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Vol 20 No 2 August 2021ISSN 2752-3918Offi cial Journal of the Institute of Animal Technology and European Federation of Animal Technologists●Refi nement for Oral administration ● New Special Interest section● ABTA 2021 Award winning article● Congress 2021 Posters – Part 1IAT JournalAnimal Technology and Welfare

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85August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareVol 20 No 2 August 2021EditorialJas 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 iEditorial Jas Barley, Chair of the Editorial BoardEmulsifi ed gels: a refi ned vehicle for accurate and rapid oral administration of lipid based preparations to rats Vidit Satokar, Mark Vickers, Pania Bridge-Comer, Wayne Cutfi eld and Benjamin Albert The effect of different biosecurity, feed and frequency of breeding on the fecundity and fertility of Zebrafi sh (Danio rerio) Joe Warmsley, Paul Barwood, Visila Moiche and Carole Wilson SPECIAL INTEREST SECTION Electronic instrumentation of a swingletree for equid pull load monitoring: a contribution for the welfare and performance of working donkeysJoão Paulo Coelho, João Brandão Rodrigues, Luís Queijo, Higor Vendramini Rosse, Francisco Albuquerque, Andrew Judge, Fiona Cook and Chris GarrettPAPER SUMMARY TRANSLATIONSFrench, German, Italian, Spanish ANDREW BLAKE TRIBUTE AWARD 2021Refi nements in head plate mouse nesting: using composite nests to enhance welfareZoe WindsorTECH-2-TECH Interpreting water monitoring results in laboratory rodent facilitiesLorna CleverleyEnrichment for laboratory Zebrafi sh Chloe Stevens 1111041261491451359592

86Animal Technology and Welfare August 2020POSTER 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 ii155161165175172168Scientific, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and educationKumud Kant Awasthi Thinking outside of the tunnel for non-aversive mouse handlingJoanne Moore and Mark WickertPOSTERSDevelopment of a feeding device to reduce reliance on field trials to test novel poultry red mite controlsFrancesca Nunn, Kathryn Bartley and Alasdair Nisbet PREPARE for better Science: guidelines for animal researchAdrian J Smith, R Eddie, Elliot Lilley, Kristine Hansen and Trond BrattelidRefining cages for social housing of non-human primates on ADME studies William Archibald and Colin GlynnControlling humidity – improved breeding and validity of researchKaren Ekkelund PetersenLaboratory primate enrichment ideasJoe Peploe and Chris MacaulayHome cage monitoring: investing in the futureJoanne Moore and Hilary LancasterUsing physiotherapy to successfully manage Chronic Atrophic hindlimb lameness in the Beagle dog – a case studySamantha Shanks177184188

87August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfare

88Animal Technology and Welfare August 2020If 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 5th November 2021. Simon CummingHonorary Secretary

89August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarevOFFICERSPresidentDr 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 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 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 vMembers of CouncilCarmen Abela, Kally Booth, Steven Cubitt,Simon Cumming, Haley Daniels, Glyn Fisher,Nicky Gent, Alan Graham, Diane Hazelhurst, Linda Horan, Sam Jameson, Elaine Kirkum, Adele Kitching, Robin Labasse, Theresa Langford, Sylvie Mehigan, Steve Owen, Alan Palmer, Allan Thornhill, John Waters, Lynda Westall, Carole Wilson, Adrian WoodhouseJas Barley (Chair), Nicky Gent, Patrick Hayes,Diane Hazelhurst, Elaine Kirkum, Carole Wilson,Lynda WestallBranch and BING Liaison Officer:Kally Booth MIAT RAnTechEFAT Representatives:Glyn Fisher, Robin Labasse MIAT RAnTech, Alan PalmerWebsite Coordinator:Allan Thornhill FIAT RAnTechAnimal Welfare Group:John Waters (Chair), C Alba, Kally Booth, Nicky Gent, Sam Jameson, Sylvie Mehigan, Steve OwenBoard of Educational Policy:Steven Cubitt (Chair), Adele Kitching (Secretary), Tina O’Mahoney Communications Group:Adrian Woodhouse (Chair), Carmen Abela, Kally Booth, Sam Jameson, Elaine Kirkum, Teresa Langford, Tara Mclaughlin, Sylvie Mehigan, Allan Thornhill, Lynda Westall

90Animal Technology and Welfare August 2020BRANCH SECRETARIES 2021Cambridge: 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.© 2021 Institute of Animal TechnologyAll rights reserved. No par t of this publication may be reproduced without per mission 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 viCPD Officer: Alan Palmer MIAT RAnTechRegistration and Accreditation Board:Glyn Fisher (Chair), Ken Applebee, Charlie Chambers, John Gregory, Cathy Godfrey, Kathy Ryder, Wendy Steel, Stuart StevensonObserver: Ngaire Dennison (LAVA)Congress Committee:Alan Graham (Chair), Haley Daniels, Adele Kitching,Allan Thornhill, John WatersEquality, Diversity and Inclusion Officer:Haley Daniels MBA MSc MIAT RAnTech CIPDUK Biosciences ASG Representative/Home Office:Alan Palmer MIAT RAnTechIndex to AdvertisersAS-ET ...........................................................164Avid plc ..........................................................87CLAST ...................................................170-171Datesand Ltd .................................................IFC Institute of Animal Technology ............88, 93, 134, 142-143, 144, 164, OBC IPS Product Supplies Ltd ................................IBCLBS Serving Biotechnology Ltd .........................91Somni Scientific ...........................................110Tecniplast UK Ltd ............................................94BRANCH SECRETARIES 2021Cambridge: 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.© 2021 Institute of Animal TechnologyAll rights reserved. No par t of this publication may be reproduced without per mission 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 vi

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92Animal Technology and Welfare August 2020August 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 that has 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 report. 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 r educing 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 structur ewhich 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 sur gery, presented a very real danger to bothanimals and staff. Disease in laboratory animal units was often a recurring 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 article by Seonagh Henderson of the University of Glasgow, ona novel technique of cage cleaning which hasa positive effect on the welfare of laborator y 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 ixAnimal Technology and Welfare August 2021How does an issue of the Journal evolve? If I am truthful the answer should be on the wing and a prayer. In reality, it very much depends on what material is available. I aim for a minimum of 80 pages of Animal Technology and Animal Welfare related material and 16 pages of announcements, advertisements etc. In a perfect world it would be good to always have one issue’s worth of material in reserve but in almost 12 years as Editor I have yet to achieve that, although the situation is improving. Ideally all technology papers will take welfare into account but it may not be the main theme. Others will deal purely with welfare issue’s but we also publish papers on management, ethics and other subjects that affect either technologists or the animals in their charge. Formal papers are sent to two external peer reviewers who will comment on the technology/science, experimental design where appropriate, statistics, the style of writing, etc. This is not an ad hoc affair, each reviewer is a specialist in the subject, for example the paper involving donkeys and the device known as a swingletree went to an expert in donkey welfare and a bioengineer. Paper author details are not supplied to peer reviewers so that material is judged at face value and nobody can be accused of prejudice or a vested interest. Peer reviewers are sent details as to which criteria they should be assessing papers on and they report their comments back to me. Details of who reviewed the papers are not supplied to any author so again there is absolute fairness. Once peer review is completed authors will be informed of any comments that need addressing and our decision as to whether or not we will publish their paper subject to any corrections required. A peer reviewer will also comment on the quality of writing for example correct use of English grammar, spelling and avoidance of repetition of the same phrase, etc. Sometimes it is apparent that the author is affected by dyslexia or a similar condition and in this situation I will often correct the English myself taking care to maintain the author’s ‘voice’ – after all the material is by the author not Jas Barley. Particularly when we have first time authors, I want the publishing experience to be exciting and pleasurable and to encourage them to write again without any lowering of standards. We are a professional journal and must always appear as such. Every article both formal and informal then needs be formatted ready for typesetting, checking spelling and punctuation, checking accuracy of information, that diagrams, etc are legible and many other criteria that are important in maintaining the quality of the material we publish. As you will see from the content of this issue material is varied and no one person can be an expert on everything. Fortunately the Institute of Animal Technology has a wide range of expertise and if I do not know someone with the appropriate expertise to review material I know someone who does. Council members are a terrific resource when it comes to finding experts as are our Vice Presidents. The peer reviewers for this issue’s articles were based as far apart as the USA and Glasgow and I am indebted to everyone of them. Content this time includes a paper on a refinement of oral dosing in rats from a team at the University of Auckland, New Zealand. This is the first submission from New Zealand for many years and which I hope is the first of many future EditorialJas BarleyChair of the Editorial Board

93August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfarepublications. Joe Warmsley and colleagues at University College London have supplied some interesting observations on the possible effects of various environmental and husbandry practices on fecundity and fertility of Zebrafi sh. The new special interest section offers a paper from Portugal on how they developed instrumentation of a swingletree used with donkeys. We are all probably aware that donkeys are a major source of transport in many countries around the world and this work appears to have terrifi c potential for ensuring that animals are not overburdened when pulling loads. We have had a paucity of Tech-2-Tech articles in recent issues despite the fact that the Animals in Science Education Trust (AS-ET) sponsors a prize for the Tech-2-Tech article considered to be the best in each years volume. However in this issue I am able to offer 4 articles ranging in subject from the basis of Animal Welfare and its enhanced public concern via Training and Education in India, Interpreting water monitoring results, enrichment for Zebrafi sh and another aspect of non-aversive mouse handling. Also included is the Andrew Blake Tribute award winning entry for 2021 by Zoe Windsor and the fi rst of the posters from the IAT Virtual Congress 2021. This issue covers 14 different subjects and 8 different species. EditorialAnimal Technologists – the key workers for medical researchCALL FOR PAPERSl take an active part in the leading annual meeting for Animal Technologistsl 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 October 2021Contact: congress@iat.org.ukCongress2022CONGRESS Invitation to Participate29th March – 1st April

94Animal Technology and Welfare August 2020ISSUE2 - VISION+ - 210X297.indd 1 05/11/2018 18:14:29find out more on www.tecniplastuk.com Or call us on 0345 050 4556

95August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAbstract Oro-gastric gavage is used to accurately administer nutritional substances or drugs to animals. However it induces stress and has a substantial risk of mishap. Incorporation into edible gels is difficult for lipid-based preparations. We repor t a new methodology for producing emulsified oil-enriched gels, their effectiveness in pilot studies and subsequent larger experimental studies. Emulsified fish oil-enriched gels were produced using non-polar starch. Multiple gel types were made incorporating 0.05ml or 1ml oil doses, oxidised or unoxidised oil and with or without raspberry flavouring. The palatability and safety were assessed with i) 8 gel types in female SD rats consuming a chow diet (40 treatments) and ii) 3 gel types in rats consuming a high fat diet (45 treatments). Subsequently, palatability and safety were further assessed in a large cohort of pregnant rats (n=155; 4,242 treatments). Across both studies, all gels were eaten completely, whether the rats consumed a chow or high-fat diet. There was a 5-day period of acclimatisation. Raspberry flavoured gels were consumed more quickly than unflavoured gels. Rats exhibited positive behaviour towards receiving the gels and there were no ill-health effects. In subsequent experimental studies 4,242 doses were given to pregnant rats and all were completely consumed. Oil-enriched emulsified gels represent an easily administered highly acceptable, reliable and safe method of lipid delivery to rats, that we propose is superior to oro-gastric gavage. Keywords: refinement, stress, pregnancy, Animal Welfare, emulsified gels, lipid administrationIntroductionAdministration of nutritional supplements or drugs to animals can be challenging and the method of administration may represent a potential experimental confounder. Commonly, oral administration of agents represents the most physiologically (and translationally) appropriate method. However, oral administration is associated with important challenges. Animals may not consume a substance placed in their cage or may do so slowly or incompletely. This can be solved by incorporating it into food or water but the volume of substance consumed will scale with appetite or thirst, so that it cannot be precisely controlled. Some substances have additional challenges. For example, polyunsaturated fatty acid rich oils such as marine oils are highly prone to oxidation1,2 and oxidation is known to alter their effects on health.1,3,4 Thus, incorporation into food, would risk unacceptable loss of quality that could mask important health effects, or cause inadvertent harm. Oro-gastric gavage; passing a tube through the mouth into the stomach, is an appealing solution as it allows rapid administration of a carefully controlled dose. However in rats, even when expertly performed, gavage is invasive and involves restraint of the animal, causing stress.5 Furthermore, mishap may lead to trauma, aspiration or even death6-8 and the cumulative risk of a mishap occurring becomes much higher if gavage is repeated daily for the same animal, in one study reaching 56%.9 Administration of viscous substances by gavage may magnify risks due to the potential for the substance to coat the outside of the tube and lead to aspiration and the need for larger bore Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid based preparations to ratsVIDIT SATOKAR,1 MARK VICKERS,1 PANIA BRIDGE-COMER,1 WAYNE CUTFIELD,1,2 and BENJAMIN ALBERT1,21 Liggins Institute, University of Auckland, Auckland, New Zealand 2 A Better Start – National Science Challenge, University of Auckland, Auckland, New ZealandCorrespondence: b.albert@auckland.ac.nzAugust 2021 Animal Technology and WelfareISSUE2 - VISION+ - 210X297.indd 1 05/11/2018 18:14:29find out more on www.tecniplastuk.com Or call us on 0345 050 4556

96Animal Technology and Welfare August 2020gavage tubes which may be more traumatic.10 Viscous substances could stick within the syringe and tubing leading to incomplete delivery. Stress has immediate effects such as increasing heart rate and blood pressure,11 increasing corticosterone secretion12-14 and inducing insulin resistance14 which can all act as confounders in cardiometabolic studies. Of note, it has been recommended that gavage is avoided in toxicity studies13 as it may increase toxicity8 and death due to mishap could be misattributed. In addition, stressful stimuli in pregnancy has been shown to lead to lower birthweight15-17 which is a known risk factor for the development of cardiometabolic disorders in later life18 which may be in part the consequence of changes in maternal care.19 We planned a series of supplementation studies in rat pregnancy examining effects of unoxidised fish oil on offspring metabolism and of oxidised fish oil on toxicity. Thus prior to conducting these upcoming studies, we aimed to develop a refined method of administration to avoid the need for oro-gastric gavage but retain its benefits (controlled dose and rapid consumption). Previous studies have incorporated drugs or nutritional substances into flavoured gels20,21 which may be an enriching rather than aversive experience for the animals consuming them. Moreover, use of a gel vehicle has been recommended as a refined method of substance administration by a multidisciplinary, multiagency joint working group focussed on Animal Welfare.22 However as gels are water based, lipid treatments are difficult to incorporate, especially when given at high doses. Thus we developed a method of emulsifying fish oil into gels and conducted a series of pilot studies in rats to ensure they were consumed quickly and completely, both in the context of a standard diet and a highly palatable high-fat diet. We also observed their effects on the health of the animal.MethodsAnimal EthicsEthical approval was granted by the Animal Ethics Committee at the University of Auckland (approval #R001936) and the studies were performed in accordance with all appropriate institutional and international guidelines and regulations of animal research.RatsAdult female Sprague-Dawley (SD) rats were sourced from the Vernon Jenson Unit at the University of Auckland. All animals were individually housed under standard conditions in an open top cage at 25°C with a 12-h light: 12-h dark cycle. Specific details around age, weight and experimental diets are detailed for each protocol below. Given our past experience in dietary studies using female SD rats and the tight regulation of caloric intake in these animals, an n = 5 per group was considered sufficient for the Pilot studies.Production of flavoured edible oil-enriched gelsWe devised a protocol to produce 5ml oil-enriched gels from 100ml of gel solution. A variety of different gel types were produced with varying quantities of fish oil and flavoured jelly crystals and their macronutrient content was calculated (Table 1). As fish oil and water are immiscible, we used a non-polar starch emulsifier; N-Creamer 46 (Ingredion ANZ Pty Ltd, Auckland, New Zealand) to produce an oil-in-water emulsion. The final gel solution had a 4% concentration of non-polar starch. 4g of non-polar starch was dissolved in 50ml of water at 70°C using a magnetic stirrer over a period of 90 minutes. This solution was then poured into a blender and the fish oil was added dropwise and pulsed to facilitate emulsification. This was continued until all the oil was added, until a uniform emulsion was achieved. Then, 8.6g of gelatine (Ward McKenzie Pty Ltd, Victoria, Australia) and the raspberry flavoured jelly crystals (Cerebos Greggs Ltd, New Zealand) were mixed with 20ml of 50°C water (water at higher temperatures was not used due to the potential to cause the fish oil to oxidise). The oil and water emulsion and the gelatine/jelly mixture were both added to a volumetric flask which was mixed vigorously but with care to avoid foaming and then topped up with water to 100ml. The flask sat in a 50°C water bath to prevent the solution from setting. 5ml of mixture was transferred into individual moulds of ice trays, which were labelled, covered and refrigerated overnight (4°C). The following day the individual gels were removed and stored at 4°C. To aid separation of the gels, the ice tray was placed in a bath of warm water for 20 seconds, before the gels were scooped out with a small spatula.Control gels were produced similarly, excluding the step of adding the oil, unflavoured gels were produced without adding the raspberry flavoured jelly powder to the mixture and double raspberry gels used twice the quantity of raspberry powder (Table 1).To reduce the potential for oxidation and to prevent mould growth, we stored gels refrigerated in small sealed containers and in the dark, prior to use and created fresh gels every 3 days.Pilot Study 1 HypothesisAdult female rats will consume oil-enriched gels completely and within 60 minutes, whether they contain 0.05ml (a comparable dose to human consumption23) or 1ml (used in a previous rat study24) of fish oil, the oil is oxidised or unoxidised and whether the gel is flavoured or unflavoured.Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid-based preparations to rats

