and reﬁ ning husbandry processes in mouse colony management. The observed changes in this projectdemonstrate the signiﬁ cant impact that small changes can make. Moreover, the commitment to honesty and improvement underscores the ethical responsibility of Animal Technicians to prioritise Animal Welfare and strivefor excellence in animal research practices.AcknowledgementWith special thanks to Hayley Carr for the statistical analysis and everyone in the Biological Support Unit for their hard work.References1 Bryda, E.C. (2013). The Mighty Mouse: the impact of rodents on advances in biomedical research. Mo Med, 110(3), 207-211. PMID: 23829104; PMCID: PMC3987984.Technicians and Vets: a partnership for animal welfareCALL FOR POSTERSCongress2025CONGRESS Invitation to Participate4th March – 7th Marchl take an active part in the UK’s leading annual meeting for our industryl submit a poster – ﬁnal date Friday 31st January 2025*l consider giving an oral presentation of your poster as part of the main scientiﬁc programme, it could also be accepted within the ﬁrst time presenter’s category – closing date Friday 29th November 2024*l send your ideas today on the Submission form available from www.iat.org.uk (*posters will not be accepted for display at Congress unless they have been properly submitted and approved by the Congress Committee)l two best posters will receive a prize based on the criteria: animal welfare / scientiﬁc contentl plus claim up to 10 CPD points – details given on acceptance of submissionContact: congress@iat.org.ukANNIVERSARY1950 � 20252 Baumann, C.W., Kwak, D., & Thompson, L.V. (2018).Assessing onset, prevalence and survival in mice using a frailty phenotype. Aging (Albany NY), 10(12), 4042-4053. doi: 10.18632/aging.101692. PMID: 30562163; PMCID: PMC6326660.3Baumann, C.W., Kwak, D., & Thompson, L.V. (2020).Phenotypic Frailty Assessment in Mice: Development, Discoveries, and Experimental Considerations. Physiology, 35(6), 405-414.4Wilkinson, M.J., Selman, C., McLaughlin, L., Horan,L., Hamilton, L., Gilbert, C., Chadwick, C., & Flynn, J.N. (2020). Progressing the care, husbandry andmanagement of ageing mice used in scientiﬁ c studies.Lab Anim, 54(3), 225-238. doi: 10.1177/ 0023677219865291. PMID: 31403890; PMCID: PMC7301645.5Hawkins, P., Morton, D.B., Burman, O., Dennison, N.,Honess, P., Jennings, M., Lane, S., Middleton, V.,Roughan, J.V., Wells, S., & Westwood, K.; UK JointWorking Group on Reﬁ nement BVAAWF/FRAME/RSPCA/UFAW. (2011). A guide to deﬁ ning and implementing protocols for the welfare assessment of laboratoryanimals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Reﬁ nement. Lab Anim, 45(1), 1-13. doi: 10.1258/la.2010.010031. PMID: 21123303.

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133August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2024 Animal Technology and WelfareIntroductionThe Charles River (CRL) 4Rs Mission strives to advance science by improving upon the 3Rs principle which was introduced by Russell and Burch1 in 1960 and focussed on enhancing animal wellbeing through emphasis on an imperative R – Responsibility.The four terms are:– Replacement: to avoid or replace the use of animals.– Reduction: to minimise the number of animals.– Reﬁnement: minimise pain, stress and suffering.– Responsibility: leading progressive change regarding Animal Welfare.Non-invasive sampling methods for genotyping: improvements towards the 4r principleSIVATHARSINI THASIAN-SIVARAJAH, REBECCA MOHR, MARIA WALTER, JOHN GBADEGOYE and MIRIAM HOPFECharles RiverCorrespondence: John.Gbadegoye@crl.com In research involving genetically modiﬁed mice which is usually to perform genotyping using invasive ear or tail biopsies in 92% of cases,2 there is a growing shift towards non-invasive sampling techniques such as oral swabs, hair, faeces and tears. Our European Union (EU) Charles River genetic testing laboratory has successfully tested and proven the efﬁcacy of genotyping using oral swabs (from mice and rabbits) and hair (from mice), aligning with the 4R principle and European regulations. The advantages of using non-invasive methods such as oral swabs and hair instead of biopsies is shown in Figure 1.Figure 1. • 4Rs of Animal Welfare ➞ Reﬁnement• Non-invasive alternative• Decreased risk of cross-contamination*• Large scale automated workﬂow• Turnaround time of 1-3 days• Suitable for every kind of PCR analysis* with oral swabs onlyBeneﬁts• Standard Genotyping analysis• Sampling if second invasive biopsy is not possible• Homozygous lines and old animals• Animals identiﬁed with ear tags/tattoos• Conﬁrmation before/during experimentsApplications