97August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareStudy DesignEight gel types with oxidised or unoxidised oil, with or without raspberry flavouring (17g in 100ml) and with 0.05ml or 1ml of oil were prepared as described above (Table 1). The oxidised fish oil was produced by bubbling oxygen through a large glass bottle of oil for 1 month under fluorescent lighting (peroxide value: 48.8 meq/kg, p-anisidine value: 4.5).24 The peroxide and p-anisidine values were determined according to the European Pharmacopoeia 8.0 method.25Five adult female SD rats (age: 106 ± 1 days) weighing 245 ± 2.8 g were used for the study (Figure 1) and individual rats were considered the experimental unit. Rats were fed a standard chow diet (2018 Teklad global 18% protein rodent diets, Envigo, USA) ad libitum. The food hopper was topped up to 100g daily. Gels were placed in the food hopper in the morning at the same time and position every day for 10 days. Each rat received each gel type once, in an order determined using a random sequence generator (www.random.org). Timers were set from the time the gels were placed with the animals and continuously observed for the first hour and then reviewed hourly until no gel was found in the cage. The timers were stopped when the gel was completely eaten by the rat and the time taken to eat the gel completely was recorded. After 24 hours, if the gel was incompletely eaten, the percentage of gel remaining was visually estimated within the following categories: <25%, ≥25% but <50%, ≥50% but <75%, ≥75%, untouched). Food consumption was recorded daily when the animals were weighed and inspected for wellbeing. Signs of stress or ill-health including abnormal respiration or motor postures, piloerection, fur loss, changes in the eyes, weight loss or fearful or aggressive behaviour towards the researcher were recorded as well as the Rat Grimace Scale score.26 All observations were made by the same researcher.Pilot Study 2HypothesisAdult female rats that are fed a highly palatable high-fat diet (HFD) will consume fish oil-enriched gels completely and within 60 minutes of placement, independently of whether the gel has a standard content of raspberry flavouring or double flavouring. Study Design Three raspberry flavoured gel types were prepared: a control gel with no fish oil and standard raspberry jelly content (17g in 100ml) and 2 low dose unoxidised fish oil (0.05ml) gels, one containing standard raspberry jelly and one with double-strength raspberry jelly (34g in 100ml) (Table 1). Five adult female SD rats (age: 84 ± 1 days) weighing 228.8 ± 7.9 g were individually housed as detailed above with individual rats considered the experimental unit. They were fed a commercially available HFD (D12451, Research Diets Inc., New Brunswick, NJ, USA) containing 45% kcal as fat ad libitum for three days before initiating with the control gel (gel with flavouring but no oil). Food was topped up to 100g daily. Table 1. Components of 100 ml of gel solution and nutritional composition of 5ml gels RJC; raspberry flavoured jelly crystals.* Fish oil as received from the manufacturer, or that had been intentionally oxidised. ** approximate water volume added, to produce a final volume of 100 ml in a volumetric flaskPilot Study 1 Pilot Study 2Gel Type0.05ml oil0.05ml oil + RJC1ml oil1ml oil + RJCControl + RJC0.05ml oil + RJC0.05ml oil + 2x RJCComponents of 100ml of gel solution Nonpolar starch (g) 4 4 4 4 4 4 4Gelatin (g) 8.6 8.6 8.6 8.6 8.6 8.6 8.6Raspberry jelly crystals (g) - 17 - 17 17 17 34Study Oil* (ml) 1 1 20 20 - 1 1Water** (ml) 85 70 70 50 70 70 50Nutritional composition of 5ml gels Energy (kJ) 5.4 21.9 38.4 54.9 20.1 21.9 38.3Protein (g) 0.19 0.25 0.19 0.25 0.25 0.25 0.32Carbohydrate (g) 0.19 1.09 0.19 1.09 1.09 1.09 1.99Fat (g) 0.046 0.046 0.92 0.92 0 0.046 0.046Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid-based preparations to rats

98Animal Technology and Welfare August 2020In Pilot Study 1, the time to consume gels reduced after the fi rst two days suggesting the need for a period of acclimatisation prior to starting the experiment. Hence in Pilot Study 2, the rats initially received a control gel for three days. Subsequently, the intervention began with each of the 3 gel types administered twice in a randomised order over 6 days (order determined by random sequence generation (www.random.org)). Timers were set to record the time taken to eat the gels. Each day the rats were weighed, inspected for wellbeing and food consumption was recorded (Figure 2).26Two major experimental studies utilising oil-enriched gels during rat pregnancyFollowing the pilot studies, two major experimental studies were carried out as informed by the results of the pilot studies. The major purpose of these two experimental studies was to determine the effects of fi sh oil supplementation during pregnancy on the offspring (not reported here). However gel consumption and effects on wellbeing were assessed and are reported here.In the fi rst trial, 98 SD dams were allocated to a high-fat or a matched control diet prior to mating. Control gels were started 5 days before the fi rst attempt at mating. Once mated, dams received a gel on each day of gestation, and the lactation period, while continuing on their allocated diet. These gels were raspberry fl avoured and contained 0.05ml of unoxidised fi sh oil, or no oil (control) (Figure 1). Figure 1. A female rat holding an oil-enriched gel in its paws.14 raspberry flavoured, and were either control gels (no oil), or contained 0.05ml of fish oil that was oxidised to various levels (peroxide value 5, 10, or 40 meq/kg), or 1ml of highly oxidised fish oil (peroxide value 40 meq/kg) (Figure 1). On each day a gel was placed into the food hopper and the following day it was noted whether or not it had been completely eaten. The animals were observed daily for signs of stress and wellbeing.26. (insert fig Figure 1. A female rat holding an oil-enriched gel in its paws. Formatted: English (United Kingdom)Formatted: Font: (Default) Arial, English (United Kingdom)Formatted: English (United Kingdom)Formatted: Font: (Default) Arial, English (United Kingdom)Formatted: Font: (Default) Arial, English (United Kingdom)Formatted: English (United Kingdom)Formatted: English (United Kingdom)Formatted: Font: (Default) Arial, English (United Kingdom)Formatted: English (United Kingdom)Formatted: Font: (Default) Arial, English (United Kingdom)Formatted: English (United Kingdom)Figure 2. Flow diagram, representing the 4 studies used to assess the reliability of emulsifi ed gel treatments.15Figure 2. Flow diagram, representing the 4 studies used to assess the reliability of emulsified gel treatments.ure 1 here)(insert figure 2 here)Statistical analyses Across both studies, differences in the time taken to eat the gels completely, weight change and food consumption were compared using Formatted: English (United Kingdom)Formatted: English (United Kingdom)Formatted: Font: ( Default) Arial, Englis h ( United Kingdom)Formatted: English (United Kingdom)Formatted: Font: ( Default) Arial, Englis h ( United Kingdom)Emulsifi ed gels: a refi ned vehicle for accurate and rapid oral administration of lipid-based preparations to rats

99August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIn the second trial, 99 SD dams were fed the standard chow diet and control gels were started 5 days before the fi rst attempt at mating. Once mated, dams received a gel on each day of pregnancy. These gels were raspberry fl avoured and were either control gels (no oil) or contained 0.05ml of fi sh oil that was oxidised to various levels (peroxide value 5, 10, or 40 meq/kg), or 1ml of highly oxidised fi sh oil (peroxide value 40 meq/kg)(Figure 1).On each day a gel was placed into the food hopper and the following day it was noted whether or not it had been completely eaten. The animals were observed daily for signs of stress and wellbeing.26Statistical analyses Across both studies, differences in the time taken to eat the gels completely, weight change and food consumption were compared using the Mann-Whitney test. The change in the time taken to eat the gels across the intervention period in Pilot Study 2 was analysed using Spearman’s rank correlation coeffi cient. All statistical analyses were carried out in IBM SPSS Statistics v.26.0.0.0 (IBM Corp.). Data are presented as mean +/- standard error and signifi cance was determined as p<0.05.ResultsTable 2. Time to completely consume gels, and food consumption and weight gain in the following 24 hours in rats fed either a chow diet (Pilot Study 1) or a high fat diet (Pilot Study 2). Data are shown as mean ± standard error, n=5 per group. Data in parentheses are negative numbers. †indicates a difference between the unoxidised 0.05ml fl avoured gel and the unoxidised 0.05ml unfl avoured gel p=0.001. *p=0.03 **p=0.01 for comparison with the run in (no gel) period. ††p=0.06 ††† p=0.02 for comparison with the run in (no gel) period. ¥p=0.0003 for comparison between all gels with RJC and all gels with no RJC. ¥¥p=0.009 for comparison between all gels with 1 ml and all gels with 0.05 ml of oil. RJC; raspberry fl avoured jelly crystals.Treatment Time to consume (in min)Food consumption (g/ 24hours)Weight gain (g) in next 24 hoursPilot Study 1Run in (no gel) 20.9 ± 1.1 4.1 ± 1.4Unoxidised oil 0.05ml 47.5 ± 25.4†20.2 ± 1.2 2.0 ± 1.7Oxidised oil 0.05ml 23.3 ± 16.7 18.8 ± 1.1 5.6 ± 2.9Unoxidised oil 1ml 85.5 ± 48.7 18.2 ± 1.1 2.2 ± 3.2Oxidised oil 1ml 19.5 ± 29.5 16 ± 3.2††(4.0) ± 2.6†††Unoxidised oil 0.05ml + RJC 2.9 ± 0.4†20 ± 1.4 5.3 ± 3.4Oxidised oil 0.05ml + RJC 4.6 ± 1.1 21.5 ± 0.7 5.3 ± 3.2Unoxidised oil 1ml + RJC 10.1 ± 6.4 15.8 ± 2.4††1.0 ± 3.8Oxidised oil 1ml + RJC 13.2 ± 7.3 17.8 ± 1.6 1.0 ± 3.3Grouped Analyses: All gels with RJC 7.7 ± 2.5¥18.5 ± 1.0 3.1 ± 1.7 All gels with no RJC 51.1 ± 15.7¥17.8 ± 1.1 1.7 ± 1.4 All gels with Oxidised oil 22.4 ± 8.8 18.8 ± 0.9 2.2 ± 1.7 All gels with Unoxidised oil 36.5 ± 14.8 18.4 ± 0.9 2.5 ± 1.5 All gels with 1ml oil 39.3 ± 15.0 16.9 ± 0.9¥¥0.22 ± 1.6 All gels with 0.05ml oil 19.6 ± 8.1 20.1 ± 0.6¥¥4.5 ± 1.3Pilot Study 2Run in (no gel) 16.3 ± 0.6 1.9 ± 1.3Control (no oil + RJC) 44.8 ± 16.9 14.1 ± 4.9**3.2 ± 1.5Unoxidised oil 0.05ml + RJC 44.2 ± 33.2 14.6 ± 2.7 2.3 ± 1.6Unoxidised oil 0.05ml + 2X RJC42.4 ± 17.3 13.8 ± 1.9*5.2 ± 3.6Emulsifi ed gels: a refi ned vehicle for accurate and rapid oral administration of lipid-based preparations to rats

100Animal Technology and Welfare August 2020Pilot Study 1 There was a 2-day period of acclimatisation, as across all groups the time to fully consume the gel was substantially greater in the first 2 days (381.0 ± 177.8 min vs 29.4 ± 8.5 min, p<0.0001). This was identified as the study was being carried out and it was recognised that this did not reflect individual gel types but the novelty of receiving any gel. To prevent introducing a bias towards greater times for gels delivered on those days, the first 2 days were excluded from the analysis and repeated at the end of the study to make up the full 8 days of treatment. The analysis presented represents the 8 days after acclimatisation. All gel types were eaten completely and the mean time for complete consumption was <90 minutes for all gel types (Table 2). No animals showed any signs of stress or ill-health. Amongst the gel types containing 0.05ml of unoxidised fish oil, the time taken to fully consume them was lower if the gel was raspberry flavoured (2.9 ± 0.4 vs 47.5 ± 25.4 minutes; p=0.001). There were no other significant differences between the individual gel types as regards pattern of consumption. To increase statistical power, gel types were grouped. Overall, gel types containing raspberry flavouring were consumed more quickly than those without (7.7 ± 2.5 vs 51.1 ± 15.7 minutes; p=0.0003). There was no difference in the time to fully consume gel types Figure 3. Time taken to completely consume the gels across the period of Pilot Study 2. Data are shown as means ± standard error, n=5 per group. RJC; raspberry flavoured jelly crystals. 0123456789050100150200Day Time (min)0.05ml oil gel with RJCControl gel with no oil0.05ml oil gel with 2x RJC Figure 3.: Time taken to completely consume the gels across the period of Pilot Study 2. Data areis shownpresented as means ± standard error, n=5 per group. RJC; raspberry flavoured jelly crystals Regression analysis showed reduction in the time to fully consume gels over time for each gel type (control b=(-17.0), p=0.0004; RJC b=(-20.1), p=0.002; 2x RJC b=(-19.4), p=0.03) across all the groups. Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid-based preparations to ratsTable 3. Effect of gel consumption on daily food intake in rats. Data are shown as means ± standard error, n=5 per group. Note that for Pilot Study 1 oxidised and unoxidised gel types have been grouped together as oxidised and unoxidised oil have identical macronutrient content and did not have different effects on food intake. RJC; raspberry flavoured jelly crystals.*p<0.05 for the difference between food consumption or nutritional intake after consumption of the gel compared with baseline.containing 1ml and 0.05ml of fish oil, or between gel types containing oxidised and unoxidised fish oil. Following consumption of the gels, there were no between-gel type differences in food consumption or 24-hour weight gain. Further, grouped analyses showed that the presence or absence of raspberry flavouring or whether the oil was oxidised or unoxidised did not affect food consumption. However, food consumption was reduced after addition of the gel, compared to the lead-in period when the rats did not receive a gel (20.9 ± 1.1 g/day). This reached significance for gels containing 1ml of fish oil (with raspberry 18.0 ± 1.3 g/day; p=0.03, without raspberry 16.9 ± 1.3 g/day; p=0.03), but not for those containing 0.05ml of fish oil (with raspberry 20.0 ± 0.8 g/day; p=0.24, without raspberry 20.0 ± 1.0 g/day; p=0.38) (Table 3). Food consumption was lower following consumption of a gel containing 1ml of oil, compared with gels with 0.05ml of oil (16.9 ± 0.9 vs 20.1 ± 0.6 g/day; p=0.009). Pilot Study 2In rats fed a high-fat diet, all gel types were consumed completely within 24 hours but regression analysis indicated a reduction in the time to consume the gels over the study period (control: no oil with standard raspberry; β=(-17.0); p=0.0004, 0.05ml fish oil with standard raspberry; β=(-20.1); p=0.002, 0.05ml fish oil with double raspberry; β=(-19.4); p=0.03). This occurred over the first 5 days, so that across all groups gels were eaten much faster in the final 4 days (104.3 ± 6.7 minutes vs 17.9 ± 1.6 minutes, p<0.0001) (Figure 3). There were no between gel-type differences in the time to consume the gels, weight gain or food intake (Table 2). However, following consumption of gels there was a reduction of food intake from baseline (16.3 ± 0.6 g/Gel type Baseline food intake (g)Food intake post gel (g)Pilot Study 1: Rats consuming a chow diet0.05ml oil 20.9 ± 1.1 20.0 ± 1.00.05ml oil + RJC 20.9 ± 1.1 20.0 ± 0.81ml oil 20.9 ± 1.1 16.9 ± 1.3*1ml oil + RJC 20.9 ± 1.1 18.0 ± 1.3*Pilot Study 2: Rats consuming a high fat dietControl (no oil + RJC)16.3 ± 0.6 14.1 ± 4.9*0.05ml oil + RJC 16.3 ± 0.6 14.6 ± 2.70.05ml oil + 2x RJC 16.3 ± 0.6 13.8 ± 1.9*

101August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfareday) to 14.1 ± 4.9 g/day (p=0.01), 14.6 ± 2.7 g/day (p=0.17) and 13.8 ± 1.9 g/day (p=0.03) per day after consumption of control, raspberry and double raspberry gels respectively (Table 2). Use of oil-enriched gels in 2 major experimental studies of rat pregnancyAcross the two major experimental studies 155 of the 197 dams were successfully mated and received gels throughout pregnancy with 57 dams also receiving gels throughout lactation, a much longer period than in the pilot studies. A total of 4,242 doses of gel were provided. The dams appeared to seek out the gel treatments and began to eat them almost immediately. They showed no signs of stress upon handling and appeared comfortable with researchers. All gel types were eaten completely across all groups irrespective of whether the dam consumed a control or high-fat diet and even in those dams that received gels containing a high dose of highly oxidised fish oil.DiscussionWe have reported a method for producing oil-enriched flavoured gels using an emulsifier and shown that they represent an acceptable, reproducible and efficient method of oral oil administration in female rats. All adult female rats consumed the gels whether they were flavoured or not, enriched with 1ml or 0.05ml of oil and whether the oil was highly oxidised or not. Even when fed a highly palatable high-fat diet, the gels were still completely and quickly consumed. As there was no difference in the time taken to consume gels between those made with a standard concentration of raspberry flavouring and those with double-concentrated flavouring, the standard concentration (which has a lower sugar content) should be preferred. Importantly, the rats remained in good health and showed no visible signs of stress associated with provision of gels.Both pilot studies indicated that rats take time to accept and rapidly consume the novel gel treatment. In rats fed a chow diet, the time taken to fully consume the gels reduced after 2 days but this took 5 days when they were fed the highly palatable high-fat diet. In studies where it is critical that the gel is eaten quickly, such as when it contains a chemically unstable constituent such as fish oil, a lead-in period using control gels of at least 5 days is appropriate.We have demonstrated that gels of relatively large volume (5ml) can be completely and reliably consumed by an individual rat. Such large volume gels were necessary in order to incorporate a proportionally large amount of lipid (1ml). However, it is likely that most future studies utilising emulsified gels, could use smaller sized gels e.g. 0.5ml or 1ml and that these would be completely consumed even more rapidly.It is critical to consider the nutritional impact of any oral intervention as vehicles such as gels could affect macronutrient and total energy intake. Importantly, the gels containing a low dose of fish oil (0.05ml), comparable to human consumption had a minimal effect on food intake.23 However, when rats consumed the gels containing a high dose (1ml) of fish oil they reduced their food consumption which altered their overall macronutrient intake (data not shown). It is likely that the nutritional impact would be substantially reduced with smaller gels, so that where the volume of the supplement or drug is low, a smaller gel should be utilised (e.g 1ml). It is important to consider each of the gel components and whether they were necessary. The starch emulsifier was essential in order to incorporate the lipid into the gels. However, in studies without a lipid component this would not be required. Using gelatine in addition to the raspberry jelly powder was also necessary as without it the lipid enriched gels liquefied at room temperature. Using the proportions reported we were able to produce relatively firm, resilient gels that did not melt when brought into the animal laboratory which is key given the ambient temperatures of most small animal research facilities. The raspberry jelly flavouring was not essential, in that gels were completely eaten when unflavoured. However it led to much faster consumption which is advantageous when a gel component is chemically unstable (such as fish oil1,3). We observed no advantage of using double-strength raspberry jelly, even in the context of the high fat diet and we calculated that it substantially increased the carbohydrate content of the gel (data not shown).Welfare is a critical ethical consideration in any study involving animals. We are obligated to work towards the 3Rs i.e. replace or reduce the use of animals where possible and where animal use is essential refine methodology to improve welfare.27 The use of oil-enriched gels we report represents an important refinement to study design that directly improves Animal Welfare. Oro-gastric gavage requires a highly skilled operator and even when expertly performed is a stressful procedure with a high risk of mishap8 which can harm the animal and potentially confound the results of a study.13 It is unphysiological as it bypasses the oral phase of digestion and it has been recommended that oro-gastric gavage should not be used in toxicity studies13 In contrast, the production of gels and provision to the animals described is simple and solves the same problems that gavage does (controlled dose, and rapid administration) but instead of risking mishap and inducing stress, appeared to be an enriching experience for the rats.Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid-based preparations to rats

102Animal Technology and Welfare August 2020In the pilot studies, animals were single-caged. This was because the future studies for which the gels were developed were to include pregnant dams that would be single-caged. Rats are social animals and should be allowed to exhibit their normal social behaviours. Although being housed within sight and smell of each other can ameliorate some of the stresses associated with singleton housing, such caging should only be used when necessary. We believe the gel treatments we have developed could be used for co-housed rats. Where two animals are caged together, placement of the gels in opposite ends of the cage may be sufficient to ensure each animal is dosed. Where more precise control is necessary, temporary cage dividers could be used after gel placement until the gels are consumed.In principle, replacement of daily gavage with provision of a gel, would be expected to reduce stress. In support of this, across all the presented studies, animals did not present with visible signs of stress and appeared comfortable with researchers, which contrasts with a previous study of daily gavage in pregnancy where the animals did appear stressed.24 However, we did not directly compare these methods of administration and did not measure physiological markers of stress such as heart rate, blood pressure, and corticosterone concentration. This could be addressed in future research.The major strength of this series of studies was the very large number of total gel doses administered, all of which were completely consumed with no signs of ill-health. The large cohort of rats included pregnant, lactating and non-pregnant females. Moreover, the gels were eaten even when they contained oxidised oil or the animals were fed a highly palatable diet. However this study has limitations. Only female rats were studied and we did not study other small animals commonly used in biomedical research. There is no reason to expect that males would be less likely to fully consume the gels but it is important that reliability of consumption is assessed in other target animals such as mice. The gels were always provided in the morning, if future studies planned administration at other times of the day, when the animals were more or less active, it would be wise to conduct a brief pilot to ensure rapid consumption. Lastly, following the principle of Reduction, it was important to use a small number of animals in the pilot studies.27 While this limited statistical power to detect small differences between gel types, the sample sizes were sufficient to demonstrate the presence of an acclimatisation period and that raspberry flavoured gels were more quickly consumed than unflavoured gels. Importantly, the critical finding, that every gel was completely consumed, was corroborated by the large experimental studies. In summary, we report a methodology for oil to be incorporated into small gels with the use of an emulsifier. Such gels are highly acceptable to rats and enable a complete dose to be administered quickly. This represents an important refinement over oro-gastric gavage in rats, replacing a stressful and risky experience with an enriching one. This could be of great importance in many studies including those with behavioural outcomes or investigating the developmental origins of health and disease.18 Future animal studies examining the effects of oral nutritional supplementation or drug administration should consider the use of gels as the vehicle.FundingThese studies were funded by an HRC Emerging Researcher First Grant and an Australasian Paediatric Endocrine Care Grant from Pfizer. BBA also received the Maurice Paykel Postdoctoral Fellowship and the Rutherford Postdoctoral Fellowship.Authors’ contributionsThe gel treatment was conceived by MHV and BBA and developed by BBA and VVS. The pilot and experimental trials were conceived by MHV, BBA, and VVS carried out by BBA, VVS and PEB. Data was analysed by BBA and VVS with input from MHV, and interpreted by MHV, BBA, VVS and WSC. The draft was written by VVS and all authors contributed to and approved the final manuscript.AcknowledgementsWe acknowledge Dr Clare Reynolds, Thomas Tsiloulis and the staff of the Vernon-Jensen Unit (University of Auckland) for their valuable technical assistance.Conflicts of InterestNothing to declareReferences1 Albert, B.B., Cameron-Smith, D. Hofman, P.L., et al. (2013). Oxidation of marine omega-3 supplements and human health. Biomed Res Int 2013; 2013: 464921. 2013/06/06. DOI: 10.1155/2013/464921.2 Shahidi, F. and Zhong, Y. (2010). Lipid oxidation and improving the oxidative stability. Chem Soc Rev 2010; 39: 4067-4079.3 Benzie, I.F. (1996). Lipid peroxidation: a review of causes, consequences, measurement and dietary influences. Int J Food Sci Nutr 1996; 47: 233-261. 1996/05/01. DOI: 10.3109/09637489609012586.4 Ismail, A., Bannenberg, G., Rice, H.B., et al. (2016). Oxidation in EPA-and DHA-rich oils: an overview. Lipid Technol 2016; 28: 55-59.Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid-based preparations to rats

103August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfare5 Brown, A.P., Dinger, N. and Levine, B.S. (2005). Stress produced by gavage administration in the rat. Contemp Top Lab Anim Sci 2000; 39: 17-21. 2001/02/15.6 Bonnichsen, M., Dragsted, N. and Hansen, A.K. (2000). The welfare impact of gavaging laboratory rats. Anim Welf 2005; 14: 223-227.7 Damsch, S., Eichenbaum, G., Tonelli, A., et al. (2011). Gavage-related reflux in rats: identification, pathogenesis, and toxicological implications. Toxicol Pathol 2011; 39: 348-360.8 Balcombe, J.P., Barnard, N.D. and Sandusky, C. (2005). Laboratory routines cause animal stress. Contemp Top Lab Anim Sci 2004; 43: 42-51. 2005/01/27.9 Murphy, S.J., Smith, P., Shaivitz, A.B., et al. (2001) The effect of brief halothane anesthesia during daily gavage on complications and body weight in rats. Contemp Top Lab Anim Sci 2001; 40: 9-12. 2001/04/13.10 Turner, P.V., Pekow, C., Vasbinder, M.A., et al. (2011). Administration of substances to laboratory animals: equipment considerations, vehicle selection, and solute preparation. J Am Assoc Lab Anim Sci 2011; 50: 614-627.11 Õkva, K., Tamoseviciute, E., Ciziute, A., et al. (2006). Refinements for intragastric gavage in rats. Scand J Lab Anim Sci 2006; 33: 243-252.12 Walker, M.K., Boberg, J.R., Walsh, M.T., et al. (2012). A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice. Toxicol Appl Pharmacol 2012; 260: 65-69.13 Vandenberg, L.N., Welshons, W.V., Vom Saal, F.S., et al. (2014). Should oral gavage be abandoned in toxicity testing of endocrine disruptors? Environ Health 2014; 13: 1-7.14 Rostamkhani, F., Zardooz, H., Zahediasl, S., et al. (2012). Comparison of the effects of acute and chronic psychological stress on metabolic features in rats. J Zhejiang Univ Sci B 2012; 13: 904-912.15 Barlow, S., Knight, A. and Sullivan, F. (1978). Delay in postnatal growth and development of offspring produced by maternal restraint stress during pregnancy in the rat. Teratology 1978; 18: 211-218.16 Lobel, M., Cannella, D.L., Graham, J.E., et al. Pregnancy-specific stress, prenatal health behaviors, and birth outcomes. Health psychol 2008; 27: 604.17 Wadhwa, P.D., Sandman, C.A., Porto, M., et al. The association between prenatal stress and infant birth weight and gestational age at birth: a prospective investigation. Am J Obs Gynecol 1993; 169: 858-865.18 Pankevich, D.E., Mueller, B.R., Brockel, B., et al. (2009). Prenatal stress programming of offspring feeding behavior and energy balance begins early in pregnancy. Physiology & Behaviour 2009; 98: 94.19 Boero, G., Biggio, F., Pisu, M.G., et al. (2018). Combined effect of gestational stress and postpartum stress on maternal care in rats. Physiology & Behaviour 2018; 184: 172-178.20 Gustavsson M, Hodgkinson SC, Fong B, et al. (2010). Maternal supplementation with a complex milk lipid mixture during pregnancy and lactation alters neonatal brain lipid composition but lacks effect on cognitive function in rats. Nutr Res 2010; 30: 279-289.21 Vickers, M.H., Guan, J., Gustavsson, M., et al. (2009). Supplementation with a mixture of complex lipids derived from milk to growing rats results in improvements in parameters related to growth and cognition. Nutr Res 2009; 29: 426-435.22 Morton, D., Jennings, M., Buckwell, A., et al. (2001). Refining procedures for the administration of substances. Laboratory Animals 2001; 35: 1-41.23 Reagan-Shaw, S., Nihal, M. and Ahmad, N. (2007). Dose translation from animal to human studies revisited. FASEB J 2008; 22: 659-661. 2007/10/19. DOI: 10.1096/fj.07-9574LSF.24 Albert, B.B., Vickers, M.H., Gray, C., et al. (2016). Oxidized fish oil in rat pregnancy causes high newborn mortality and increases maternal insulin resistance. Am J Physiol Regul Integr Comp Physiol 2016; 311: R497-R504. 2016/07/08. DOI: 10.1152/ajpregu.00005.2016.25 Commission EP. European Pharmacopoeia 5.0: Vol-2. European Directorate for the Quality of Medicines & Healthcare, Council of Europe: Strasbourg, France 2004.26 Council NR. Recognition and Assessment of Stress and Distress. Recognition and Alleviation of Distress in Laboratory Animals. National Academies Press (US), 2008.27 Russell, W.M.S. and Burch, R.L. The principles of humane experimental technique. London, UK: Methuen, 1959.Emulsified gels: a refined vehicle for accurate and rapid oral administration of lipid-based preparations to rats

104Animal Technology and Welfare August 2020SummaryAn investigation into what differences in environments, both intrinsic and extrinsic, make to the success of breeding in Zebrafish (Danio rerio). Four different groups were set up across two different environments. The environmental differences included aquatic systems built by different manufacturers, one utilising a drum filtration system and the other with a more conventional sock filtration system. The room employing drum filtration was also held at a higher level of biosecurity – appearing to be free from Mycobacterium marinum, Mycobacterium haemophilium and without evidence of Pseudoloma neurophilia. The conventional room was older and had fewer biosecurity measures. Both rooms were fed the same dry diet and the more biosecure room fed rotifer as a live feed with the conventional room fed artemia. Two lines of fish were used in this trial – AB and TL and once the fish were sexually mature – 56 days post fertilisation (dpf) they were set up in breeding boxes, at varying time intervals in both rooms. The number and proportion of viable and non-viable embryos were counted to determine the level of fecundity and fertility and the differences between the two rooms and between the frequencies of breeding were also measured. It is hoped these results will help reduce both the number of fish needed and their frequency of use for breeding and improve welfare by reducing handling time while increasing embryo number collection and overall refining the breeding process to become more efficient. Additionally, this trial also highlights potential differences in results between facilities employing different husbandry and welfare practices.IntroductionZebrafish in research are frequently bred for many different purposes, whether continuing the population of lines, creation of different genetic strains or other experimental purposes. Under laboratory conditions they can breed all year round, as laboratory parameters strive to replicate a continuous summer (when they generally breed in the wild).1 Light is a trigger for breeding and eggs are usually laid at the start of the light cycle2 but there are also other cues, including olfactory from a pheromone secreted by the ovaries.3The success rate of breeding varies greatly, due to a number of changing variables.4 These can include extrinsic controllable factors such as diet5 and housing6 but sexual selection based on intrinsic visual and behavioural cues must also be taken into account as a major factor in breeding success.7 Stocking density and male to female ratios are important considerations when setting up a breeding population, for example too many males can cause increased aggression which can interfere with female oviposition,8 bio-secure therefore it can be beneficial to have a female bias.9An important factor when setting up a breeding population is sex determination, which is not fully understood in Zebrafish but is influenced by multiple factors which can cause uneven sex ratios.10 Wild Zebrafish have a sex-linked gene on the tip of chromosome 4, however this appears to have been lost in domesticated Zebrafish, which may have evolved differing sex-determination mechanisms whilst isolated in laboratories11 or may reflect that most wildtype lines of Zebrafish have one common source.Unsuccessful breeding can impact the welfare of the animals, as repeated attempts are needed, causing stress to the fish, whether the exhaustion of the act of breeding itself causing metabolic stress,6 or stress of physical manipulation when placed together for mating, which may involve netting them out of their tank and placing them in a new environment. Contact with the net, exposure to the air and the new environment can all be causes of stress which can be measured through increased cortisol levels.12 The effect of different biosecurity, feed, and frequency of breeding on the fecundity and fertility of Zebrafish (Danio rerio)JOE WARMSLEY, PAUL BARWOOD, VISILA MOICHE and CAROLE WILSONUniversity College London, UCL Zebrafish Facility, Gower Street, London WC1E 6BTCorrespondence: J.warmsley@ucl.ac.ukAnimal Technology and Welfare August 2021

105August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareUnderstanding the variables involved with the breeding of Zebrafish will lead to refinements in the process and a reduction in the required size of a breeding population. The variables looked at in this study are; • diet• habitat (involving different systems)• genetic history• breeding frequencyMethodsPairing and embryo counting For this study, fish were kept in groups of 16 and then separated into 8 pairs for breeding. At this time, they were netted out of the group tank and placed into breeding boxes, between 3pm and 5pm and the embryos collected the next day between 12am and 2pm. The number of pairs from each tank that successfully laid embryos were recorded then all embryos from the original group tank were combined and counted, a falcon tube was used to get an estimate of the number (-/+ 5%). From this, a sub-sample was taken and used to determine a fertilisation rate by counting the ratio of viable to non-viable embryos. DietAll fish in this study were fed the same combined mixed dry food diet, high in protein (approximately 50%), this is a standard diet fed to laboratory Zebrafish but does not replicate their wild natural diet, which is likely to be lower in protein. In the room with higher biosecurity (Room D), fish were fed rotifer, as a live diet, during the nursery stage (up to 28 dpf ) whilst in the conventional room (Room B) fish were additionally fed artemia throughout their life. HabitatThe conventional and more biosecure rooms are from different manufacturers of aquatic systems (see Figure 1) which affects the flow of water around the tank and also employs different types of filtration which may affect water quality and management but the systems themselves are not responsible for differences in biosecurity levels. Differences of this nature are due to differences in husbandry practice – in the room with higher levels of biosecurity (Room D) all fish are grown from embryos which have undergone a disinfection programme and all tanks and equipment undergo a rigorous sterilisation programme. The conventional room (Room B) is older and houses fish from a different husbandry regimen, where disinfection of equipment was not practiced for many years and embryos did not routinely undergo a disinfection programme. Aside from the differences in systems and husbandry practice, the two rooms have different lighting systems – Room B has no dawn and dusk period, the lights come on and go off immediately, while Room D have both a dawn and dusk period. Both rooms have 14 hours of light and 10 hours of dark.System in Room D.Genetic LineTwo different genetic lines were used; a more genetically isolated line – AB which has been genetically isolated at UCL for more than 21 years and TL which has been outbred more recently to other TL lines within the institute. They are also phenotypically different; TL are long finned and AB short finned. AB appear to be more prone to health problems, as we noted higher incidence of egg-bound females, leading to spinal conditions including lordosis System in Room B.Figure 1. Subtle differences between two different manufacturers aquatic systems. The effect of different biosecurity, feed and frequency of breeding on the fecundity and fertility of Zebrafish (Danio rerio)

106Animal Technology and Welfare August 2020and scoliosis and is probably connected to the length of time they have been inbred at UCL and is unlikely to be representative of institutes’ different AB lines.Frequency Different frequencies of breeding were also looked at and the following frequencies of set up were investigated – • weekly• fortnightly• monthly• quarterlyEach tank of fi sh was allocated a group, no fi sh were set up fewer or greater times than the group they were allocated to, for example fi sh in the once-a-week breeding group were always set up once a week and fi sh in the quarterly breeding group were only ever set up once every quarter (Table 1). ResultsSex RatioAs the two lines were grown from 5dpf, before the adult fi sh could be moved into fi nal groupings, they were sexed. Interestingly, the sex ratios seemed to be infl uenced by the different environmental conditions in the rooms. The sex ratios were uneven with the AB line favouring males whilst the opposite was true of the TL, having more females. In the case of the TL line in Room B, the lack of males meant that the desired number of repeats could not be set up (Figure 2).Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods was compared, fi sh from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). ROOM ROOM D ROOM BDiet Rotifer Brineshrimp RotiferLine AB TL AB TL AB TLWeekly AB1 AB2 TL1 TL2 A1 A2 A3 A5 A6 A7 A8Fortnightly AB3 AB4 TL3 TL4 B1 B2 B3 B5 B6 B7 B8Monthly AB5 AB6 TL5 TL6 C1 C2 C3 C5 C6 C7Quarterly AB7 AB8 TL7 TL8 D1 D2 D3 D5 D6 D7Table 1. Breeding Rotation – Groups of fi sh in breeding programme. Diet: see diet for further explanation, Line: see genetic line for further explanation, Weekly, fortnightly, monthly and quarterly indicates frequency of fi sh set up for breeding. Each code represents a group of 16 fi sh – 8 males and 8 females, for example AB1 represents 16 fi sh.11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)Figure 2. Sex ratios, appear to be affected by line and room. 11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)Figure 3. Two-way ANOVA. There was a signifi cant difference in the % of pairs laying weekly and fed with Room D diet (P<0.0001). Percentage of Pairs LayedAB (Room D) AB (Room D)TL (Room B (D Food))AB (Room B (D Food))The effect of different biosecurity, feed and frequency of breeding on the fecundity and fertility of Zebrafi sh (Danio rerio)Males Females11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)Males Females11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)Males Females11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)Males Females11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)11Set-up frequency and line differencesWhen the percentage of pairs laying at different time periods were compared, fish from the TL line consistently laid more than the AB line, irrespective of the period between set ups (Figure 3). Figure 2 – sex ratios, appear to be affected by line and room. 020406080100Weekly Fortnightly MonthlyPercentage of Pairs LayedAB (Room D) TL (Room D)