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134Animal Technology and Welfare August 2020MethodResearch ConditionsFor the data represented here, both male and female mice from different transgenic lines were used. Due toanimal wellbeing and the size of the swab head all animals were at least 16 days old while oral swab and hair follicle samples were taken. Several swab-types were tested in terms of the cotton head size, the surface structure and the quality of the genotyping results. Based on this pre-test, the optimal type of swab regarding our whole genotyping process was determined. Besides that, a thorough sampling procedure is important to acquire sufﬁ cient animal tissue for further processing. SamplingOral swab samples of the mice were taken, as shown in Figure 2. Brieﬂ y, swabs were autoclaved and brought into the animal barrier facility according to standard procedures. Mice were securely scruffed and the swab was twirled around for 5 to 19 seconds to collect the sample from the inside the cheek (Figure 2A). While swabbing it was ensured to not hurt the mice. Hair follicle samples were obtained by carefully plucking a small amount of hair (10-20 units) (Figure 2B). After sampling, mice were placed back into their cages. The oral swabs were left to dry before placing each swab andhair sample into individual tubes. Ear biopsies taken forroutine genotyping analyses from the same animals wereused as controls. Finally, the samples were shipped tothe genotyping facility. All samples were taken at AAALAC accredited CRL sites according to Animal Welfare rules and guidelines.Figure 2A and 2B. Sampling methods.type of swab regarding our whole genotyping process was determined. Besides that, a thorough sampling procedure is important to acquire sufficient animal tissue for further processing. Sampling Oral swab samples of the mice were taken, as shown in figure 2. Briefly, swabs were autoclaved and brought into the animal barrier facility according to standard procedures. Mice were securely scruffed and the swab was twirled around for 5 to 19 seconds to collect the sample from the inside the cheek (figure 2A). While swabbing it was ensured to not hurt the mice. Hair follicle samples were obtained by carefully plucking a small amount of hair (10-20 units) (figure 2B). After sampling, mice were placed back into their cages. The oral swabs were left to dry before placing each swab and hair sample into individual tubes. Ear biopsies taken for routine genotyping analyses from the same animals were used as controls. Finally, the samples were shipped to the genotyping facility. All samples were taken at AAALAC accredited CRL sites according to Animal Welfare rules and guidelines. type of swab regarding our whole genotyping process was determined. Besides that, a thorough sampling procedure is important to acquire sufficient animal tissue for further processing. Sampling Oral swab samples of the mice were taken, as shown in figure 2. Briefly, swabs were autoclaved and brought into the animal barrier facility according to standard procedures. Mice were securely scruffed and the swab was twirled around for 5 to 19 seconds to collect the sample from the inside the cheek (figure 2A). While swabbing it was ensured to not hurt the mice. Hair follicle samples were obtained by carefully plucking a small amount of hair (10-20 units) (figure 2B). After sampling, mice were placed back into their cages. The oral swabs were left to dry before placing each swab and hair sample into individual tubes. Ear biopsies taken for routine genotyping analyses from the same animals were used as controls. Finally, the samples were shipped to the genotyping facility. All samples were taken at AAALAC accredited CRL sites according to Animal Welfare rules and guidelines. The mouse was securely scruffed to prevent it from moving its head. (Figure 2A) The autoclaved oral swab was gently inserted into the oral cavity of the animal from an angle to collect the sample from inside the cheek. The oral swabs were snapped off, dried and placed into shipping tubes for transport. Hair follicle samples were obtained by carefully plucking a small amount of mouse hair (10 to 20 units), (Figure 2B) followed by placing them into shipping tubes for transport.Processing: Throughout the whole processing workﬂ ow, samples werekept inside a 96-well format to avoid potential mix up andto allow the processing of a large number of samples.Lysis and DNA extraction:The oral swabs were incubated in lysis buffer for 2 hours at 56°C. Hair and ear biopsies were incubated under the same conditions but overnight. Deoxyribonucleic acid (DNA) was extracted using Solid Phase Reversible Immobilisation (SPRI) technology. Puriﬁ ed DNA was storedat +4°C (short term) until polymerase chain reaction (PCR) analyses.Polymerase Chain Reaction (PCR) and analysisof results:The DNA extracted from the samples (oral swabs, hair and biopsies) was subjected to either conventional PCR and analysis using capillary gel electrophoresis (CE) (LabChip GX Touch, Perkin Elmer) or real-time PCR (quantitative PCR and endpoint analysis) and analysis using StepOne Cycler (ThermoFisher Scientiﬁ c). Slightly adapted conditions were established, if needed e.g. increased number of PCR cycles, template or primer concentration. Ampliﬁ cation products were analysed and compared between the different sample types.Results1. Oral swab genotyping – suitable for every kind of PCROral swabs taken from transgenic lines (KO, KI, etc.) were tested in conventional PCR with PCR amplicons ranging from 100 to 1500 bp in length (Figure 3A), real-time endpoint analysis (allele discrimination plot (qPCR: n=11 samples Figure 3B) and in zygosity testing for transgene lines (Figure 3C), leading to the correct genotyping determination and comparable signal intensity. In ourstudy we could show that >98% of oral swab samples led to clear results. Furthermore >99% of the results from oral swabs matched those from corresponding ear biopsies.2. Shipment and storage condition for oral swab – (RT (+ 20°C), + 4°C and – 20°C)The robustness of oral swabs genotyping in terms ofshipment and storage time/condition were tested for upto 25 days using capillary gel electrophoresis (CE) asPoster Presentations

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135August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePoster Presentationsanalysis method. The percentage ratio of the PCR amplicon in ng/µl to biopsy is shown in Figure 4. Signalscould be detected up to 18 days after sampling if the oral swabs were shipped and stored at room temperature (RT) (+20°C). The best results were obtained when the samples were stored and shipped at -20°C. Visible signals and evaluable results were also detected at +4°C up to 25 days after sampling.3. Swabs versus hairIn this pilot study we developed a simple, economic and efﬁ cient strategy to extract DNA from hair follicles of mice which are suitable for genotyping. When comparing oral swab and hair follicle samples, we were also able to demonstrate consistent genotyping results from hair follicles.Figure 3. Exemplary electropherogram traces – Ampliﬁ cation products from genomic DNA (ear biopsy or oral swab from the same mouse) up to 520 bp in size. IQC – internal quality control; TG – transgene (A). Allele discrimination plot from ear biopsies (left) and swab samples (right) in endpoint analysis; HE – heterozygous; WT – wild type; negative control samples (NTC) (B). Ampliﬁ cation plot of ear biopsies versus oral swab samples in quantitative real time PCR analysis (C).Figure 4. Bar chart. Value was determined from the capillary gel electrophoreses using TG-PCR fragment amplicon amount in ng/μl for biopsies versus oral swabs 4, 11, 18 and 25 days after sampling. Samples were shipped and stored at room temperature (RT: +20°C), +4°C and -20°C. Standard deviation is shown in the Figure. TG – transgene.Figure 5.Genotyping using murine hair follicle and oral swab samples. Results of capillary gel electrophoresis showing the presence or absence of transgene in both biological materials – hair follicle and oral swab from same mouse (A). Exemplary electropherogram traces – ampliﬁ cation products from genomic DNA (oral swab and hair follicle from same mouse) up to 500 bp in size (B). IQC – internal quality control; TG – transgene (A+B).A B

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136Animal Technology and Welfare August 2020ConclusionIn accordance with the 4R principle we have optimised and expanded the possibilities of genotyping mice using non-invasive methods in our automated workﬂ ow. This involves the non-invasive collection of oral swabs and hair follicles to replace stressful and painful biopsies. Since hair sticks electrostatically to instruments, there isa risk of cross contamination between hair samples fromdifferent animals. Considering the 4Rs and a growing interest in reﬁ ning genotyping sampling methods, oral swab and hair follicle sampling provide alternatives thatcan be used for large scale routine genotyping especially if no invasive biopsy is allowed (e.g. animals with ear tags or toe tattoos) or no second biopsy is possible.Non-invasive sampling methods:•alternative to ear biopsies•sampling of mice aged 16 days or older•conﬁ rmation of genotype before/during experiments•storage up to 25 days at +4°C; for longer storage -20°C advisable*•shipping at RT, on cool packs or dry ice•suitable for conventional PCR, real-time qPCR and endpoint analysis*•large scale routine genotyping leading to faster turnaround times**only tested for oral swabs so farAcknowledgementsWe would also like to thank our German and French colleagues for establishing the oral swab and hair sampling method at our Charles River breeding facilities (AAALAC accredited). Further thanks to our European Genetic Testing Services technicians for their support.References1 Russell and Burch Methuen & Co. Limited, 1960.2 Mazlan, N., López-Salesansky, N., Burn, C., Wells, D.(2013). Mouse identiﬁ cation methods and potential welfare issues: a survey of current practice in the UK. Animal Technology and Welfare Vol 13 No 1, 1-10.Technicians and Vets: a partnership for animal welfareCALL FOR WORKSHOPSl take an active part in the UK’s leading annual meeting for our industryldo you have an area of expertise? (i.e. work with a more unusual species, bio-security, management, health & safety, been involved in a new build, environmental enrichment, GA breeding, ageing animals, transport, etc)lcould you run a 1 - 2 hour interactive workshop and qualify for a free congress?l send your ideas today on the Submission form available from www.iat.org.uklﬁnal date for submissions: Friday 29th November 2024Contact: congress@iat.org.ukCongress2025CONGRESS Invitation to Participate4th March – 7th MarchANNIVERSARY1950 � 2025Poster Presentations

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137August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2024 Animal Technology and WelfareIntroductionThe test article for this study was an antibiotic to treat urinary tract infections (UTIs), one of the top recurring infectious ailments of the elderly.Whilst many UTIs respond well to antibiotics, it is not uncommon for them to resurface several months later, which is thought to be (amongst other reasons) due to infection of the bladder itself. Most treatments for UTIs involve oral administration of an antibiotic, which relies on systemic exposure to remove microbes within the bladder wall and urinary excretion of the antibiotic to remove microbes within the bladder. In clinical treatments for this test article, the urinary bladder will be catheterised and dose will be instilled; two hours later the patient will be allowed to urinate to remove residual dose. Because we cannot tell the animal not to urinate for a couple of hours, the animal was anaesthetised to facilitate catheterisation and then held under general anaesthesia for an hour after dose administration.Elements to considerFemales have a shorter urethra than males. This means bacteria are more likely to reach the bladder or kidneys and cause an infection. Therefore UTIs occur more frequently in females. Also, the anatomy of the female lends itself to this dosing route a lot easier than the anatomy of the male. In addition, the published papers on this model/route only refer to females.The study consisted of 12 female Sprague Dawley rats, 6 control and 6 treated. Animal care and use was conducted in alignment with Animal Welfare regulatory requirements in an AAALAC accredited facility.Intravesical dosing in the female Sprague Dawley ratSARAH GRAHAM CHENERY and ZAC TURNERLabcorp Early Development Laboratories LimitedCorrespondence: sarah.graham-chenery@labcorp.com and zac.turner@labcorp.com As the patients on the clinical trial will receive treatment once a week for 4 weeks, it was decided that the animals would receive treatment twice weekly for 4 weeks to give a safety margin, which is important as the objective of this study is to protect the ﬁrst patients on the clinical trial.Excessive salivation was seen on a similar (once weekly dosed) study at our Madison, WI (US) facility. Accordingly, discussions were held with the veterinary surgeon regarding holding the animals on their back headﬁrst on a slight downward incline of 10° on a purpose-made slope, which was slight enough to give the saliva a directional ﬂow out of the body but not too much to cause issues, as well as reducing dose loss due to gravity. Subsequently no issues regarding salivation were observed throughout the duration of the study.MethodsWe started the procedure by anaesthetising the animal in the animal room with a combination of 2.5% isoﬂurane and 3 litres per minute of oxygen. These measurements were deemed appropriate as the animals were subject to this procedure for 1 hour, twice a week for 4 weeks. Once the animal was satisfactorily anaesthetised, it was transferred to the surgery suite and placed in a supine position onto the 10° slope with the nose positioned in the breathing tube (Figure 1). Viscotears were applied to both eyes to ensure they were kept hydrated during the procedure. A rectal thermometer was inserted with the aid of KY jelly, which was connected to a heat blanket as part of our routine observations. The thermometer was then secured to the tail using LeukoFix™ tape to ensure that it remained in