107August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareHowever, both lines appeared to lay more consistently when paired once every two weeks (Figures 4 and 5).Weekly spawning events in the AB line were more infl uenced by environmental factors than those of the TL line (Figure 6) but overall tended to produce fewer fertilised and more unfertilised embryos than the TL line (Figure 7). DiscussionIt is clear from all the data that the more outbred TL line is more likely to lay (Figure. 3), produce a more consistent yield (Figure 5) and produces embryos with a higher fertilisation rate (Figure 6). Genetic history appears to be a major infl uence on fertility, in Zebrafi sh as well as in other species, in a process known as inbreeding depression.13 This not only affects fertility but also health, the more genetically isolated a line becomes the fewer embryos are produced, whilst creating larger numbers of non-viable embryos (Figure 7).This is important to try to understand in Zebrafi sh. It is better understood in other research species such as mice, where inbreeding depression is a well-known phenomenon. It is therefore benefi cial to use hybrid females where possible, as the genetic history of the males has no impact on breeding success.14 However, it is a possibility that the difference in fertility may be due to the phenotypical differences, as this study does not empirically show that genetic history was the cause. As the rotifer diet was only fed during the nursery stage and artemia was fed throughout the lifespan in the conventional room, the comparison is not aiming at nutritional differences but the effect of live food enrichment during adulthood. The live food diet showed a signifi cant difference to the dry food (Figure 6) but interestingly only across the different rooms. Literature suggests the fi sh fed live food throughout their 12 Figure 3 Two-way ANOVA. There was a significant difference in the % of pairs laying weekly and fed with Room D diet (P<0.0001). However, both lines seemed to lay more consistently when paired once every two weeks (Figures 4 and 5). Figure 4 Line graph showing the percentage of pairs laying over time of the AB line, comparing weekly set up to fortnightly. The weekly set up fluctuates in yield, compared to the more consistent fortnightly set up. Both appear to decrease in yield over time. 010203040506070809010015-Jan 22-Jan 29-Jan 5-Feb 12-Feb 19-Feb 26-Feb 5-Mar 12-Mar% Pairs Layed ABWeekly FortnightlyFigure 4. Line graph showing the percentage of pairs laying over time of the AB line, comparing weekly set up to fortnightly. The weekly set up fl uctuates in yield compared to the more consistent fortnightly set up. Both appear to decrease in yield over time.Figure 5. Line graph showing the percentage of pairs laying over time of the TL line, comparing weekly set up to fortnightly. The weekly set up fl uctuates in yield compared to the more consistent fortnightly set up. Both appear to increase in yield over time.13 Figure 5 Line graph showing the percentage of pairs laying over time of the TL line, comparing weekly set up to fortnightly. The weekly set up fluctuates in yield, compared to the more consistent fortnightly set up. Both appear to increase in yield over time. Weekly spawning events in the AB line were more influenced by environmental factors than those of the TL line (figure 6), but overall tended to produce fewer fertilised and more unfertilised embryos than the TL line (Figure 7). 010203040506070809010015-Jan 22-Jan 29-Jan 5-Feb 12-Feb 19-Feb 26-Feb 5-Mar 12-Mar% Pairs Layed TLWeekly FortnightlyRMD (RMD Food)RMB (RMB Food)RMB (RMD Food)020406080100% of AB pairs laying weekly13 Figure 5 Line graph showing the percentage of pairs laying over time of the TL line, comparing weekly set up to fortnightly. The weekly set up fluctuates in yield, compared to the more consistent fortnightly set up. Both appear to increase in yield over time. Weekly spawning events in the AB line were more influenced by environmental factors than those of the TL line (figure 6), but overall tended to produce fewer fertilised and more unfertilised embryos than the TL line (Figure 7). 010203040506070809010015-Jan 22-Jan 29-Jan 5-Feb 12-Feb 19-Feb 26-Feb 5-Mar 12-Mar% Pairs Layed TLWeekly FortnightlyRMD (RMD Food)RMB (RMB Food)RMB (RMD Food)020406080100% of AB pairs laying weeklyFigure 6. One-way ANOVA. There were signifi cant differencesin the percentage of AB’s pairs laying weekly depending on the room and diet.Figure 7. Bar chart showing the differences in the number of embryos collected between the two rooms, the lines within the rooms and showing fertilisation rate. Red represents infertile embryos and blue represents fertilised embryos.14 Figure 6 One-way ANOVA. There were significant differences in the percentage of AB’s pairs laying weekly depending on the room and diet. Figure 7 Bar chart showing the differences in the number of embryos collected between the two rooms, the lines within the rooms and showing fertilisation rate. Red represents infertile embryos and blue represents fertilised embryos. Discussion It is clear from all the data that the more outbred TL line is more likely to lay (Figure. 3), produce a more consistent yield (Figure 5) and 05001000150020002500AB TL AB TLMore Biosecure ConventionalAverage Number of EmbryosAverage Number of Embryos% Pairs Layed AB % Pairs Layed TLThe effect of different biosecurity, feed and frequency of breeding on the fecundity and fertility of Zebrafi sh (Danio rerio)

108Animal Technology and Welfare August 2020adulthood should be more fecund, probably because of the enrichment a live food diet can give, which allows for more natural behaviour, whilst perhaps providing a better nutritional diet than dry food.15 This study suggests other factors influence fecundity, as well as access to live feed, as fecundity was raised in Room B, as opposed to Room D even when the fish were fed the non-live diet in Room B.It also appears that there were a higher number of embryos in the conventional room for both the AB and TL lines. There are many possible reasons for this difference; variation in lighting (both colouration of the tank and the position of the breeding box in relation to the light),16 water flow differences and the separate filter systems. However, there was a higher fertilisation rate in the more bio-secure room perhaps due to the reduced pathogen load, allowing for healthier fish.17 There were differences between breeding frequencies (Figures 4 and 5) with the fortnightly showing a more consistent yield, whilst those set up weekly fluctuated, this was clear in the AB line (Figure 4). The fluctuation may be due to stress and exhaustion of frequently being set up to breed and suggests more time may be needed to recover from a breeding session. When setting up to breed, contact with the net, exposure to the air and the new environment are all causes of stress12 and it can take time to fully recover from this stress.18 Breeding too often can cause metabolic stress to the females which can also have lasting impacts.6It is also important to consider when setting up a breeding population that Zebrafish sex ratios are rarely even (Figure 1). Sex determination in Zebrafish is still not fully understood, there are many variables which can contribute to it but it is known that these include extrinsic factors such as water temperature as well as intrinsic, such as partial chromosome loss.19 As a result of this, the desired number of repeat set-ups for fish mating were not always achieved because of shortages of either males or females within the groups.Further WorkThis study is planned to continue until the fish are 18 months old, which is the recommended maximum age for a breeding population.2 It will then be possible to assess how each population’s embryo number and fertilisation rate deteriorate over time. We have a new rotifer culture system which will now allow us to feed all of our adult fish rotifer in addition to their dry food diet, we can therefore view the effect of the nutritional differences between rotifer and artemia. We are also to trial gut loading of the rotifer with different nutritional compounds in order to refine the live food diet, to improve both overall health and breeding. It is important to note that in this study, we only used paired breeding boxes but it is important and necessary to assess other methods such as larger breeding boxes or trays inserted into the tank. These other breeding methods may introduce more variables and may have differing effects, for example when placing breeding trays into home tanks, the male to female ratios will have impacts and the effects of the social hierarchy will have greater impacts. Concluding RemarksZebrafish have great plasticity, they have evolved to survive in a wide range of habitats, they are both ectothermic and poikilothermic, meaning their body temperature is dictated by their environment and they do not require a fixed body temperature to maintain their health. It is also likely to mean that it will be difficult for us to find the perfect welfare and husbandry standards for them and this trial shows that there are many variables that must be considered, not just in breeding fish but in all aspects of their husbandry and welfare.AcknowledgementsWe acknowledge the help of our colleagues Jenna Hakkesteeg, Elise Hitchcock and Heather Callaway in both the practical work involved in this study but also in the preparation of this paper.References1 Spence, R. Smith, C. (2006). Mating preference of female zebrafish, Danio rerio, in relation to male dominance. Behavioural Ecology, Vol 17, No. 5, 779-783.2 Nasiadka, A. Clark, M.D. (2012). Zebrafish Breeding in the Laboratory Environment. ILAR Journal, Vol 53, No. 2, 161-168.3 van den Hurk, R. Lambert, JGD. (1983). Ovarian steroid glucuronides function as sex pheromones for male zebrafish, Brachydanio rerio. Canadian Journal of Zoology, Vol 61, No. 11, 2381-2387.4 Tsang, B. Zahid, H. Ansari, R. Chi-Yeung Lee, R. Partap, A. Gerlai, R. (2017). Breeding Zebrafish: A Review of Different Methods and a Discussion on Standardization. Zebrafish, Vol 14, No. 6, 561-573.5 Markovich, ML. Rizzuto, NV. Brown, PB. (2007). Diet Affects Spawning in Zebrafish. Zebrafish, Vol 4, No. 1, 69-74.6 Kurtzman, M. Craig, M. Grizzle, B. Hove, J. (2010). Sexually segregated housing results in improved early larval survival in zebrafish. LabanimNY, Vol 39, No.6, 183-189.7 Pyron, M. (2003). Female preferences and malemale interactions in zebrafish (Danio rerio). Canadian Journal of Zoology, Vol 81, No. 1, 122-125.The effect of different biosecurity, feed and frequency of breeding on the fecundity and fertility of Zebrafish (Danio rerio)

109August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfare8 Spence, R. Smith, C. (2005). Male territoriality mediates density and sex ratio effects on oviposition in the zebrafish, Danio rerio, Animal Behaviour, Vol 69, No.6, 1317-1323.9 Castranova, D. Lawton, A (2011). The Effect of Stocking Densities on Reproductive Performance in Laboratory Zebrafish (Danio rerio). Zebrafish, Vol 8, No. 3, 141-146.10 Lawrence, C. Ebersole, JP. Kesseli, RV. (2008). Rapid growth and out-crossing promote female development in zebrafish (Danio rerio). Environmental Biology of Fishes, Vol 81, No.2, 239-246.11 Wilson, CA. High, SK. McCluskey, BM. Amores, A. Yan, YL. Titus, TA. Anderson, JL. Batzel, P. Carvan, MJ. Schartl, M. Postlethwait, JH. (2014). Wild Sex in Zebrafish: Loss of the Natural Sex Determinant in Domesticated Strains. Genetics, Vol 198, No. 3, 1291-1308.12 Ramsay, JM. Feist, GW. Varga, ZM. Westerfield, M. Kent, ML. Schreck, CB. (2009). Whole-body cortisol response of zebrafish to acute net handling stress. Aquaculture, Vol 297, No. 1-4, 157-162.13 Charlesworth, D. Willis, JH. (2009). The genetics of inbreeding depression. Nature Reviews Genetics, Vol 10, No.1, 783-796.14 Monson, CA. Sadler, KC. (2009). Inbreeding Depression and Outbreeding Depression Are Evident in Wild-Type Zebrafish Lines. Zebrafish, Vol 7, No.2, 189-197.15 Lawrence, C. Sanders, E. Henry, E. (2012). Methods for Culturing Saltwater Rotifers (Brachionus plicatilis) for Rearing Larval Zebrafish. Zebrafish, Vol 9, No. 3, 140-146.16 Spence, R. Ashton, R. Smith, C. (2007). Oviposition Decisions Are Mediated by Spawning Site Quality in Wild and Domesticated Zebrafish, Danio rerio, Behaviour, Vol 144, No. 8, 953-966.17 Crim, MJ. Riley, LK. (2012). Viral Diseases in Zebrafish: What Is Known and Unknown, ILAR Journal, Vol 53, No. 2, 135-143.18 Pavlidis, M. Digka, N. Theodoridi, A. Campo, A. Barsakis, K. Skouradakis, G. Samaras, A. Tsalafouta, A. (2013). Husbandry of Zebrafish, Danio rerio, and the Cortisol Stress Response. Vol 10, No.4, 524-531.19 Liew, W.C. Orbán, L. (2013). Zebrafish sex: a complicated affair. Briefings in Functional Genomics, Vol 13, No. 2, 172-187.The effect of different biosecurity, feed and frequency of breeding on the fecundity and fertility of Zebrafish (Danio rerio)

110Animal Technology and Welfare August 2020Watch it in actionby visiting:somniscientific.com/accuracyTHE SOMNI AMD 3+Somni Scientific’s AMD 3+ is the ultimate in Inhalant Anaesthesia gas delivery. This pressurized anaesthesia system can deliver accurate adjustable flowrates to 3 stations simultaneously. Each station is equipped with a flowmeter and toggle switch that allows the user to leave flows preset or adjust to their needs. Unlike any other system on the market this allows users the greatest flexibility for their procedures. Accuracy(Accurate Multiple Delivery)COMPATIBLE WITH EXISTING UK/EU:ProductsAccessoriesFittingsINHALATION ANAESTHESIA EQUIPMENT AND SERVICESOMNI Scientific is centered on the animal welfare and research community with a focus on clinical accuracy, clinician/technician safety, economic performance and intuitive functionality.SOMNI PROVIDES UNPARALLELED CUSTOMER SERVICE, CLINICAL AND TECHNICAL SUPPORT.(T) 0800 0129101 (D) 01872 248890 (M) 07798 969805 enquiries@somniscientific.com www.somniscientific.co.uk

111August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareSPECIAL INTEREST SECTIONElectronic instrumentation of a swingletree for equid pull load monitoring: a contribution for the welfare and performance of working donkeysJOÃO PAULO COELHO,1,2 JOÃO BRANDÃO RODRIGUES,3 LUÍS QUEIJO,1HIGOR VENDRAMINI ROSSE,1 FRANCISCO ALBUQUERQUE,1 ANDREW JUDGE,3 FIONA COOKE3and CHRIS GARRETT31 Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal 2 Research Centre in Digitalization and Intelligent Robotics (CeDRI), Campus de Santa Apolónia, 5300-253 Bragança, Portugal3 The Donkey Sanctuary, Devon EX10 0NU, Sidmouth, UKCorrespondence: jpcoelho@ipb.pt Abstract Equids play a fundamental role in supporting livelihoods in many parts of the world. Being able to access the animal’s welfare, especially while performing tasks that involve high levels of physical effort such as those found in agroforestry activities, is of utmost importance. The Donkey Sanctuary, a UK-based international charitable institution, has designed a project that aims to develop a set of tools to evaluate the working conditions of donkeys and mules worldwide. This requires the measurement of several different parameters, including the force exerted by an animal to pull a load during work. This article presents the stages of design, development and implementation of a device capable of carrying out these measurements with minimal human intervention and with negligible impact on the task operating conditions. Data obtained from real fi eld conditions validates the devised measurement method.Keywords: working equids, Animal Welfare, embedded systems, electronic instrumentation, data acquisition, force measurementIntroductionEquids are still kept for working purposes and relied upon as a major resource in many parts of the world and are essential to agricultural and industrial activities, both as pack and traction animals.1,2 They also play a key role in strengthening human livelihoods through their contribution to economic, environmental and social capital, particularly in low and middle-income countries, where animal energy represents a vast and extremely important sustainable power resource.3Equids also make a wider, systemic contribution to the sustainability of agroforestry-based economies, through the amelioration of soil degradation and near optimal transfer of consumed biomass into workforce and natural fertiliser.4 This, in turn, supports food security and economic self-reliance through a reduction in the consumption of external resources.5 These reasons have, over recent years, contributed towards an emerging trend for the use of animal traction, as an alternative or complementary option to motorised traction, in small and medium sized farms in developed countries. The use of animal traction has also proved to be economically viable in other activities such as forest or urban surrounding management.6August 2021 Animal Technology and WelfareWatch it in actionby visiting:somniscientific.com/accuracyTHE SOMNI AMD 3+Somni Scientific’s AMD 3+ Accuracy(Accurate Multiple Delivery)COMPATIBLE WITH EXISTING UK/EU:ProductsAccessoriesFittingsSOMNI Scientific is centered on the animal welfare and research community with a focus on clinical accuracy, clinician/technician safety, economic performance and intuitive functionality.SOMNI PROVIDES UNPARALLELED CUSTOMER SERVICE, CLINICAL AND TECHNICAL SUPPORT.

112Animal Technology and Welfare August 2020Although there is very little doubt about their importance as a working force, the use of equids should always respect their appropriate physical limits, dignity and ensuring their health and welfare. The correct and efficient use of working equids clearly depends on how they are attached to the implement they are pulling, the weight distribution of the materials they are carrying, the quality of equipment used and also how well the animals have been trained and managed throughout their lives.7 The way an animal is harnessed and hitched to a plough, cart, wagon or saddle can affect their overall health, welfare and ability to execute the required tasks efficiently. A poorly designed or ill-fitted, harness will cause inefficient transfer of power from the animal to the equipment, leading to decreased working efficiency and output, discomfort, fatigue and in many cases cutaneous and musculoskeletal injuries.8The gathering of evidence based scientific knowledge regarding working equids, is considered a powerful tool in improving the health and welfare of working equids in developing countries, as well as for animals used in modern animal traction work.The Donkey Sanctuary, a UK based, international charity working with donkeys and mules worldwide, designed a research project focussed on the analysis and characterisation of different harness models. The aim of this project was to better understand the interactions between working equids and different equipment while performing a set of distinct load pulling tasks. A central aspect of this project was the monitoring of the force exerted by the animals and this paper aims to describe the methodology used to obtain such data resorting to the instrumentation of a custom-made swingletree.In the context of animal traction processes, a swingletree is a device used to balance the load during the pull and allows the harness to move freely with the equid without causing abrasions. By embedding the load cell and related electronics into the swingletree, it enables the measurement of pulling forces without disrupting the task carried out by the equid. In this frame of reference, this paper describes a novel solution devised to continuously and, with minimal human intervention, monitor the force exerted by an animal during work. Related workStrain gauges are transducers that can directly translate deformation variation into changes in electrical resistance. Despite their simplicity, these types of devices are ubiquitous and can be found in a myriad of different industrial, technical and scientific state-of-the-art applications. For example Huang and Ying (2017) resort to three-axis strain gauges to measure printed circuit board warping due to reflow based soldering processes.9 In Anaf et al (2020), the same type of sensors are used to monitor the wood behaviour in real-time measurements.10 Biomedics is also an area where those types of sensors are frequently employed.11The use of strain gauges can also be found in the context of health monitoring and animal behaviour evaluation. For example, Chien and Chen (2018),12 uses those type of transducers to detect the number of eggs in hens’ nests and Sturges et al (2019)13 resort to those types of sensors to measure the intracranial pressure in dogs.In the equid context, a very expressive number of the scientific articles found in the literature focussed on the analysis and influence of several distinct variables on the pressure exerted by the saddle over the sport horses’ backs, in response to the demands of the equine industry. The variation in pressure over the animal skin, when subjected to different types of loads is traditionally measured using commercially available saddle pressure measuring system composed of an array of piezoelectric sensors.14-17 When compared to strain gauges, piezoelectric sensors have a high-pass frequency behaviour which makes them unsuited to steady-state or low frequency load measurements as is the case of the current work. Moreover, since the majority of the available studies are conducted regarding the influence of riding saddles and validation of rehabilitation exercises,18 little has been explored about the injury caused by the misuse of equipment used by working equids.19 This fact adds an increased value to this work since it focusses on the development of a measuring device that can be used to explore those lines of research.It is worth noticing that commercial dynamometers are unable to be used in the actual framework due to several reasons: first, the measurement system must be able to withstand harsh environmental conditions that are found under real working conditions. Standard solutions found for measuring static forces are typically built to be used within laboratory conditions and not in dusty environments, subject to collisions and high variations of temperature and humidity. Second, the measurement device must not interfere with the task to be monitored. The typical shape of a dynamometer involves a load cell connected to a measurement unit through an electrical cable. This arrangement is not suited for the required application since the cables would make the measurement process difficult to manage and may even interfere with the working capacity of the equids or the normal execution of the task under real conditions. Moreover, being able to provide the type of power supply required by some of the commercial solutions, can present several logistic and technical challenges since the measurements must be carried out in remote rural locations. Finally, other factors that prevented the choice of a commercial solution are related to economic cost and ease of use. Regarding the latter, it is worth noting that equid owners themselves may need to carry Special Interest Section – A contribution for the welfare and performance of working donkeys