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138Animal Technology and Welfare August 2020Poster Presentationsplace. A pulse oximeter was attached to the forepaw of theanimal, which enabled monitoring of the heart rate and oxygen saturation. After the animal was prepared, the catheter was lubricated with Instillagel and placed into theurethra in the direction of the tail (Figure 2), then rotated upwards until the catheter was parallel with the tail. After an hour of administration, the last set of observations were performed, the dose was withdrawn slowly back into the 1 ml syringe and the rectal thermometer was removed.RecoveryTo aid recovery post-administration, we placed the animalinto its own recovery cage, which was then put on top of a heat mat. Once the animal had recovered from thegeneral anaesthetic, it was given post-operative recoveryhydrogel, ¼ teaspoon of sunﬂ ower seeds and a grape. This enrichment was chosen to encourage the animal toeat and drink, to stay hydrated and for enjoyment purposes.After four weeks of treatment the animals were sacriﬁ ced and subjected to necropsy examination and histopathology of the urinary tract (kidneys, ureters, urinary bladder and urethra).ResultsThere were no effects on organ weights; however there was a slight effect on bodyweight. This was not a response to the test article but was considered to be associated to the anaesthesia/dosing procedure, as during the ﬁ rst four days of the study the controls lost a mean of 3g and the test gained a mean of 1g.There were some minor ﬁ ndings from the urinary bladderthat came back in the toxicology report; one of them being some congestion for one control but no test animals. The other ﬁ nding was isolated cases of focal areas of oedema (one control and two test animals). This was also considered to be associated with the administration procedure. There were no signs of adverseeffects from the procedure seen post-dose or at dose site observations.ConclusionsThis dose administration method was well tolerated by female Sprague Dawley rats, which were treated for 1 hour, twice a week for 4 weeks, with minimal irritation of the urinary bladder epithelium.AcknowledgementsHelen Hornsey, Emily Jones, Stuart Hazlewood, Kate Read, Manuela Teti, Peter Rees and Duncan Patten.ReferencesBrown, C. (2011). Urethral catheterization of thefemale rat. Lab Animal (NY). 40(4), pp.111-112.doi: https://doi.org/10.1038/laban0411-111.https://www.nhs.uk/conditions/urinary-tract-infections Figure 1. Anaesthesia workstation including dosing equipment.ObservationsObservations were performed every 5 minutes to monitor the animal’s heart rate, oxygen saturation, body temperature, isoﬂ urane/oxygen rate, oral secretions as well as applying Viscotears and supplemental care as necessary.Figure 2. A22G x 1" soft Teﬂ on intravenous administrationcatheter inserted into the external urethral oriﬁ ce. Illustration by Duncan Patten of Labcorp.Equipment1. Isoﬂ urane2. Drapes3. Viscotears4. KY Jelly5. Rectal thermometer6. LeukoFix™ tape7. Aldasorber scavenging canister8. Instillagel9. Hydrogel (diet gel recovery)10. Grapes11. Sunﬂ ower seeds12. Pulse oximeter13. 1-mL syringe14. Heat pad15. 10° Slope

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139August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIntroductionPublic engagement is one of The Crick’s strategic priorities, by looking to engage and inspire the public with discovery and science. As much of our life-changing science involves the use of animals, we aim to ensure we are being open and transparent about the way we use animals in science and the beneﬁ ts they bring. To support this commitment, The Crick is one of over 100 signatories to the Understanding Animal Research (UAR) Concordat on Openness. As more organisations pledge to be open about the work that they do involving animals, the various methods by which we can communicate and engage with the public become more widely known and institutions are able to learn from each other and trial what works most effectively for their operating model, including what is practical and achievable, whilst still making an impact.Here we highlight some of the different ways in which TheCrick has engaged with the public to promote openness in animal research, including the different mediums and formats used and different audiences we have reached. We aim to show the variety of methods that are available to organisations, how staff of various seniority can be involved in outreach, the opportunities for cross-departmental collaboration, and to demonstrate how a diverse and varied approach to openness can be more effective.Public-facing Website – customisable, accessible, familiarThe public-facing website at The Crick is a key part of howwe can communicate with the public. The Crick website has a dedicated section on animal research, explaining how animals play a key role in our science (Figure 1). We Public Engagement and Openness in animal research at The CrickJAMIE REDDEN, EWELINA KOZLOWSKA, CLARE BRAZILL-ADAMS, CAROLINE ZVEREV and SARAH HART-JOHNSONThe Francis Crick InstituteCorrespondence: Jamie.redden@crick.ac.uk also publish our annual returns of procedures, as well asnon-technical summaries of the projects licensed to carryout work at The Crick.The use of public-facing websites can be tailored toinclude as much or as little information as an organisationis able or willing to provide, and often already exist withinan organisation in some form. For smaller organisations,there are easily accessible hosting solutions andapplications; a method that requires an initial time commitment but following this is relatively low cost and low time commitment to maintain.Social media – low cost, accessible,widely usedThe Crick uses multiple social media platforms to engagewith the public, including Instagram, LinkedIn, X (formerlyTwitter) and You Tube. Social media is widely used by thepublic of all ages and backgrounds and at a relatively lowcost (often free) is also accessible to most organisationsand their staff. The support available from others in theindustry who use these platforms is also readily available,Public-facing Website – customisable, accessible, familiar The public-facing website at the Crick is a key part of how we can communicate with the public. The Crick website has a dedicated section on animal research, explaining how animals play a key role in our science (figure 1). We also publish our annual returns of procedures, as well as non-technical summaries of the projects licensed to carry out work at the Crick. Figure 1 The Francis Crick institute public facing web page for animal research The use of public-facing websites can be tailored to include as much or as little information as an organisation is able or willing to provide, and often already exist within an organisation in some form. For smaller organisations, there are easily accessible hosting solutions and applications; a method that requires an initial time commitment, Figure 1. The Francis Crick Institute public facing web page for animal research.August 2024 Animal Technology and Welfare

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140Animal Technology and Welfare August 2020Poster Presentationscreating opportunities to have real time engagement with the public, as well as learn from other establishments and collaborate through social media events.Public exhibits and events – creative,eye-catching free accessThe Crick is a multi-disciplinary institute, covering a widerange of research topics, and we can use the scale of the building and the diversity of skills to create public exhibits and events.Whilst large-scale exhibits are great for generating interest, they may not always be feasible. However smaller exhibits or events are still a way of engaging the public especially those local to the establishment, in an eye-catching and varied way showing the work behind the science.Craft and graftThe Craft and Graft exhibit aimed to highlight the scale and variety of the science that goes on behind the scenes at The Crick. The public exhibit included videos (Figure 2) and interactive stations to demonstrate how specialist technicians, including Animal Technicians, make sciencehappen.Outwitting cancerThe Outwitting Cancer exhibit was an immersive installation and series of ﬁ lms that discussed how scienceat The Crick contributes to cancer research. Part of this exhibit discussed the use of animal models in cancer research, including how and why they are used (Figure 3)but also the investments being made to migrate to non-animal models where possible.Behind the scenesOne example of how The Crick has used social media to promote openness in animal research is by collaborating with UAR, who shadowed two Animal Technicians for the day inside the animal facilities (Figure 4) to produce materials for use on social media. This video showed how our Animal Technicians care for a variety of species including ferrets and mice and the day-to-day tasks they carry out.Figure 2. Craft and Graft exhibit video showing Animal Technicians in aquatics working with Zebraﬁ sh.Figure 3. Outwitting Cancer discussion on the use of animals in cancer research.Figure 4. You Tube video showing Animal Technicians handling ferrets.

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141August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePoster PresentationsMeet a scientistPrior to the COVID-19 pandemic, The Crick was able to run public events such as Meet a Scientist where members of the public could engage with them and discover their work. This event then relocated to social media via Instagram Stories, where scientists took part in Instagram features on the ofﬁ cial Crick Instagram page, sharing behind the scenes insights and answer questions submitted by the public.After the events, visitors to The Crick website are given the chance to revisit previous take overs and catch up on stories from around The Crick (Figure 6).Another way of using social media and engaging with thepublic but also with the wider scientiﬁ c community is by participating in social media events, which are often widely publicised and participated in. Some examples of how we have engaged with these events are as follows:Figure 5. Instagram stories take over for Meet a Scientist.Figure 6. Previous Meet a Scientist features available on The Crick website.Figure 7. Mollie Millington, manager of the aquatic facilityin a You Tube video for Fish in Research Week.Fish in Research WeekFish in research week is an initiative by Understanding Animal Research (UAR), where facilities are encouraged to share information on ﬁ sh/aquatic species in research.As part of this event, The Crick shared a video from ourAquatic facility, describing the species we house and howthey are used. This video was made publicly available on You Tube (Figure 7) and was an excellent way to highlight the use of ﬁ sh and frogs, the use of which is lesser known to the public.Be Open About Animal Research Day (BOARD)BOARD is a yearly Europe wide initiative lead by the European Animal Research Association (EARA). Here organisations are encouraged to share their animal research activities on social media using the hashtag #BOARD. This provides all participating organisations with support from others, as well as the chance to learn and inspire further engagement. As part of #BOARD23 Turner laboratory from The Crick shared how they are

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142Animal Technology and Welfare August 2020Poster Presentationsusing opossums in research into sex chromosomes (Figure 8). Opossums are a unique species and provide an interesting topic for engagement. Learning and conclusions– Committing to public engagement comes with challenges but can be approached from various angles depending on an organisation’s goals.– The availability of resources does not have to be a barrier to public engagement. Larger organisations with more staff may be able to designate more time.The availability of a media or public engagement team within the establishment can aid in producing materials and designated funding can facilitate generation of materials. However there are a wide range of methods and approaches that can be utilised in many ways and can be manipulated to each organisation’s goals, as well as, available resources.–Public engagement and openness are best lead by staff who are happy to act as champions on behalf of their organisation, rather than as a mandatory initiative.Staff from all career stages and backgrounds can participate, with enthusiasm being the only common trait. Establishments should show an open commitment to the public and their staff, supporting ideas and activities. This may include open calls for contributions aimed at all staff, support by line managers, time allowances for staff to attend or participate in events, provision of training on outreach and recognition of staff for their contributions.– Don’t be afraid to ask for help! Openness can be daunting but there is a community both internally andexternally that can be utilised.AcknowledgementsThank you to all Crick staff that have contributed to activities, including technicians and Crick labs that shared their research, whether through appearing invideos, helping to coordinate or plan events and producematerials, as well as, The Crick Education and Public Engagement Teams for their support and encouragement of the BRF to participate in Crick engagement activities and for providing ideas and resources to bring these to life.Thank you to BRF Senior Management for supporting their staff to engage with the public and for fostering a culture of openness.Thank you to UAR, who have supported The Crick with outreach ideas, such as You Tube videos and social media events and to other organisations such as EARA that have developed initiatives The Crick has been able to engage in.Figure 8. #BOARD23 video on the use of opossums at the Crick.

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143August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIntroductionRat tickling is a positive handling technique promoted bythe NC3Rs https://nc3rs.org.uk/3rs-recources/rat-tickling.The website shared links to an online course from Purdue University, developed by Dr. Megan LaFollette. The course, with continuous assessment throughout, once completed successfully, provides certiﬁ cation in rat tickling. Labcorp successfully trialled and adopted rat tickling in early 2021. Rat tickling was performed in most studies.However it was not recommended to do this to pregnant animals, as advised by the NC3Rs. From the positive results seen, we then questioned if rat tickling could be used for pregnant females as this had previously been not recommended on the website.After discussion with our veterinarians, Named Animal Care and Welfare Ofﬁ cer (NACWO) and the study director, agreement was given and we trialled rat tickling on an embryo-foetal study.The objectives of the embryo-foetal developmental toxicitystudies are designed to detect adverse effects on the development of the embryo and foetus consequent to exposure of the dam from implantation to hard palate closure. The pregnant dams are typically dosed on days 6 to15 of gestation.Materials and methodsIn total, 28 female Han® Wistar rats were ordered at a minimum age of 11 weeks and 175g. These animals weretime-mated, arriving on day 2 post coitum (pc). Animal care and use was performed in conformance withthe Guide for the Care and Use of Laboratory Animals in an AAALAC-accredited facility.Rat tickling in gestation femalesALICE LOMAX, DAREN LURKINS and HELEN HORNSEYLabcorp Early Development Laboratories LimitedCorrespondence: daren.lurkins@covance.comTechnicians started the rat tickling procedure 3 days prior to the commencement of dosing, on day 4 pc. We followed the guidance from the NC3Rs’ website, which at the time was to:1. Make light quick movements with your ﬁ ngers on the back of the neck for a couple of seconds (Figure 1).1. Make light quick movements with your fingers on the back of the neck for a couple of seconds (figure 1).2. Pick the rat up for the flip, by placing your index finger in front of the rat’s collar bone and your thumb and middle finger under its armpits.3. Then give a flick of the wrist (figure 2).Figure 1 first stage of rat ticklingFigure 1. First stage of rat tickling.2. Pick the rat up for the ﬂ ip, by placing your index ﬁ nger in front of the rat’s collar bone and your thumb and middle ﬁ nger under its armpits.3. Then give a ﬂ ick of the wrist (Figure 2).4. Once the rat is on its back, tickle its belly for approximately 15 seconds and repeat the process for a total of 2 minutes.–Once oral gavage dosing commenced, rat tickling wasperformed once a week prior to the weekly detailed examination procedure.August 2024 Animal Technology and Welfare