113August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfareout the measurement. Hence, the measurement system cannot introduce additional obstacles to the operating conditions of everyday tasks. Furthermore, the device must be simple to operate and should not rely on the existence of smartphone terminals or third-party communication infrastructures.Due to all these design constraints, a custom made swingletree, with an integrated dynamometer, was designed, implemented and tested in real-life operating conditions. Material and methodsThe study took place in the Animal Traction Interpretation Centre (CITRAN), in Galiza, Spain, during June 2019. Three healthy adult Zamorano-Leones donkeys (10, 13 and 13 years old), living under similar management and working conditions, were used in the experimental field trials. At the commencement of the study, individual bodyweights were accurately obtained (354, 345 and 340 kg). All donkeys presented an ideal body condition.The objective was to understand if working donkeys exerted different force for the same task, depending on the type of collar used. For that, three different collars were used (a complete adjustable full collar, a breast collar and a new prototype collar developed by the harness makers involved in the project) while pulling a modern light plough along a flat transect, comprised of two segments of 75 metres each.Pulling forces exerted by the animals were recorded using a custom-made dynamometer whose design, implementation and metrological characterisation is the main objective of this article.During the process, a team of welfare professionals and veterinarians continually monitored the health and welfare of the animals. The workload effort was evaluated through the heart rate, measured using an Equine Polar System® attached to the donkey’s harness.UK Animal Welfare legislation was followed during this study.20 Non-invasive techniques were used to assess the equids involved. The study was conducted in accordance with the Declaration of Helsinki21 and the protocol approved by the executive board of The Donkey Sanctuary, UK.Instrumented swingletree: architecture and implementationThe objective of this study was to perform the instrumentation of a swingletree to measure the mechanical stress exerted by donkeys or other equids, during their normal work while pulling different load types. The use of a measurement system in real operating conditions poses several challenges that must be considered. In the current case, one of these challenges is related to the extreme conditions to which the device will be subjected during the measurement process. In particular, vibrations caused by collisions, high thermal amplitude, dust and debris as well as high levels of humidity.In addition to the harsh environmental conditions, it is necessary to ensure that the deployment in the field of the measurement system does not conflict with the normal operating conditions. That is, the complexity of using the dynamometer should not cause additional difficulties or even increase the animal’s rigging time when compared to the use of conventional equipment.It is also an important design condition that Animal Welfare is not disturbed. Furthermore, the measurement device must be able to cope with technical conditions such as being able to be battery operated, withstand at least 8 hours of continuous activity and be easy to be managed by regular workers in the field.For the system to be wear-resistant and able to withstand extreme operating conditions and not increase the burden associated with the execution of normal tasks performed by working equids, any external electrical wiring must be avoided or kept to a minimum.The use of wireless technology in order to avoid the use of connecting cables between the load cell and the data logging system was initially considered. Indeed, wireless data transmission methods have already been presented in the literature as a way to keep the transducer away from the data acquisition and recording system. For example, DiGampaola et al (2017) present a wireless strain gauge measurement system resorting to near-field communication using RFID techniques.22 Harnett et al (2011) describes a strain gauge based wireless sensor network applied to stream flow measurement in environmental research.23 Wireless measurement of Computer Numerically Controlled (CNC) tools cutting forces using strain gauges has been reported by de Oliveria et al (2020) and Chakavarthi et al (2018) resort to an RFID based technology to keep track of the strain in metals.24,25 Also, in the domain of civil engineering, Kumar and Hossain (2018) and Furkan et al (2020) report the development of wireless sensing systems for monitoring the health of civil structures.26,27.However, none of those solutions were suited to be included in the current study. On one hand, all those methods require a two-part solution: the emitter, which includes the load cell and the receiver which must be handled or operated by the worker during the task. On the other hand, due to mechanical constraints, the swingletree steel structure prevents radio frequency Special Interest Section – A contribution for the welfare and performance of working donkeys

114Animal Technology and Welfare August 2020(RF) data transmission. At the same time, the current solution must be self- contained in the sense that it should not require the user to have access to any type of third-party technology, such as hand-held devices or information network infrastructures. This is due to the fact that it is expected to use this dynamometer in some developing countries where those technologies are not readily available.In this frame of reference, a custom-made measurement system was designed and developed where a load cell, together with the data acquisition and recording electronics, was completely integrated into a regular-sized swingletree. This compact approach leads to a robust plug-and-play measurement solution that can be easily deployed in the fi eld, even by non-technical staff, under real operating conditions. Overall system designConceptually, a swingletree consists of a rod or bar made of wood or metal to guarantee the traction balance produced by a draught animal when pulling a load. In the current framework, an additional technological layer must be added to this simple device to enable the measurement and recording of the pulling forces occurring during a typical donkey’s working day. The traditional swingletree was completely redesigned to integrate the embedded electronics. For this reason, this section presents a thorough description of the mechatronic project component. Specifi cally, the details associated with the swingletree structure will be presented within subsection Mechanical Structure with further subsections to describe the features concerning the electronic instrumentation, the embedded system and its fi rmware. Finally, the subsection Integration and Casing reports the solution devised to integrate both the mechanical and electronic components.16 Figure 1. Diagram of a single animal hitched to a load where the maincomponents of the harnessing structure can be viewed. The distance between points A and B, drawn over the croup’s hips, is used to define the length of the swingletree.In practice, the swingletree is a crossbar built around a piece of hard wood or steel metal. There is a ring at the centre of the swingletree where the load, cart or implement can be connected. At the same time, the end of both traces are attached to grooves or rings at the extremities of the swingletree.As previously stated, the aim of this work is to develop a swingletree able to measure and store data about the donkey pulling forces during a common workday. To accomplish this, a new swingletree structure had to be designed and built to allow the inclusion of the entire electronic Figure 1. Diagram of a single animal hitched to a load where the main components of the harnessing structure can be viewed. The distance between points A and B, drawn over the croup’s hips, is used to defi ne the length of the swingletree.Mechanical structureFigure 1 represents the typical way of hitching a donkey to a load using a collar harness. When pulling a load the animal’s legs and shoulders displace forwards and backwards leading to movement, in opposite directions of both traces. The swingletree enables the independent movement of the traces whilst ensuring that the draft on both sides of the harness remains balanced. That is, it turns the cyclically moving motion of the donkey into a steadier source of pulling power, by negating the movement around its shoulders.The size of the swingletree is related to the size of the equid that it will be used for. The minimum size should correspond to the distance between points A and B of the hips in the equid’s croup. This will allow the traces to move freely around the equid’s body and will avoid friction. Considering the average size of working equids worldwide, it was decided to design the swingletree with 60 cm as a standard measure for this study.The swingletree will be connected to the collar via the traces and to a light plough weighing around 40 kg.In practice, the swingletree is a crossbar built around a piece of hard wood or steel metal. There is a ring at the centre of the swingletree where the load, cart or implement can be connected. At the same time, the end of both traces are attached to grooves or rings at the extremities of the swingletree.As previously stated, the aim of this work is to develop a swingletree able to measure and store data about the donkey’s pulling forces during a common workday. To accomplish this, a new swingletree structure had to be designed and built to allow the inclusion of the entire electronic component which will be described under Electronic Instrumentation and Hardware.Special Interest Section – A contribution for the welfare and performance of working donkeys

115August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfare17 component which will be described under Electronic Instrumentation and Hardware. The overall shape and dimensions devised for this element are presented in Figure 2. The swingletree has an end-to-end length of 60 cm and includes a centred enclosure, with a volume of around 240 cm3, where the load cell is fitted. Figure 2. Representation of the most relevant dimensions of the swingletree (expressed in millimeters). For the construction of the swingletree main structure, pipes of EN 10219 carbon steel with a 40 mm square profile was used. This material exhibits Figure 2.Representation of the most relevant dimensions of the swingletree (expressed in millimeters).19 Figure 3. Simulation results where a static force of 4500 N is applied to the two interface rings. Having defined the mechanical structure of the swingletree, the following section will describe the electronic instrumentation chain. Electronic instrumentation and hardware The load cell is the fundamental component on the electronic instrumentation chain. For this reason, care must be taken to choose an appropriate device to match the application. In this work, the transducer selected is an S-Shape load cell rated to a nominal mechanical load Figure 3.Simulation results where a static force of 4500 N is applied to the two interface rings.The overall shape and dimensions devised for this element are presented in Figure 2. The swingletree has an end-to-end length of 60 cm and includes a centred enclosure with a volume of around 240cm3, where the load cell is fi tted.For the construction of the swingletree main structure, pipes of EN 10219 carbon steel with a 40mm square profi le was used. This material exhibits a yield strength of around 275 N/mm2 and a linear density of 3.3 kg/m. The tube walls have a thickness of 3mm and Figure 3 shows the simulation results where a static force of 4500 N is applied to the two interface rings. The choice of this force value for the simulation is based on the fact that it is the upper limit force of the load cell used.As seen on the colour map, there are some points where the pressure exerted slightly exceeds the yield strength of the material. However, this is not critical since due to the size of the target donkeys for the current study, we are expecting forces lower than half of this threshold value. It is worth noting that pipes with thicker walls can be used in case one wants tocarry out force measurements on stronger animals such as horses. Having defi ned the mechanical structure ofthe swingletree, the following section will describe the electronic instrumentation chain.Electronic instrumentation and hardwareThe load cell is the fundamental component on the electronic instrumentation chain. For this reason, care must be taken to choose an appropriate device to match the application. In this work, the transducer selected is an S-Shape load cell rated to a nominal mechanical load capacity of 500 kilogramme-force (kgf). This device is identifi ed by the company reference CZL-301. The reason for the selection of a 500 kgf load cell was a somewhat conservative decision. First, there is only empirical knowledge on the nominal loads pulled by equids and second, this value has a large dispersion and strongly depends on the breed of the animal as well as the task performed. Moreover, the price is similar to lower range transducers such as those that are in the range of 300 kgf. As it will be shown later, the load measurement resolution achieved with 21 Figure 4. CAD image showing the integration of the load cell into the swingletree. Note the central ring, where the load, cart or implement can be attached. The most relevant metrological features of the load cell can be read from the manufacturer’s calibration certificate which is delivered with the product. In particular, the sensitivity is equal to 2.0044 mV/V and it exhibit an accuracy and repeatability, relative to the full-scale value, equal to 0.02% and 0.0017% respectively. The temperature is a very important disturbance factor in any load cell and, in this case, the manufacturer defines the effect of temperature on both zero and span to be equal to Figure 4. CAD image showing the integration of the load cell into the swingletree. Note the central ring, where the load, cart or implement can be attached.Special Interest Section – A contribution for the welfare and performance of working donkeys

116Animal Technology and Welfare August 2020this conservative selection is more than reasonable. Figure 4 shows using a 3D generated Computer-Aided Design (CAD) image, how this load cell will be fi tted into the swingletree structure. One of its ends is bolted to the frame and the other has a swivel which will be used to attach the load to be pulled.The most relevant metrological features of the load cell can be read from the manufacturer’s calibration certifi cate which is delivered with the product. In particular, the sensitivity is equal to 2.0044 mV/V and it exhibits an accuracy and repeatability, relative to the full-scale value, equal to 0.02% and 0.0017% respectively. The temperature is a very important disturbance factor in any load cell and, in this case, the manufacturer defi nes the effect of temperature on both zero and range to be equal to 0.0019% of the full-scale for each 10°C of increment in temperature. The input and output impedance are within ± 5 Ω around the nominal value of 350 Ω. Bandwidth is not provided but it is expected to depend heavily on the mechanical inertia of the primary element to which the strain gauge bridge is attached. However, its value is expected to be higher than the dynamic behaviour of the current process which will be below the range of one hertz.The full instrumentation and data acquisition system developed for the current work is presented in Figure 5. The load cell signal conditioning is mainly performed by the HX711 integrated circuit. A regulated voltage of 2.6 V is supplied to load cell strain gauge bridge and the unbalance voltage generated due to the mechanical deformation is delivered to an analogue amplifi er with a gain of 128 V/V. In this context, and taking into consideration the transducer characteristics enumerated above, the maximum unbalanced voltage at the amplifi er input will be equal to 2.0044 × 1.8 ≈5.2 mV and the full-scale voltage at the amplifi er output will be 128 × 5.2 which leads to a value near 667 mV.This signal will be input to a differential input 24-bit Σ-Δ analogue-to-digital (A/D) converter. According to theHX711 datasheet, the maximum absolute allowable differential voltage at the A/D input is half the bridgevoltage AVCC. In the current case, this value is ±1.3 Vwhich means that it is possible to perform measurements over the entire dynamic range of the load cell without attaining saturation.The data is delivered to the microcontroller unit (MCU) using a synchronous serial communication interface where two wires, one for data and the other for the clock signal, are used. Since we are dealing with a Σ-Δ converter, the use of an anti-aliasing fi lter is not a fundamental requirement.28 In fact, this type of A/D converters, oversample the input signal to a rate much higher than that of the Nyquist frequency. Moreover, the data is decimated and passed through a digital fi lter before being available to the output. This also enables the quantisation noise to be moved away from the frequency band of interest. However, in the current setup, a fi rst-order low-pass balanced fi lter is added. As can be seen from the schematic diagram of Figure 5, this fi lter is implemented using two 100 Ω resistors and a 0.1 µF ceramic capacitor. Note that the bridge impedance will have a major infl uence on the corner frequency of the fi lter which, in this case and with the components used, is near 2 kHz.Figure 5.Conceptual diagram of the hardware instrumentation and data acquisition system.24 Figure 5. Conceptual diagram of the hardware instrumentation and data acquisition system. Removal of unwanted signal artifacts and spurious noise is carried out at different levels. Initially, the 50 Hz noise induced by the power lines is mitigated by the electromagnetic interference (EMI) shielding promoted by the swingletree metallic casing and by the HX711 integrated notch filter and, secondly, using a digital low-pass FIR filter embedded in the MCU firmware. A detailed description about the firmware and its functionality will be provided in Firmware and Signal processing. Special Interest Section – A contribution for the welfare and performance of working donkeys

117August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareRemoval of unwanted signal artifacts and spurious noise is carried out at different levels. Initially, the 50 Hz noise induced by the power lines is mitigated by the electromagnetic interference (EMI) shielding promoted by the swingletree metallic casing and by the HX711 integrated notch filter and secondly, using a digital low-pass FIR filter embedded in the MCU firmware. A detailed description about the firmware and its functionality will be provided in Firmware and Signal processing. The MCU selected for this application is the popular Atmel’s ATmega328. This is a general-purpose microcontroller featuring an 8-bit RISC processor with a modified Harvard architecture. In the present design, the MCU will run with a 3.3 V supply voltage using its internal clock defined at 8 MHz. This device will gather data from several peripherals and populate a secure-digital (SD) card with the time-tag provided by a real-time clock (RTC) and the tensile strength reported by the HX711.There are several reasons that lead to the choice of an SD card for storing the data: on one hand, the metallic structure of the swingletree prevents the use of RF signals. On the other hand, the measurement system is not reliant on the existence of any third-party communication infrastructure or even on the use of smartphones or other handheld devices. The data communication between the MCU and the SD card is carried out using the SPI protocol where the former acts as the master and the latter as the slave.The time-tag is delivered to the MCU, upon request, by a DS1308 RTC integrated circuit. The current date and time are sent to the MCU using the I2C communication protocol. This device will also produce a regular 1 Hz digital signal that is used to wake up the microcontroller such that a new sample can be registered in the SD card. Data retention is ensured by a coin type manganese lithium battery, CR2032, with a nominal voltage of 3.0 V and a charge capacity of 210 mAh.The choice of this particular RTC was mainly driven by the requirement of having all the integrated circuits operating with the same supply voltage of 3.3 V. This voltage is provided by a DC-DC buck converter built around the Texas Instruments’ LM3671 and three passive external components: one inductor and two capacitors. This voltage regulator can provide up to 600 mA of output current, while also ensuring current overload and thermal shutdown protection.One of the design requirements was that the system must be battery operated in order to make it portable and self-contained. This aspect was achieved using an 18650 Li-Ion cell with a charge capacity of 2600 mAh. The battery can be charged in situ since the developed electronic circuit board includes a charge regulator, in this case handled by the TP4056 integrated circuit. Overcharge, over discharge and overcurrent protections are provided by the DW01A integrated circuit.A key design requirement was the capacity to sustain the operation for a period of at least one full working day. The total quiescent current of the measuring system is around 20 mA, raising to near 58 mA during the active state. The current consumption during the active state is slightly above the double of the one observed during the idle state. However, this active state only occurs during less than 10% of the time leading to an average current consumption, during a full operating cycle below 25 mA. That is, an average power consumption of around 83 mW.Since the battery capacity is rated to 2600 mAh, with a nominal output voltage of 3.6 V, it exhibits an energy content of 9620 mWh which, according to the average circuit consumption, leads to a theoretical autonomy close to 100 hours which is in line with the practical results measured. This result has superseded the initial autonomy design constraint by a factor of ten. The next section will focus on the description of the firmware that runs inside the MCU and the signal processing tasks carried out.Firmware and signal processingThis system was devised to be used by non-technical persons. For this reason, a plug and play approach was considered and the interface with the user was made as simple as possible. Therefore there is just one switch for controlling the on/off status and a pair of light emitting diodes (LED) to inform the user if the system is active or if an error has occurred.The firmware that runs continuously on the MCU is presented as a flowchart in Figure 6. It is divided in three different routines: the main routine that deals with the hardware detection and ports instantiation; an interrupt service routine (ISR) that runs whenever a rising edge, generated by the FT/OUT pin of the DS1308, takes place; and an error routine.The user can activate the system just by turning on the key switch. This will start the firmware that runs a set of hardware interfacing procedures beginning with I/O ports definition, checking the status of the HX711 and RTC and creating a new file in the SD card root. The filename is defined as the concatenation of the current date and an index number. This index is an integer number that is automatically incremented if the storage device already has a file with the same name. Currently, due to using an FAT16 structure, the filename can only have eight characters plus the extension. For this reason, a maximum of 100 files can be created in a single day which, in practice, has been demonstrated to be more than adequate.Special Interest Section – A contribution for the welfare and performance of working donkeys