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144Animal Technology and Welfare August 2020– Extra care was taken when using the ﬂ ip method because of the rat’s physical condition. Tickling was slightly higher on the belly especially later in pregnancy. – Tickling procedure ceased on day 20 pc when parturition checks commenced. – The Magenta® Bat4 (Magenta Electronics Ltd) wasused to collect the results, demonstrating that gestating females responded positively to rat tickling.–According to the NC3Rs’ website, positive calls occurat 35 to 75kHz, while negative calls occur around 22kHz.– Behaviour of each animal was also assessed to evaluate if they responded positively.ResultsResults obtained, clearly displayed that the gestationfemales enjoyed the interaction.Most readings were heard at 35 to 50kHz (Figure 3).No impact was found on the pregnant females at necropsy. There was no impact on foetus implantation compared to background control data.Discussion– The females reacted positively to the rat tickling. Technicians could hear noises of chirping on the Magenta Bat4 detector at 35 to 50kHz. It was fascinating for technicians to hear these positive sounds. – The dams also responded by licking the handler’s ﬁ ngers and once placed back in the cage, would run back to the handler’s hand to be picked up again. – Some females could also be heard chirping when removed from the cage prior to any procedure being performed, using the Magenta Bat4 detector.– Technicians commented that the animals in general were very relaxed for the oral gavage procedure.– Due to the positive ﬁ ndings, we have introduced rat tickling to a range of our reproductive study designs. 4. Once the rat is on its back, tickle its belly for approximately 15 seconds and repeat the process for a total of 2 minutes.Figure 2 the rat has been flipped and ready for the rat tickling procedure. Figure 2. The rat has been ﬂ ipped and ready for the rat tickling procedure. • Behaviour of each animal was also assessed to evaluate if they responded positively.ResultsResults obtained clearly displayed that the gestation females enjoyed the interaction. Most readings were heard at 35 to 50kHz (figure 3).Figure 3 results showing that most had positive reactions.Positive response86%Negative response 14%Day 20 PC ResultsFigure 3. Results showing that most had positive reactions.Day 20 PC ResultsNegative response 14%Positive response 86%Poster Presentations

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145August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAugust 2024 Animal Technology and WelfareIntroduction– Tropicamide is supplied as a 1% solution in a sealed bottle with a dropper lid. This is a short-acting anticholinergic agent used to induce dilation of the pupil.– Pupil dilation is required to facilitate ophthalmoscopy examinations.– Ophthalmoscopy examinations are carried out in drug development trials to detect any abnormalities within the eyes at deﬁ ned stages of a study.– Assessments include a comprehensive examination of the globe structures (e.g. cornea, anterior chamber,lens and ocular fundus) by using binocular indirect ophthalmoscope and/or slit lamp.– The current method of application is with a dropper bottle. However this can be difﬁ cult in pigs because it is not possible to manoevre the head in the way required to allow drops to fall into the eye (Figure 1).Reﬁ ned method of Tropicamide application in minipigsKEAL GRACEYLabcorp Early Development Laboratories LtdCorrespondence: keal.gracey@labcorp.com–When administered via dropper method, animals oftenexhibit signs of discomfort, such as head thrashing and vocalisation, while the technician attempts to steady the head in order to get close enough to the eyeto instill the drop.− A new method of application has been developed to streamline the process of Tropicamide application making it easier for technicians and animals. The new process allows Tropicamide to be sprayed onto the eye, eliminating the need for the head to be manoevred (Figure 2).− There is a lack of research into the application of Tropicamide via spray in pigs in a preclinical setting. Research in humans however showed that the spray application is a viable method.1 − When administered via dropper method, animals often exhibit signs of discomfort, such as head thrashing and vocalization, while the technician attempts to steady the head in order to get close enough to the eye to instill the drop. − A new method of application has been developed to streamline the process of Tropicamide application, making it easier for technicians and animals. The new process allows Tropicamide to be sprayed onto the eye, eliminating the need for the head to be maneuvered (Figure 2). Figure 1 movement restrictions of pigs Figure 2 spray applicaon of tropicamide Figure 1. Movement restrictions of pigs. − When administered via dropper method, animals often exhibit signs of discomfort, such as head thrashing and vocalization, while the technician attempts to steady the head in order to get close enough to the eye to instill the drop. − A new method of application has been developed to streamline the process of Tropicamide application, making it easier for technicians and animals. The new process allows Tropicamide to be sprayed onto the eye, eliminating the need for the head to be maneuvered (Figure 2). Figure 1 movement restrictions of pigs Figure 2 spray applicaon of tropicamide Figure 2. Spray application of Tropicamide.

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146Animal Technology and Welfare August 2020ObjectiveThe objective of this experiment was to investigate the efﬁ cacy of Tropicamide when administered using a new spray method.Materials and methods–The study design was to compare pupil dilation across2 administration methods (spray vs. drop).– A pump-action spray, speciﬁ cally designed for ophthalmic use, replaced the removable dropper on the bottle of Tropicamide.– 30 animals were allocated to one of 3 separate groups. Each group (A/B/C) was allocated a different application method: A) One drop in both eyes, B) One drop in the left eye and one spray in the right eye, or C) One spray in both eyes.– A second and third round of investigations took place wherein two sets of 30 animals were administered Tropicamide using only the spray method.– Human data showed that spray application was effective on open or closed eyes;2 as such, whether the animal’s eyelid was open or closed upon spray application was recorded at all occasions.–Animals were held by a competent handler as shown in Figure 3, with the front legs placed over the handler’sarms.– The administering technician directed the spray nozzle towards the animal’s eye and pressed down once on the spray pump (Figure 2).– The handler then turned to show the administering technician the other side of the animal’s head and the process was repeated (Figure 4).– It was not necessary to manipulate the animal’s head or to turn the animal around in the handler’s arms.–The method of Tropicamide application was unknown to the veterinarian that performed the ophthalmology examinations.– Whether or not the pupil was sufﬁ ciently dilated for examination was recorded.– The animal was returned to the home pen and rewarded with a small edible treat (Figure 5).− Human data showed that spray application was effective on open or closed eyes2; as such, whether the animal’s eyelid was open or closed upon spray application was recorded at all occasions.− Animals were held by a competent handler as shown in figure 3, with the front legs placed over the handler’s arms.Figure 3 handling of animals with front legs placed over handler’s arms− The administering technician directed the spray nozzle towards the animal’s eye and pressed down once on the spray pump (figure 2).Figure 3. Handling of animals with front legs placed over handler’s arms. − The handler then turned to show the administering technician the other side of the animal’s head and the process was repeated (figure 4).Figure 4 handling of animals with front legs placed over handler’s arms (from behind)− It was not necessary to manipulate the animal’s head or to turn the animal around in the handler’s arms.− The method of Tropicamide application was unknown to the veterinarian that performed the ophthalmology examinations.− Whether or not the pupil was sufficiently dilated for examination was recorded.Figure 4. Handling of animals with front legs placed over handler’s arms (from behind). − The animal was returned to the home pen and rewarded with a small edible treat (figure 5).Figure 5 small edible treat given after Tropicamide applicationConclusionsThe spray application method of Tropicamide administration was successful in all cases. The method worked on open or closed eyes and the time taken to complete the procedure was reduced considerably. The revised method had a positive impact on Animal Welfare and proved to be considerably easier for technicians, handlers and the animals involved.Figure 5. Small edible treat given after Tropicamide application. Poster Presentations

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147August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareConclusionsThe spray application method of Tropicamide administration was successful in all cases. The method worked on open or closed eyes and the time taken to complete the procedure was reduced considerably. The revised method had a positive impact on Animal Welfare and proved to be considerably easier for technicians, handlers and the animals involved.References1Bartlett, J.D., Wesson, M.D., Swiatocha, J., et al.(1993). Efﬁ cacy of a pediatric cycloplegic administeredas a spray. J Am Optometric Association; 64(9):617-21.2Portes A, Barbosa A, de Mello G, et al. (2012). Tropicamide 1% Mydriatic Effect: Comparison BetweenSpray in Closed Eyes and Eye Drops in Open Eyes. J Ocular Pharmacol Thera; 28(6):632-635.Poster PresentationsBulletin16 • August 2024 Introduction to the Mental Health First Aid (MHFA)We are aware of an increasing pressure on Animal Techs within the industryand we are delighted to share a new initiative from the IAT Equality, Diversity and Inclusion group led by Haley Daniels.Mental health problems can affect any of us at any time. One in four of us will experience a mental ill-health at some point in our lives and this increases amongst people in caring professions. Anxiety, stress and depression are the most common mental health struggles and can lead to other health issues.If you are experiencing any of these issues and you feel that you would beneﬁ t from a one-to-one chat, the IAT have a group of people all with experience of Animal Technology, all of whom who are mental health ﬁ rst aid trained. Via the IAT members’ website and using this QR Code – you can request a conﬁ dential and unbiased conversation with any of the mental health ﬁ rst aiders who will be able to listen and guide you to seek further assistance if required. Technicians and Vets: a partnership for animal welfareCALL FOR PAPERSl take an active part in the UK’s leading annual meeting for our industryl present a paper and qualify for free attendance at Congressl make this your debut presentation year – ﬁrst time presenter papers are only 20 minutes long and as well as a free congress there is a prize for the one judged to be the bestl send your ideas today on the Submission form available from www.iat.org.ukl ﬁnal date for submissions: Friday 29th November 2024Contact: congress@iat.org.ukCongress2025CONGRESS Invitation to Participate4th March – 7th MarchANNIVERSARY1950 � 2025

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148Animal Technology and Welfare August 2020IntroductionEdinburgh University have actively looked at re-homing any animals which have not undergone any invasive procedures.Bioresearch and Veterinary Services (BVS) are now looking at rehoming rats and mice. To date we have found that rats are more easily rehomed. Rehoming of laboratory animals NEIL ODEYThe University of Edinburgh, Bioresearch and Veterinary ServicesCorrespondence: Neil.Odey@ed.ac.uk Introduction Edinburgh University have actively looked at re-homing any animals which have not undergone any invasive procedures. Bioresearch and Veterinary Services (BVS) are now looking at rehoming rats and mice. To date we have found that rats are more easily rehomed. A rehome group/committee was established during February 2023 A rehome group/committee was established during February 2023.Why?3Rs – replacement, reduction, refinement From a Culture of Care point of view there has been an incredibly positive response from the technicians, vets and scientists to the rehoming of rats. Introduction Edinburgh University have actively looked at re-homing any animals which have not undergone any invasive procedures. Bioresearch and Veterinary Services (BVS) are now looking at rehoming rats and mice. To date we have found that rats are more easily rehomed. A rehome group/committee was established during February 2023 Why?3Rs – replacement, reduction, reﬁ nement From a Culture of Care point of view there has been anincredibly positive response from the technicians, vetsand scientists to the rehoming of rats.Why?3Rs – replacement, reduction, refinement From a Culture of Care point of view there has been an incredibly positive response from the technicians, vets and scientists to the rehoming of rats. 3Rs – replacement, reduction, reﬁ nement Why?3Rs – replacement, reduction, refinement From a Culture of Care point of view there has been an incredibly positive response from the technicians, vets and scientists to the rehoming of rats. incredibly positive response from the technicians, vets rehoming_Request form_template.docxUniversity of Edinburgh Re-homing owner’s declaration form.docx University of Edinburgh Re-homing NVS declaration form.docxAnimal Technology and Welfare August 2024

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152Animal Technology and Welfare August 2020Animal Technology and Welfare August 2024The purpose and use of singly housed animals in scientiﬁ c research MARK HARRINGTON and GEORGE ZIMMERMANCentral Biomedical Service, Imperial College LondonCorrespondence: m.harrington@imperial.ac.ukReasons for singly housing animalsThere are few reasons why an animal must be housed on its own. Some of the main factors are either behavioural, health or research based.1,2Behavioural as reasons to singly house animals:− Fighting, mostly caused by territorial aggression or barbering (overgrooming).− Social issues can also be a cause for single housing due to gender related behaviours and sexual incompatibility. Health concerns as reasons to singly house animals:– Animals needing isolation from others due to infectious pathogens. – Animals needing isolation from others to recover in a more stable environment from situations such as surgery, injury or sickness.Research purposes as reasons to singly house animals:– Studies needing animals isolated, to enable the correlation of data with the animal, without interreferences from external inﬂ uences such as other cage mates.Strategies for reducing stress in singly housed animalsWhen using a singly housed system there is the need to compensate for the lack of available social interactions.1,2 This can be done by providing extra and unique forms of enrichment and modifying the cage housing to better suit the occupants.Example of alternatives to single housing:− Improving/rotating the enrichment provided to the animals. – Providing a social companion animal.– Allow visual/olfactory contact with the conspeciﬁ c animals.– Providing larger cages to encourage more exploratory behaviour. – Regrouping animals back together when possible.– Use of cage monitoring camera system. Cages or pen walls allowing good visual contact (Figure 1) with the surrounding environment and equipped with sheltered area large enough to hold all animals within the group.3Cages or pen walls allowing good visual contact (figure 1) with the surrounding environment and equipped with sheltered area large enough to hold all animals within the group3. Figure 1 good visual contact Refined rat housing (figure 2) which allows the animals to rear and climb4. Figure 1. Good visual contact.