118Animal Technology and Welfare August 2020The last action of the main routine is to turn the MCU into sleep-mode. This feature is used to reduce the overall energy footprint of the device. The microcontroller is awoken, with time rate of one second, by an external interrupt signal generated by the RTC. This triggers the execution of the ISR routine which, in turn, is responsible for acquire, process and record the value of the mechanical tension applied to the load cell.The actual value of tensile strength is delivered by the HX711 using a two’s complement 24-bit format. The update rate of the A/D registry is carried out with a frequency of 10 Hz and the actual mechanical tension data is saved on the SD card and obtained from the average of the last 10 samples.On the assumption that a tension force that acts over the load cell has a positive sign while a compression force will have a negative one, and since a 2.6 V voltage is used to supply the strain gauge bridge, the load cell unbalance voltageνd, expressed in µV, will be equal to:νd= ±0.106 x F #(1)where F denotes the force, measured in newtons (N), applied to the load cell.As already referenced, this differential voltage is amplifi ed before being fed to the A/D converter. The gain factor considered in this application was 128 V/V which results in a voltage at the amplifi er output being proportional to 13.6 µV for each newton of force applied to the load cell.Since the A/D converter has a 24-bit resolution which is too high for the current application, a downgrading of the resolution was made by reducing the 24-bit data to half. With 12 bits, the resolution is now equal to 0.635 mV per bit which leads to the ability of detecting load disturbances on the order of magnitude of 40 N.Figure 6.Firmware fl owchart running on the MCU.29 Figure 6. Firmware flowchart running on the MCU. The user can activate the system just by turning on the key switch. This will start the firmware that runs a set of hardware interfacing procedures beginning with I/O ports definition, checking the status of the HX711 and RTC and creating a new file in the SD card root. The filename is defined as the concatenation of the current date and an index number. This index is an integer number that is automatically incremented if the storage device already has a file with the same name. Currently, due to using an Special Interest Section – A contribution for the welfare and performance of working donkeys

119August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIn order to further reduce high-frequency disturbances, the measured signal is low- pass fi ltered by a second-order IIR Butterworth fi lter with a cut-off frequency of 0.45 Hz. The fi lter was implemented using a direct-form II with one second-order structure as illustrated in Figure 7.Error parsing is carried out by a third routine that drives the error LED to blink at different rates and patterns according to the type of error found. Five main different types of errors are considered, with each one coded with a different light blinking pattern. This pattern is composed by the concatenation of three bits where each bit is encoded using a line coding scheme where different duty-cycle light pulses are used to represent the two different bit states: 0 or 1. Each bit has a 1000 milliseconds (ms) duration, the bit 0 is defi ned as having a 20% duty-cycle while the bit 1 is considered to have a duty-cycle of 80%. The three-bit pattern of a give error is repeated cyclically with a period of four seconds. For example, the error regarding the initialisation failure of the HX711 is encoded as “000” which will result in a sequence of three short burst pulses separated by 1000 ms delay. On the other hand, an SD card write failure is defi ned by “101” which gives rise to two long pulses separated by a short one.Being able to assemble all the different mechanical components and electronic circuits in one robust solution that could endure the harsh environments found during agroforestry tasks was a particular important condition. The following section will deal with the description of the solutions found to integrate all the components into one fi nal robust solution.Integration and casingHaving the means of acquiring data with the lowest disruption possible throughout the monitoring process is always a challenging task. This is even more crucial when developing a measurement system targeted to non-technical users and, most importantly, not com- promising the normal execution of the equid’s work. It must be stressed that agroforestry processes take place in harsher environmental conditions than those found in many other circumstances such as in common industrial facilities for example. For this reason, robustness and physical integrity are key factors when devising a solution to be used by agroforestry workers in everyday tasks. In the context of the current work, the performance of the electronic instrumentation measurement chain strongly depends on its ability to endure severe environmental and vibration conditions such as large thermal loads, humidity, dust and considerable physical impacts. For this reason, the correct layout of the electronics, in conjunction with an appropriately designed case, will enable the data acquisition equipment to withstand the conditions found in normal operating conditions.Figure 8 presents a 3D view of the enclosure devised to host the printed circuit-board with all its electronics. As can be seen in Figure 8a, interaction with the user is very simple and composed of only a few LEDs and a turn-key switch. The box slides into the tubular structure of the swingletree as shown in Figure 8b. The status of the LEDs is visible through acrylic lenses embedded in each of the four holes in the metallic structure. Moreover, during operation, the key can be removed from the swingletree switch and a rubber cap can be placed to cover the hole that was left open avoiding the entry of dust, humidity and other debris.The electronics enclosure box was 3D printed with a ‘Stratasys Eden 260V’ 3D printer, (manufactured by ‘Object Eden’). This machine operation is based on a Poly- Jet technology and the material used in the fi nal solution is identifi ed by the manufacturer using the reference RGD525. According to the product datasheet, this material is able to withstand temperatures up to 32 cut-off frequency of 0.45 Hz. The filter was implemented using a direct-form II with one second-order structure as illustrated in Figure 7. Figure 7. Digital filter structure embedded in the microcontroller firmware. Error parsing is carried out by a third routine that drives the error LED to blink at different rates and patterns according to the type of error found. Five main different types of errors are considered, with each one coded with a different light blinking pattern. This pattern is composed by the concatenation of three bits where each bit is encoded using a line coding scheme where different duty-cycle light pulses are used to represent the two different bit states: 0 or 1. Each bit has a 1000 milliseconds (ms) duration, the bit 0 is defined as having a 20% duty-cycle while the bit 1 is considered to have a duty-cycle of 80%. The three-bit pattern of a give error is repeated cyclically with a period of four seconds. For example, the Figure 7.Digital fi lter structure embedded in the microcontroller fi rmware.Special Interest Section – A contribution for the welfare and performance of working donkeys

120Animal Technology and Welfare August 202075°C. For the current prototype, this temperature was considered the upper working limit which is also near the maximum-operating temperature of the enclosed electronics. Figure 9 shows the fi nal version of the swingletree deployed in the fi eld where it has been used to measure the force exerted by the donkey during ploughing.In the section that follows, details regarding the load cell calibration process will be presented. This process allowed to obtain, in an empirical way, the transducer calibration curve considering the nominal excitation conditions of the strain gauge bridge.Calibration procedureIn order for the system to be calibrated, the load cell was clamped into a Shimadzu’s AGS-X series universal test frames as can be seen in Figure 10. In particular, the trials were conducted using the 10 kN version of this machine. Tensile loads from 0 N up to 4750 N, with a 250 N step resolution, were applied to the measurement system and the raw values delivered by the A/D converter were recorded. It should be noted that, every time a target load was reached, the traction force was kept constant for a time interval of fi ve seconds after which the A/D delivered value was recorded.35 Figure 8. In (a) a 3D view of the solution and in (b) its integration in the swingletree structure. The electronics enclosure box was 3D printed with a “Stratasys Eden 260V” 3D printer, (manufactured by “Object Eden”). This machine operation is based on a Poly- Jet technology and the material used in the final solution is identified by the manufacturer using the reference RGD525. According to the product datasheet, this material is able to withstand temperatures up to 75 ºC. For the current prototype, this temperature was considered the upper working limit which is also near the maximum-operating temperature of the enclosed electronics. Figure 9 shows the final version of the swingletree deployed in the field where it has been used to measure the force exerted by the donkey during ploughing. (a) (b)Figure 8.In (a) a 3D view of the solution and in (b) its integration in the swingletree structure.Figure 9.The swingletree with all the electronic instrumentation operating in the fi eld.36 In the section that follows, details regarding the load cell calibration process will be presented. This process allowed to obtain, in an empirical way, the transducer calibration curve considering the nominal excitation conditions of the strain gauge bridge. Figure 9. The swingletree with all the electronic instrumentation operating in the field. Calibration procedure In order for the system to be calibrated, the load cell was clamped into a Shimadzu’s AGS-X series universal test frames as can be seen in Figure 10. In particular, the trials were conducted using the 10 kN version of this machine. Tensile loads from 0 N up to 4750 N, with a 250 N step resolution, were applied to the measurement system and the raw values delivered by the A/D converter were recorded. It should be noted that, Special Interest Section – A contribution for the welfare and performance of working donkeys

121August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe interval plot, obtained after a set of trials, is presented in Figure 11. The marking dots identify the measurement average values, which are complemented by the addition of a ±3σ limit bars.From the calibration curve, there is a clear linear relationship between both the dependent and independent variables. In particular, a least squares approximation of a fi rst-order parametric model to the obtained data, leads to:MA/D= 0.2193 x F + 9.5952 #(2)where MA/D is the value provided by the A/D converter and F is the traction force measured in newtons.For this model, the Pearson correlation coeffi cient is 0.9999966 and, as can be seen from Figure 12, the model error, relative to the full-scale value, is below 2.2%.This mathematical relation, between the tensile force and the value provided by the A/D converter, is used in the fi rmware to map the data from the HX711, after being digitally fi ltered, into traction force.An initial set of experiments was carried out in order to evaluate the performance of this instrumentation method in the fi eld. The next section describes the methodology adopted in the experimental trials. Samples of the measured data will be provided followed by a discussion of the results obtained.Results and discussionAlthough some data is provided, this section is, by no means, intended to provide an exhaustive analysis of the data gathered during the fi eld assays. Indeed, only a small subset of the experimental data will be used in order to characterise and validate the dynamometer design described in the previous section.Throughout this section, the data obtained from the set of experiments carried out under the conditions described in Materials and Methods will be presented. This data will be used to infer about the measurement’s consistency and repeatability of the devised swingletree dynamometer under real operating conditions.38 Figure 10. Calibration setup for the swingletree load cell.Figure 11. Calibration curve obtained from the set of experimental tests.For this model, the Pearson correlation coefficient is 0.9999966 and, as can be seen from Figure 12, the model error, relative to the full-scale value, is below 2.2%.Figure 10.Calibration setup for the swingletree load cell.38 Figure 10. Calibration setup for the swingletree load cell.Figure 11. Calibration curve obtained from the set of experimental tests.For this model, the Pearson correlation coefficient is 0.9999966 and, as can be seen from Figure 12, the model error, relative to the full-scale value, is below 2.2%.Figure 11. Calibration curve obtained from the set of experimental tests.Traction Force/NNormalised A/D value39 Figure 12. Modelling error, relative to the full-scale value, resulting from the linear approximation described by equation (2).This mathematical relation, between the tensile force and the value provided by the A/D converter, is used in the firmware to map the data from the HX711, after being digitally filtered, into traction force.An initial set of experiments was carried out in order to evaluate the performance of this instrumentation method in the field. The next section describes the methodology adopted in the experimental trials. Samples of the measured data will be provided followed by a discussion of the results obtained.Figure 12.Modelling error, relative to the full-scale value, resulting from the linear approximation described by equation (2).Traction Force/NRelative Error%Special Interest Section – A contribution for the welfare and performance of working donkeys

122Animal Technology and Welfare August 2020The plot presented in Figure 13 represents the pulling force delivered by the three different donkeys during the fi rst one-minute timeframe. As highlighted in the beginning of this section, the analysis of the acquired data is outside the scope of this paper. For this reason we will concentrate instead on the metrological results of the dynamometer.To test for data consistency, a series of trials were carried out and ANOVA tests were used to analyse data. In this case, we will test the null hypothesis that there is no statistical signifi cance between the data acquired from a set of two different trials performed under the same conditions. That is, the same donkey, with the same load, same collar and following the same route. Each trial is composed of sixty samples which concerns a one-minute data registration. It is worth noting that the donkeys were allowed to rest between trials to reduce variability resulting from donkey tiredness. Indeed, a new trial only began 41 Figure 13, Force delivered by three different donkeys, Zimbro, Santiago and Galiego during a one-minute test period using the same collar. To test for data consistency, a set of trials were carried out and ANOVA tests were used to analyse data. In this case, we will test the null hypothesis that there is no statistical significance between the data acquired from a set of two different trials performed under the same conditions. That is, the same donkey, with the same load, same collar and following the same route. Each trial is composed of sixty samples which concerns a one-minute data registration. It is worth noting that the donkeys were allowed to rest between trials to reduce variability resulting from donkey tiredness. Indeed, a new trial only began when the heart rate was around 44 bpm, which is considered the normal rate at rest. Figure 13.Force delivered by three different donkeys, Zimbro, Santiago and Galiego during a one-minute test period using the same collar.Count Sum Average VarianceTrial #1 60 32365 599.35 51526.97Trial #2 60 29222 541.14 35111.41Table 1.Data summary regarding two trials, under the same operating conditions, performed by the donkey Zimbro.Variation SS df MS F p-value FcritBetween groups 91479.02 1 91479 2.1 0.14913 3.9307Within groups 4591834 118 43319Total 4683313 119Table 2. ANOVA table obtained from two trials done with Zimbro.when the heart rate was around 44 bpm, which is considered the normal rate at rest.Table 1 presents the statistics summary regarding the data gathered from two trials conducted with the donkey Zimbro and Table 2 show the respective one-way ANOVA table assuming an α value equal to 0.05.From Table 2, it is possible to see that the F value is lower than that of Critical Frequency (Fcrit). For this reason, the null hypothesis is accepted which lead to the conclusion that the means of the two datasets are statistically equivalent.The same procedure was carried out for the remaining two donkeys (Santiago and Galiego). Table 3 and Table 4show the ANOVA results computed using the dataset of two trials with Santiago. Moreover, Tables 5 and 6 refer to the ANOVA obtained from the Galiego set of trials.Count Sum Average VarianceTrial #1 60 31824 578.61 20710.96Trial #2 60 30558 555.59 21348.57Table 3.Summary regarding two trials, under the same operating conditions, performed by the donkey Santiago.Special Interest Section – A contribution for the welfare and performance of working donkeys

123August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe analysis of the Santiago and Galiego ANOVA tables is consistent with the one formulated for Zimbro. That is, there is a 95% confidence that the means of both trials are equal which validates the repeatability of the measuring process under field conditions.ConclusionAnimal Welfare is directly related to the conditions in which the animal lives. Moreover, in cases where equids are used as a workforce, it is essential to ensure that they are working within their physical capacity while respecting their health and welfare boundaries. For this reason, it is fundamental to be able to quantify and provide insights into the load profile of donkeys within their working environment while carrying out tasks. So far, this information is not available in the literature due to the lack of commercial measuring tools targeting this specific application. To address this problem, a new strategy was devised to measure the forces delivered during draft work and without influencing the tasks performed by the animals. This alternative measuring strategy has been documented within this paper and concerns the development and instrumentation of a swingletree. The developed device can measure the pull forces and record their values as a time-series on an SD card for subsequent offline data analysis.As the system must be replicated in order to be used in different parts of the world, it is necessary to ensure that Source SS df MS F p-value FcritBetween groups 14583.93 1 14584 0.7 0.41 3.93Within groups 2271214 118 21030Total 2285798 119Table 4. ANOVA table obtained from two trials carried out with Santiago.Source SS df MS F p-value FcritBetween groups 156208 1 156208 3.6 0.06 3.93Within groups 4747753 118 43170Total 4904961 119Table 5. Data summary regarding two trials, under the same operating conditions, performed by the donkey Galiego.Count Sum Average VarianceTrial #1 60 30840 550.72 41563.93Trial #2 60 26658 476.03 44777.03Table 6. ANOVA table obtained from two trials completed with Galiego.the economic cost is low, both in terms of technology and its application. Moreover, the exact conditions that may affect the use of the technology in some countries are unknown, which steer the required solution towards being autonomous and not relying on any third-party technologies or information infrastructures such as smartphones, Wi-Fi or any other wireless communication standard. These constraints have led to the solution documented throughout the Instrumentation section. Besides these requisite design conditions, the system must additionally be able to withstand the extreme conditions found in agricultural environments. Materials and methods presents the results obtained from a set of trials carried out in typical field condition. From the results obtained, it was possible to verify that the designed measurement system is able to provide data consistency.Several trials have already been conducted and many data points gathered within different work operating conditions, such as different types of loads and distinct collars worn by the donkeys. In future, it is intended to gather the same type of data from other places in the world where donkeys have a major footprint in the economic ecosystem.AcknowledgementThe authors would like to acknowledge the APTRAN – Portuguese Association of Animal Traction and AGATRAN – Galician Association of Animal Traction, for the support provided during the field work.Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.Special Interest Section – A contribution for the welfare and performance of working donkeys