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153August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePoster PresentationsReﬁ ned rat housing (Figure 2) which allows the animals to rear and climb.4The animals are provided with extra enrichment and cagemodiﬁ cations to mitigate the effects of single housing.All our cages have several items for standard enrichment:• plastic and cardboard tunnels• tissues (can be shredded or not) • wooden chew blocks • wood chip bedding and• paper strands (used for nesting) • rodent houses We also try to think of different ways to present enrichment. In singly housed cages, within certain research groups, there are extra forms of enrichment (Figure 4) used like building block pieces and marbles, which help to encourage natural exploration and digging behaviours and are changed regularly to try and avoid boredom and encourage stimulation. Cages or pen walls allowing good visual contact (figure 1) with the surrounding environment and equipped with sheltered area large enough to hold all animals within the group3. Figure 1 good visual contact Refined rat housing (figure 2) which allows the animals to rear and climb4. Figure 2. Reﬁ ned rat housing. Automated home cage monitoring system (Figure 3) totrack individual mice in group housed environments without compromising Animal Welfare.5Figure 2 refined rat housing Automated home cage monitoring system (figure 3) to track individual mice in group housed environments without compromising animal welfare5. Figure 3 automated home cage monitoring system Examples of singly housed animals at Imperial College London Researchers use headplate to monitor brain activity. This is helpful in Alzheimer’s disease to study the correlation between sleep and associated phenotypes of the disease with two strategies: − monitoring sleep in an animal and recording brain activity. Figure 3. Automated home cage monitoring system. Examples of singly housed animals at Imperial College LondonResearchers use headplate to monitor brain activity. Thisis helpful in Alzheimer’s disease to study the correlation between sleep and associated phenotypes of the diseasewith two strategies:– Monitoring sleep in an animal and recording brain activity.– Targeting speciﬁ c cells of brain region known for theirinvolvement in Alzheimer disease and sleep regulation. Mice are isolated after surgery for two main reasons:– Safer recovery. – Sleep behaviour can be affected by the presence of another individual or individuals in the cage. which help to encourage naturel exploration and digging behaviours and are changed regularly to try and avoid boredom and encourage stimulation.Figure 4 environmental enrichmentIn some of the singly housed caging the bottle holder is placed differently to standard caging. The water bottle inserts into the front to eliminate the possibility of overextending, while drinking and to reduce unnecessary pressure on the head and neck due from the Figure 4. Environmental enrichment. In some of the singly housed caging the bottle holder is placed differently to standard caging. The water bottle inserts into the front to eliminate the possibility of overextending, while drinking and to reduce unnecessary pressure on the head and neck due from the attached headplate (Figure 5) while this is primarily for surgery recovery and for ease of access, as the water bottle is on the outside there is no need for a designated cut out to ﬁ t the water bottle in the cage lid, providing more internal space for the animal to feel comfortable and to provide a wider range of enrichment.Male rabbits are used for cardiovascular disease research as they have increased chances of contracting the disease.

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154Animal Technology and Welfare August 2020Rabbits are territorial, therefore there is the need to put in place a singly housed system to prevent ﬁ ghting and injury as a consequence of long separation time due to research procedures (e.g. surgery, food control systems).To minimise the effects of singly housing, the animals are placed in neighbouring pens which are separated with mesh dividers. This allows them to see, smell and partially touch each other, without being able to scratch or bite. (Figure 6)Our approach to singly housing animalsSingle housing advice is embedded within SOPs and good practices. Moreover, when planning experiments withsingly housed animals, we always encourage researchersto think:− How do you feel about singly housed animals in research?− What other things could you/we take into considerationto mitigate the effects of singly housing?− What other endeavours do you think could replace singly housing?References1 Implementing strategies to reduce singly housed male mice. Azkona & Caballero. Laboratory Animals Volume 53, Issue 5, October 2019.2 Housing and husbandry. NC3Rs’ website. https://nc3rs.org.uk/3rs-resources/housing-and-husbandry-mouse3 Housing and husbandry: Guinea pig. NC3Rs’ website,23 December 2020. https://nc3rs.org.uk/3rs-resources/housing-and-husbandry-guinea-pig#anchor_54Mazhary., H. & Hawkins., P. (2019). Applying the 3Rs: A Case Study on Evidence and Perceptions Relating to Rat Cage Height in the UK. Animals. 9. 1104. 10.3390/ani9121104.5Bains, R.S., Cater, H.L., Sillito, R.R., Chartsias, A., Sneddon, D., Concas, D., Keskivali-Bond, P., Lukins, T.C., Wells, S., Acevedo, Arozena, A., Nolan, P.M. andArmstrong, J.D. (2016). Analysis of Individual MouseActivity in Group Housed Animals of Different InbredStrains Using a Novel Automated Home Cage AnalysisSystem. Front. Behav. Neurosci. 10:106. doi: 10.3389/fnbeh.2016.00106.attached headplate (figure 5) while this is primarily for surgery recovery and for ease of access, as the water bottle is on the outside there is no need for a designated cut out to fit the water bottle in the cage lid, providing more internal space for the animal to feel comfortable and to provide a wider range of enrichment.Figure 5 bottle holderMale rabbits are used for cardiovascular disease research as they have increased chances of contracting the disease. Figure 5. Bottle holder. Rabbits are territorial, therefore there is the need to put in place a singly housed system to prevent fighting and injury as consequence of long separation time due to research procedures (e.g. surgery; food control systems).To minimise the effects of singly housing, the animals are placed in neighbouring pens, which are separated with mesh dividers. This allows them do see, smell and partially touch each other, without being able to scratch or bite (figure 6).Figure 6. Rabbit pens. Poster Presentations