124Animal Technology and Welfare August 2020FundingThe author(s) received no financial support for the research, authorship and/or publication of this article.References1 Sanctuary TD. Achieving Agenda 2030: How the welfare of working animals delivers for development, International Coalition for Working Equids. International Coalition for Working Equids (ICWE); 2019.2 Tarricone S, Karatosidi D, Marsico G. (2013). Modern use of donkeys. Iranian Journal of Applied Animal Science. 2013;3:13-7.3 Geiger, M., Hockenhull, J., Buller, H., Engida, G.T., Getachew, M., Burden FA, et al. (2020). Understanding the Attitudes of Communities to the Social, Economic, and Cultural Importance of Working Donkeys in Rural, Peri-urban, and Urban Areas of Ethiopia. Frontiers in Veterinary Science. 2020;7.4 Spugnoli, P., Dainelli, R. (2012). Environmental comparison of draught animal and tractor power. Sustainability Science. 2012;8(1):61-72.5. Stringer, A. (2014). Improving animal health for poverty alleviation and sustainable livelihoods. Veterinar y Record. 2014;175(21):526-9.6 Rodrigues, J.B., Schlechter, P., Spychiger, H., Spinelli, R., Oliveira, N., Figueiredo, T. (2017). The XXI century mountains: sustainable management of mountainous areas based on animal traction. Open Agriculture. 2017;2(1).7 Terrestrial Animal Health Code (2019). Chapter 7.12: Welfare of working equids. for Animal Health WO, editor 2019.8 Heleski, C., Mclean, A., Swanson, J. (2015). Practical methods for improving the welfare of horses, donkeys, mules, and other working draft animals in developing areas. Improving Animal Welfare: A Practical Approach: 2nd Edition. 2015:328-48.9 Huang, C-Y., Ying, K-C. (2017). Applying strain gauges to measuring thermal warpage of printed circuit boards. Measurement. 2017;110:239-48.10 Anaf, W., Cabal, A., Robbe, M., Schalm, O. (2020). Real-Time Wood Behaviour: The Use of Strain Gauges for Preventive Conservation Applications. Sensors. 2020;20(1).11 Fastier-Wooller, J., Phan, H-P., Dinh, T., Nguyen, T-K., Cameron, A., Ochsner, A., et al. (2016). Novel Low-Cost Sensor for Human Bite Force Measurement. Sensors. 2016;16(8).12 Chien Y-R, Chen Y-X. (2018) An RFID-Based Smart Nest Box: An Experimental Study of Laying Performance and Behavior of Individual Hens. Sensors. 2018;18(3).13 Sturges, B.K., Dickinson, P.J., Tripp, L.D., Udaltsova, I., LeCouteur, R.A. (2019). Intracranial pressure monitoring in normal dogs using subdural and intraparenchymal miniature strain-gauge transducers. Journal of Veterinary Internal Medicine. 2019;33(2):708-16.14 de Cocq, P., Clayton, H.M., Terada, K., Muller, M., van Leeuwen, J.L. (2009). Usability of normal force distribution measurements to evaluate asymmetrical loading of the back of the horse and different rider positions on a standing horse. The Veterinar y Journal. 2009;181(3):266-73.15 de Cocq, P., van Weeren, P.R., Back, W. (2006). Saddle pressure measuring: Validity, reliability and power to discriminate between different saddle-fits. The Veterinar y Journal. 2006;172(2):265-73.16 Peinen, K.V., Wiestner, T., Rechenberg, B.V., Weishaupt, M.A. (2010). Relationship between saddle pressure measurements and clinical signs of saddle soreness at the withers. Equine Veterinary Journal. 2010;42:650-3.17 Ramseier, L.C., Waldern, N.M., Wiestner, T., Peinen, K.G-v., Weishaupt, M.A. (2013). Saddle pressure distributions of three saddles used for Icelandic horses and their effects on ground reaction forces, limb movements and rider positions at walk and tölt. The Veterinar y Journal. 2013;198:e81-e7.18 Ekstrom, R.A., Osborn, R.W., Hauer, P.L. (2008). Surface Electromyographic Analysis of the Low Back Muscles During Rehabilitation Exercises. Journal of Orthopaedic {\&} Sports Physical Therapy. 2008;38(12):736-45.19 Davis, T. Collar (2007). Pressure Mapping. Saddle, Harness & Horse Collar Maker. The Museum of English Rural Life/The University of Reading/Redlands Road/Reading/RG1 5EX/UK merl@reading.ac.uk/ www.reading.ac.uk/merl / 20 Department for Environment F, Affairs R. ANIMAL WELFARE ACT 2006. 2006. https://www.legislation.gov.uk/ukpga/2006/45/contents.21 Declaration of Helsinki – WMA – The World Medical Association (2013). https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki22 DiGiampaolo, E., DiCarlofelice, A., Gregori, A. (2017). An RFID-Enabled Wireless Strain Gauge Sensor for Static and Dynamic Structural Monitoring. IEEE Sensors Journal 2017;17(2):286-94.23 Harnett, C.K., Schueler, M.T., Blumenthal, N.R., Hopf, K.L., Fox, J.F., Pulugurtha, S. (2011). Calibration and Field Deployment of Low-Cost Fluid Flow-Rate Sensors Using a Wireless Network. IEEE Transactions on instrumentation and measurement. 2011;60(2):633-41.24 de Oliveira, A.J., Silva, D.M.L., da Silva, J.I.D., de Castro Silveira, Z. (2020). Design and experimental set-up of a hybrid dynamometer applied to a fourth axis of the vertical machining center. International Journal of Advancesd Manufacturing Technology. 2020;110(7-8):2155-68.25 Chakaravarthi, G., Logakannan, K.P., Philip, J., Rengaswamy, J., Ramachandran, V., Arunachalam, K. (2018). Reusable Passive Wireless RFID Sensor for Special Interest Section – A contribution for the welfare and performance of working donkeys

125August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareStrain Measurement on Metals, Materials Science, IEEE Sensors Journal. 26 Kumar R, Hossain A. (2018). Experimental Performance and Study of Low Power Strain Gauge Based Wireless Sensor Node for Structure Health Monitoring. Wireless Personal Ccommunications 2018;101(3):1657-69.27 Furkan, M.O., Mao, Q., Livadiotis, S., Mazzotti, M., Aktan, A.E., Sumitro, S.P., et al. (2020). Towards rapid and robust measurements of highway structures deformation using a wireless sensing system derived from wired sensors. Journal of Civil Structural Health Monitoring. 2020;10(2):297-311.28 Rogillio, B., Martin, L.A. (2007). Understanding the Aliasing Effects of Delta-Sigma Analog to Digital Converters. Sandia Corporation; 2007.Special Interest Section – A contribution for the welfare and performance of working donkeys

126Animal Technology and Welfare August 2020PAPER SUMMARY TRANSLATIONSCONTENU DE LA REVUEGels émulsionnés : un moyen perfectionné pour l’administration orale précise et rapide de préparations à base de lipides aux rats VIDIT SATOKAR, MARK VICKERS, PANIA BRIDGE-COMER, WAYNE CUTFIELD, BENJAMIN ALBERTCorrespondance: b.albert@auckland.ac.nz Résumé Le gavage oro-gastrique est utilisé pour administrer avec précision des substances nutritionnelles ou des médicaments aux animaux. Cependant, il provoque du stress et présente un risque substantiel d’accident. L’incorporation dans des gels comestibles est difficile pour les préparations à base de lipides. Nous signalons une nouvelle méthodologie pour la production de gels émulsionnés enrichis en huile, leur efficacité dans des études pilotes et des études expérimentales ultérieures de plus grande envergure. Des gels émulsionnés enrichis en huile de poisson ont été produits à partir d’amidon non polaire. Plusieurs types de gel ont été fabriqués avec des doses d’huile de 0,05 ml ou de 1 ml, de l’huile oxydée ou non oxydée et avec ou sans arôme de framboise. La palatabilité et l’innocuité ont été évaluées avec i) 8 types de gel chez les rats SD femelles consommant un régime alimentaire standard (40 traitements) et ii) 3 types de gel chez les rats consommant un régime alimentaire riche en matières grasses (45 traitements). Par la suite, la palatabilité et l’innocuité ont fait l’objet d’une évaluation plus poussée parmi une vaste cohorte de rates gravides (n=155 ; 4 242 traitements). Dans les deux études, tous les gels ont été consommés entièrement, que les rats aient consommé un régime alimentaire standard ou riche en matières grasses. Il y a eu une période d’acclimatation de 5 jours. Les gels aromatisés à la framboise ont été consommés plus rapidement que les gels non aromatisés. Les rats ont manifesté un comportement positif vis-à-vis de la réception des gels et il n’y a eu aucun effet nocif sur la santé. Dans le cadre des études expérimentales ultérieures, 4 242 doses ont été administrées à des rates gravides et toutes ont été consommées entièrement. Les gels émulsionnés enrichis en huile représentent une méthode d’administration de lipides aux rats facile, hautement acceptable, fiable et sûre, que nous proposons et qui s’avèrent supérieurs au gavage oro-gastrique. Mots-clés: perfectionnement, stress, gestation, bien-être animal, gels émulsionnés, administration de lipidesAnimal Technology and Welfare August 2021

127August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareL’impact de différentes mesures de biosécurité, d’alimentation et de fréquence de reproduction sur la fécondité et la fertilité du poisson-zèbre (Danio rerio)JOE WARMSLEY, PAUL BARWOOD, VISILA MOICHE, CAROLE WILSONCorrespondance: j.warmsley@ucl.ac.uk Résumé Une étude portant sur l’impact des différences au sein des environnements, à la fois intrinsèques et extrinsèques, qui est observé sur le succès de la reproduction parmi les poissons-zèbres (Danio rerio). Quatre groupes différents ont été établis au sein de deux environnements différents. Les différences environnementales comprenaient les systèmes aquatiques conçus par différents fabricants, l’un utilisant un système de filtration à tambour et l’autre doté d’un système de filtration à manchon plus conventionnel. La salle utilisant la filtration à tambour a également été maintenue à un niveau plus élevé de biosécurité – apparaissant comme étant dépourvue de Mycobacterium marinum, de Mycobacterium haemophilium et ne présentant aucune preuve de Pseudoloma neurophilia. La salle conventionnelle était plus ancienne et comportait moins de mesures de biosécurité. Les deux salles ont été alimentées avec le même régime sec et la salle la plus biosécurisée a été alimentée avec des rotifères en tant que nourriture vivante tandis que la salle conventionnelle a été alimentée avec des artémies. Deux lignées de poissons ont été utilisées dans cet essai – AB et TL, et une fois les poissons matures sexuellement - 56 jours après la fécondation (dpf), elles ont été placées dans des boîtes d’élevage, à des intervalles de temps variables dans les deux salles. Le nombre et la proportion d’embryons viables et non viables ont été comptés afin de déterminer le taux de fécondité et de fertilité, et les différences entre les deux salles et entre les fréquences de reproduction ont également été mesurées. Il est espéré que ces résultats contribueront à réduire à la fois le nombre de poissons nécessaires et leur fréquence d’utilisation pour la reproduction, et à améliorer le bien-être en réduisant le temps de manipulation tout en augmentant la collecte du nombre d’embryons et en améliorant globalement le processus de reproduction pour le rendre plus efficace. En outre, cet essai met également en évidence les différences potentielles dans les résultats observés dans les établissements utilisant différentes pratiques d’élevage et de bien-être. ★ ★ ★Instrumentation électronique d’un palonnier pour la surveillance de la charge de traction équine : une contribution au bien-être et à la performance des ânes qui travaillent JOÃO PAULO COELHO, JOÃO BRANDÃO RODRIGUES, LUÍS QUEIJO, HIGOR VENDRAMINI ROSSE, FRANCISCO ALBUQUERQUE, ANDREW JUDGE, FIONA COOKE ET CHRIS GARRETTCorrespondance: jpcoelho@ipb.ptRésumé Les équidés jouent un rôle essentiel dans la contribution aux moyens de subsistance dans de nombreuses régions du monde. Il est de la plus haute importance d’être en mesure d’accéder au bien-être de l’animal, en particulier lorsqu’il accomplit des tâches nécessitant un effort physique élevé, comme celles que l’on retrouve dans les activités d’agroforesterie. Le Donkey Sanctuary, une organisation caritative internationale basée au Royaume-Uni, a conçu un projet qui vise à développer un ensemble d’outils permettant d’évaluer les conditions de travail des ânes et des mules dans le monde entier. Cela nécessite la mesure de plusieurs paramètres différents, y compris la force exercée par un animal pour tirer une charge dans le cadre de son travail. Cet article présente les étapes de conception, de développement et de mise en œuvre d’un dispositif capable d’effectuer ces mesures avec une intervention humaine minimale et avec un impact négligeable sur les conditions d’exécution de la tâche. Les données obtenues à partir de conditions réelles sur le terrain valident la méthode de mesure définie. Mots-clés: équidés qui travaillent, bien-être animal, systèmes intégrés, instrumentation électronique, acquisition de données, mesure dynamométriquePaper Summary Translations

128Animal Technology and Welfare August 2020INHALTVERZEICHNISEmulgierte Gele – ein verbessertes Mittel zur präzisen und schnellen oralen Verabreichung von Präparaten auf Lipidbasis an RattenVIDIT SATOKAR, MARK VICKERS, PANIA BRIDGE-COMER, WAYNE CUTFIELD, BENJAMIN ALBERTKorrespondenz: b.albert@auckland.ac.nz Abstract Die orogastrale Schlundsonde wird verwendet, um Tieren Nahrungsstoffe oder Medikamente gezielt zu verabreichen. Dies ist jedoch mit Stress verbunden und birgt ein erhebliches Risiko für Zwischenfälle. Die Einarbeitung lipidbasierter Präparate in essbare Gele ist schwierig. Wir berichten über eine neue Methodik zur Herstellung von emulgierten, mit Öl angereicherten Gelen und deren Wirksamkeit in Pilotstudien und anschließenden größeren experimentellen Studien. Es wurden emulgierte, mit Fischöl angereicherte Gele unter Verwendung von unpolarer Stärke hergestellt. Es wurden mehrere Arten von Gel mit Öl-Dosierungen von 0,05 ml oder 1 ml, oxidiertem oder nicht oxidiertem Öl und mit oder ohne Himbeergeschmack hergestellt. Geschmackliche Akzeptanz und Sicherheit wurden mit i) 8 Gelarten bei weiblichen SD-Ratten, die Standardfutter erhielten (40 Behandlungen), und ii) 3 Gelarten bei Ratten, die fettreiches Futter erhielten (45 Behandlungen), bewertet. Anschließend wurden die geschmackliche Akzeptanz und die Sicherheit in einer großen Kohorte trächtiger Ratten (n = 155; 4242 Behandlungen) weiter untersucht. In beiden Studien wurden alle Gele vollständig gefressen, unabhängig davon, ob die Ratten Standard- oder fettreiches Futter erhielten. Es gab eine 5-tägige Akklimatisierungsphase. Gele mit Himbeergeschmack wurden schneller gefressen als Gele ohne Geschmack. Die Ratten zeigten ein positives Verhalten gegenüber der Aufnahme der Gele und es gab keine negativen gesundheitlichen Auswirkungen. In nachfolgenden Versuchen wurden 4242 Dosen an trächtige Ratten verabreicht, die alle vollständig gefressen wurden. Mit Öl angereicherte emulgierte Gele stellen eine einfach zu verabreichende, äußerst verträgliche, zuverlässige und sichere Methode der Lipidverabreichung an Ratten dar, die unserer Auffassung nach der orogastralen Schlundsonde überlegen ist. Schlagwörter: Verbesserung, Stress, Trächtigkeit, Tierschutz, emulgierte Gele, LipidverabreichungPaper Summary Translations

129August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareDie Auswirkung unterschiedlicher Biosicherheitsmaßnahmen, Fütterung und Brutfrequenzen auf die Fruchtbarkeit/Fertilität von Zebrafischen (Danio rerio)JOE WARMSLEY, PAUL BARWOOD, VISILA MOICHE, CAROLE WILSONKorrespondenz: j.warmsley@ucl.ac.uk Abstract Es wurde eine Untersuchung darüber angestellt, inwiefern Unterschiede in der Umgebung sowohl intrinsischer als auch extrinsischer Art zu Zuchterfolgen bei Zebrafischen (Danio rerio) beitragen. Vier verschiedene Gruppen wurden in zwei unterschiedlichen Umgebungen untergebracht. Die Umgebungsunterschiede betrafen von verschiedenen Herstellern gebaute aquatische Anlagen – eine mit einem Trommelfiltrationssystem, die andere mit einem konventionellen Strumpffiltrationssystem. Der Raum, in dem die Trommelfiltration zum Einsatz kam, wies auch ein höheres Maß an Biosicherheit auf – er schien frei von Mycobacterium marinum, Mycobacterium haemophilium und ohne Anzeichen von Pseudoloma neurophilia zu sein. In dem älteren konventionellen Raum gab es weniger Biosicherheitsmaßnahmen. In beiden Räumen wurde das gleiche Trockenfutter verfüttert, wobei im biologisch sichereren Raum Rädertierchen als Lebendfutter zum Einsatz kamen, während im konventionellen Raum Artemia verfüttert wurde. Es wurden zwei Fischlinien in diesem Versuch verwendet – AB und TL. Die Fische wurden bei Eintritt der Geschlechtsreife – 56 Tage nach der Befruchtung (dpf) – in beiden Räumen in unterschiedlichen Zeitabständen in Zuchtboxen eingesetzt. Die Anzahl und der Anteil lebensfähiger und nicht lebensfähiger Embryonen wurden gemessen, um den Grad der Fruchtbarkeit/Fertilität zu bestimmen. Die Unterschiede zwischen den beiden Räumen und zwischen den Brutfrequenzen wurden ebenfalls gemessen. Es besteht die Hoffnung, dass diese Versuchsergebnisse dazu beitragen, sowohl die Anzahl als auch die Häufigkeit der benötigten Fische für die Zucht zu reduzieren und das Wohlergehen der Tiere zu verbessern, indem die Handhabungszeit verkürzt und gleichzeitig die Zahl der gewonnenen Embryonen erhöht und der Zuchtprozess insgesamt durch die Weiterentwicklung effizienter wird. Darüber hinaus hebt diese Studie auch potenzielle Unterschiede in den Ergebnissen zwischen Einrichtungen mit unterschiedlichen Haltungs- und Tierschutzpraktiken hervor. ★ ★ ★Elektronische Instrumentierung eines Schwengels zur Zuglast-Überwachung bei Equiden – ein Beitrag zum Tierschutz und Leistungsvermögen von Arbeitseseln JOÃO PAULO COELHO, JOÃO BRANDÃO RODRIGUES, LUÍS QUEIJO, HIGOR VENDRAMINI ROSSE, FRANCISCO ALBUQUERQUE, ANDREW JUDGE, FIONA COOKE UND CHRIS GARRETTKorrespondenz: jpcoelho@ipb.ptAbstract Equiden spielen in vielen Teilen der Welt eine fundamentale Rolle bei der Sicherung des Lebensunterhalts. Die Gewährleistung des Wohlbefindens der Tiere ist von größter Bedeutung, insbesondere bei der Ausführung von Aufgaben, die ein hohes Maß an körperlicher Anstrengung erfordern, wie z. B. bei land- und forstwirtschaftlichen Aktivitäten. The Donkey Sanctuary, eine in Großbritannien ansässige internationale gemeinnützige Institution, hat ein Projekt zur Entwicklung verschiedener Werkzeuge konzipiert, um die Arbeitsbedingungen von Eseln und Maultieren weltweit zu bewerten. Dies bedingt die Messung unterschiedlicher Parameter, einschließlich der Kraft, die ein Tier beim Ziehen einer Last während der Arbeit aufwendet. In diesem Artikel werden die Phasen der Konzeption, der Entwicklung und des Einsatzes eines Geräts vorgestellt, das diese Messungen mit minimalen menschlichen Eingriffen und mit vernachlässigbaren Auswirkungen auf die Betriebsbedingungen der Aufgabe durchführen kann. Die unter realen Feldbedingungen gewonnenen Daten validieren die entwickelte Messmethode. Schlagwörter: Equiden als Arbeitstiere, Tierschutz/Wohlbefinden, eingebettete Systeme, elektronische Instrumentierung, Datenerfassung, KraftmessungPaper Summary Translations

130Animal Technology and Welfare August 2020INDICE DELLA REVISTAGel emulsionati: un veicolo raffinato per la somministrazione orale accurata e rapida di preparati a base di lipidi ai ratti VIDIT SATOKAR, MARK VICKERS, PANIA BRIDGE-COMER, WAYNE CUTFIELD, BENJAMIN ALBERT Corrispondenza: b.albert@auckland.ac.nzAbstract La sonda oro-gastrica viene utilizzata per somministrare con precisione sostanze nutritive o farmaci agli animali. Tuttavia, induce stress e presenta un notevole rischio di incidenti. L’incorporazione nei gel commestibili è difficile per le preparazioni a base di lipidi. Segnaliamo una nuova metodologia per la produzione di gel emulsionati arricchiti di olio, la loro efficacia in studi pilota e successivi studi sperimentali più ampi. I gel emulsionati arricchiti con olio di pesce sono stati prodotti utilizzando amido apolare. Sono stati realizzati diversi tipi di gel incorporando dosi di olio da 0,05 ml o 1 ml, olio ossidato o non ossidato e con o senza aroma di lampone. La palatabilità e la sicurezza sono state valutate con i) 8 tipi di gel in ratti Sprague Dawley femmina che consumavano una dieta di chow (40 trattamenti) e ii) 3 tipi di gel in ratti che consumavano una dieta ricca di grassi (45 trattamenti). Successivamente, la palatabilità e la sicurezza sono state ulteriormente valutate in un’ampia coorte di ratte gravide (n=155; 4242 trattamenti). In entrambi gli studi, tutti i gel sono stati consumati completamente, a prescindere se i ratti consumavano una dieta di chow o una ricca di grassi. C’è stato un periodo di acclimatazione di 5 giorni. I gel al gusto di lampone sono stati consumati più rapidamente dei gel non aromatizzati. I ratti hanno mostrato un comportamento positivo verso la somministrazione dei gel e non ci sono stati effetti negativi sulla salute. In successivi studi sperimentali sono state somministrate 4242 dosi a ratte gravide e sono state tutte completamente consumate. I gel emulsionati arricchiti di olio rappresentano un metodo di facile somministrazione di lipidi ai ratti altamente accettabile, affidabile e sicuro, che giudichiamo superiore alla sonda oro-gastrica.Parole chiave: raffinamento, stress, gravidanza, benessere animale, gel emulsionati, somministrazione di lipidi ★ ★ ★L’effetto di una biosicurezza, alimentazione e frequenza di allevamento diversa sulla fecondità e fertilità del pesce zebra (Danio rerio)JOE WARMSLEY, PAUL BARWOOD, VISILA MOICHE, CAROLE WILSONCorrispondenza: j.warmsley@ucl.ac.uk Abstract Un’indagine sulle differenze di ambiente, sia intrinseche che estrinseche, che determinano il successo dell’allevamento del pesce zebra (Danio rerio). Sono stati istituiti quattro diversi gruppi in due ambienti differenti. Le differenze ambientali prevedevano sistemi acquatici costruiti da diversi produttori: uno che utilizzava un sistema di filtrazione a tamburo e l’altro con un sistema di filtrazione a calza più convenzionale. Anche la camera adibita alla filtrazione a tamburo era tenuta a un livello di biosicurezza più elevato e appariva priva di Mycobacterium marinum, Mycobacterium haemophilium e senza evidenza di Pseudoloma neurophilia. La camera convenzionale era più vecchia e aveva meno misure di biosicurezza. Entrambe le camere sono state alimentate con la stessa dieta secca. La camera più biosicura è stata alimentata con rotifero come mangime vivo e la camera convenzionale con artemia. In questa prova sono state utilizzate due linee di pesci - AB e TL - e una volta che i pesci erano sessualmente maturi, 56 giorni dopo la fecondazione (dpf), sono stati disposti in box di allevamento, a intervalli di tempo variabili in entrambe le camere. Sono stati contati il numero e la percentuale di embrioni vitali e non vitali per determinare il livello di fecondità e fertilità e sono state anche misurate le differenze tra le due camere e tra le frequenze di allevamento. Si auspica che questi risultati aiutino a ridurre sia il numero di pesci necessari che la loro frequenza di utilizzo per l’allevamento e a migliorare il benessere riducendo i tempi di gestione, aumentando la raccolta del numero di embrioni e perfezionando in generale il processo di allevamento per renderlo più efficiente. Inoltre, questo studio evidenzia potenziali differenze nei risultati tra strutture che impiegano diverse pratiche di zootecnia e benessere.Paper Summary Translations

131August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareStrumentazione elettronica di un bilancino per il monitoraggio del carico di trazione degli equidi: un contributo per il benessere e le prestazioni degli asini da lavoro JOÃO PAULO COELHO, JOÃO BRANDÃO RODRIGUES, LUÍS QUEIJO, HIGOR VENDRAMINI ROSSE, FRANCISCO ALBUQUERQUE, ANDREW JUDGE, FIONA COOKE E CHRIS GARRETTCorrispondenza: jpcoelho@ipb.ptAbstract Gli equidi svolgono un ruolo fondamentale in termini di sostegno dei mezzi di sostentamento in molte parti del mondo. È estremamente importante poter accedere al benessere degli animali, soprattutto durante lo svolgimento di attività che comportano elevati livelli di sforzo fisico, come quelli delle attività agroforestali. Il Donkey Sanctuary, un ente internazionale di beneficenza con sede nel Regno Unito, ha ideato un progetto che mira a sviluppare una serie di strumenti per valutare le condizioni di lavoro di asini e muli in tutto il mondo. Questo progetto prevede la misurazione di diversi parametri, inclusa la forza esercitata dall’animale per tirare un carico durante il lavoro. Questo articolo presenta le fasi di progettazione, sviluppo e realizzazione di un dispositivo in grado di effettuare queste misurazioni con il minimo intervento umano e con impatto trascurabile sulle condizioni operative delle attività. I dati ricavati da condizioni reali sul campo corroborano il metodo di misurazione ideato.Parole chiave: equidi da lavoro, benessere animale, sistemi integrati, strumentazione elettronica, acquisizione di dati, misura della forzaPaper Summary Translations

132Animal Technology and Welfare August 2020INDICE DE LA REVISTAGeles emulsionados: un vehículo refinado para una administración oral rápida y precisa de preparados basados en lípidos para ratasVIDIT SATOKAR, MARK VICKERS, PANIA BRIDGE-COMER, WAYNE CUTFIELD, BENJAMIN ALBERTCorrespondencia: b.albert@auckland.ac.nzResumen La alimentación por sonda orogástrica se utiliza para administrar de forma precisa sustancias nutritivas o fármacos a animales. Sin embargo, esta técnica provoca estrés y tiene un alto riesgo de percances. La incorporación en geles comestibles es complicada para los preparados basados en lípidos. Presentamos una nueva metodología para producir geles emulsionados enriquecidos con aceites e informamos de su eficacia en estudios piloto y otros estudios experimentales de mayor envergadura. Se produjeron geles emulsionados enriquecidos con aceites utilizando almidón no polar. Se crearon distintos tipos de geles incorporando dosis de aceite de 0,05 ml o 1 ml, aceite oxidado o sin oxidar y con o sin sabor a frambuesa. Se evaluó la palatabilidad y la seguridad con i) 8 tipos de geles en ratas SD hembra con una dieta a base de pienso para roedores (40 tratamientos) y ii) 3 tipos de geles en ratas con una dieta alta en grasas (45 tratamientos). Posteriormente, se evaluó de nuevo la palatabilidad y la seguridad en un grupo grande de ratas embarazadas (n = 155; 4242 tratamientos). En ambos estudios, todos los geles fueron consumidos en su totalidad, tanto si las ratas seguían una dieta a base de pienso para roedores o alta en grasas. Se siguió un periodo de adaptación de 5 días. Los geles con sabor a frambuesa fueron consumidos más rápidamente que los geles sin sabor. Las ratas mostraron un comportamiento positivo a la hora de recibir los geles y no padecieron ningún efecto adverso. En estudios experimentales posteriores, se administraron 4242 dosis a ratas embarazadas y todas fueron consumidas. Los geles emulsionados enriquecidos con aceites representan un método seguro, fiable y altamente aceptable para la administración de lípidos a ratas, y que pensamos que es superior a la alimentación por sonda orogástrica. Palabras clave: refinamiento, estrés, embarazo, bienestar animal, geles emulsionados, administración de lípidos Paper Summary Translations

133August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareLos efectos de los distintos tipos de bioseguridad, alimentación y frecuencia de crianza en la fecundidad y fertilidad del pez cebra (Danio rerio)JOE WARMSLEY, PAUL BARWOOD, VISILA MOICHE, CAROLE WILSONCorrespondencia: j.warmsley@ucl.ac.uk Resumen Una investigación sobre las diferencias en entornos, tanto intrínsecas como extrínsecas, que influyen en posibilitar la cría del pez cebra (Danio rerio). Se crearon cuatro grupos distintos en dos entornos distintos. Las diferencias de ambiente incluían sistemas acuáticos creados por distintos fabricantes, uno utilizando un sistema de filtrado de tambor y otro con un sistema más convencional de filtrado de calcetín. La sala que utilizaba el filtrado por tambor también se sometió a un nivel más alto de bioseguridad; que parecía no contener Mycobacterium marinum, Mycobacterium haemophilium y sin indicios de Pseudoloma neurophilia. La sala convencional era más antigua y contaba con menos medidas de seguridad. En ambas salas se administró la misma dieta de materia seca, la sala con mayor bioseguridad recibió rotíferos como alimento vivo, mientras que la sala convencional recibió artemia. Se utilizaron dos líneas de peces en esta prueba, AB y TL, y una vez los peces eran sexualmente maduros, 56 días después de la fertilización (dpf), se instalaron en cajas de cría en varios intervalos de tiempo en ambas salas. Se contó el número y la proporción de embriones viables y no viables para determinar el nivel de fecundidad y fertilidad y también se evaluaron las diferencias entre las dos salas y las frecuencias de cría. Se espera que estos resultados ayuden a reducir el número de peces necesarios y su frecuencia de uso para críar, así como a mejorar el bienestar reduciendo el tiempo de manipulación, a la vez que se aumenta el número de embriones recogidos y se refina de manera general del proceso de cría para que sea más eficiente. Asimismo, este ensayo también destaca las posibles diferencias en los resultados entre instalaciones que utilizan distintas prácticas zootécnicas y de bienestar. ★ ★ ★Instrumentación electrónica de un balancín para el control de arrastre de carga equino: Una contribución al bienestar y al rendimiento de burros de trabajo JOÃO PAULO COELHO, JOÃO BRANDÃO RODRIGUES, LUÍS QUEIJO, HIGOR VENDRAMINI ROSSE, FRANCISCO ALBUQUERQUE, ANDREW JUDGE, FIONA COOKE Y CHRIS GARRETTCorrespondencia: jpcoelho@ipb.ptResumen Los equinos tienen un papel fundamental a la hora de ayudar en las tareas diarias en muchas partes del mundo. Poder evaluar el bienestar del animal, especialmente durante trabajos que requieren un gran esfuerzo físico como los de las actividades agroforestales, es de gran importancia. The Donkey Sanctuary, una institución benéfica internacional con sede en Reino Unido, ha creado un proyecto que trata de desarrollar una serie de herramientas para evaluar las condiciones laborales de burros y mulas de todo el mundo. Esto requiere el cálculo de distintos parámetros, entre los que se incluye la fuerza ejercida por un animal para arrastrar una carga durante su trabajo. Este artículo presenta las fases de diseño, desarrollo e implementación de un dispositivo capaz de realizar estas mediciones con una mínima intervención humana y con un impacto insignificante en las condiciones de las tareas. Los datos obtenidos de condiciones de campo reales validan el método de evaluación diseñado. Palabras clave: equinos de trabajo, bienestar animal, sistemas integrados, instrumentos electrónicos, adquisición de datos, medición de fuerzaPaper Summary Translations

134Animal Technology and Welfare August 2020

135August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareRefinements in head plate mouse nesting: using composite nests to enhance welfareZOE WINDSORUniversity College London, Biological Services, Institute of Neurology, Denny Brown Laboratory, Queen Square, London WC1N 3BG UKCorrespondence: z.windsor@ucl.ac.ukzWinner of the 2021 Andrew Blake Tribute Award ANDREW BLAKE TRIBUTE AWARD 202147ANDREW BLAKE TRIBUTE AWARDENTRIESTime for change? Practicalities ofimplementing non-aversive methodsfor handling miceJOHN WATERSMammalian Behaviour and Evolution Group, InstituteUniversity of Liverpool, Leahurst Campus, NestonCorrespondence: kimmy@liv.ac.ukAndrew Blake Tribute Award 2017 winning entryAbstractRecent studies have shown that the method choice forhandling laborator y mice is important to animalwelfare. In 2015, 60% of all animals used in HomeOffice procedures were laboratory mice. Given thelarge number of mice within global facilities, mousewelfare should be a high priority. Improved handling notonly leads to more consistent scientific data, it canalso lead to improved animal welfare. Historically micehave been picked up by their tail, a method that hasbeen passed down to generations of technologists andis widely accepted as a method of handling. Hurst andWest (2010) showed that picking up mice by their tailinduces aversion and high anxiety. By contrast, usingalternatives, such as a tunnel or cupped on the openhand, leads to voluntary approach to the handler, lowanxiety and animals that more readily accept somephysical restraint. Hurst and West’s findings wereconsistent across strains and sex of laboratory mice,handlers with differing levels of experience anddifferent light periods (light/dark). From a welfareperspective, the response from the mice on a dailybasis is a positive step in the right direction.The evidence presented by Hurst and West indicatesthat a change to the standard method for picking upmice would provide improved welfare for millions ofmice worldwide. However, this will only happen if thenon-aversive methods are taken up by facilities. To besuccessful, the practicalities of these methods need tobe demonstrated and appropriate instruction providedfor technical staff and researchers to aidimplementation of the methods.First, I gained information from talking to technologists,conducted a survey of their responses concerning keyissues and established practical details ofimplementation in our own facility. I then played a majorpart in designing and constructing a video-basedtutorial with accompanying commentary,todemonstrate the handling methods and their use incommon la boratory situations from a practical,technologist’s viewpoint. This includes ways to avoidcommon problems in implementation to reduce stressin both animals and the technologists carrying out thehandling. Although my main focus was practicalimplementation from an animal technologist’sperspective, as this will have the greatest impact onimplementation, the tutorial provides impor tant trainingmaterial for technologists and researchers alike.With valuable suppor t from NC3Rs to provide a web-based resource, truly available to all (nationally andinternationally), the tutorial is now hosted on theNC3Rs’ website (http://www.nc3rs.org.uk/mouse-handling-tutorial). Statistics gained from NC3Rs for thefirst six weeks since its launch show that the tutorial isalready being used in a wide range of countries inaddition to the UK. Feedback from fellow technologistsin the United States has further enhanced the hope thatanimals outside of the UK will also benefit from the non-aversive methods of handling that have been developed.April 2017 Animal Technology and WelfareSponsored by the Association of the BritishPharmaceutical IndustryAPRIL latest:Animal Technology and Welfare 24/9/20 07:28 Page 47IntroductionThe provision of nesting material is widely regarded to be beneficial for the welfare of laboratory mice and can be used as both a measure of welfare1 and a method of providing enrichment.2 When given the opportunity, mice will choose to build nests using multiple different materials.3 Studies indicate that providing mice with choices which mimic the natural environment allows laboratory mice to build the best quality nests4 and mice have also been shown to go to considerable effort to gather and combine multiple nesting materials to construct a nest.5 These types of nests are referred to as composite nests and are typically higher quality nests than those built of only one material. Given that nest building can be used as a measure of welfare, high quality nests may indicate that welfare is enhanced,6 as mice are given the opportunity to perform natural behaviours and create nests as they would in the wild.7 Many animal facilities commonly provide only one nesting material due to ease, standardisation and costs. In many cases mice are observed to shred other enrichment items within the cage such as cardboard tunnels and houses and incorporate them into the nest, indicating a preference for additional or alternative materials. Thus, allowing lab mice the opportunity to construct a nest with more than one material may be beneficial to their welfare and in turn, have positive implications for the reproducibility of the scientific output.8Special consideration must be given to head plate mice which invariably face restrictions on enrichment items such as nesting material, as long and fibrous products are liable to become tangled around the head implant device and cause entrapment or injury. Naturalistic nesting materials such as shredded paper are commonly used in laboratories and considered superior for nest building as they frequently result in high quality nest construction.4 In the case of head plate mice however, nesting materials containing long fibres pose a considerable risk to safety from entanglement and therefore cannot be used. Only materials made up of short fibres are recommended for head plate mice. It has been demonstrated that single short fibre materials can be used to construct high quality nests by head plate mice, however it is not known whether a combination of short fibre materials can be used to construct a higher quality nests than one single short fibre material.9 Allowing the opportunity to construct a high-quality nest is important for all mice. However there are several factors which amplify the need for enhanced welfare in head plate mice. Many head plate mice are singly housed due to the requirements of the study (eg. whisking studies in which grooming from cage mates may partially or completely remove the whiskers) and therefore will experience a higher level of stress than August 2021 Animal Technology and Welfare

136Animal Technology and Welfare August 2020their group housed counter parts.6 Whilst group housing is optimal, providing biologically relevant environment can help ameliorate the negative effects of single housing and increase comfort.10 A high quality nest is an easily applicable and effective way to enhance the quality of the home cage11 as nests provide essential opportunities to shelter from the environment and regulate body temperature.12 The environment also impacts aspects of post-operative recovery, as mice in enriched cages experience less post-operative pain and self-medicate less than mice in barren cages.13 Composite nests therefore have the potential to enhance welfare through both mimicking natural nest building behaviour and ameliorating some of the effects of single housing and surgery. I hypothesize that the simple addition of a second nesting material will allow for the construction of a higher quality composite nest which will have the potential to refine basic husbandry and enhance head plate mouse welfare in the laboratory. Methods This study was carried out by the Welfare Group at UCL and falls below the threshold of what is considered a regulated procedure under the Animals (Scientific Procedures) Act 1986.14 All work is performed under the authorisation of one of UCL’s Animal Welfare Ethical Review Boards, and at all times studies were undertaken in accordance with the ‘Code of practice for the housing and care of animals bred, supplied or used for scientific purposes’ as published by the Home Office in 2014.15The study subjects:Fifteen singly housed C57BL/6J mice (3M; 12F). The three test groups were: Pure Comfort White (PCW, n=5), Nestlets (Control) (n=5) and Composite (a combination of both PCW and Nestlets) (n=5). The age range of the mice was 10-56 weeks at the time of the study (with a mean age of 43 weeks). All mice had a head plate installed 4-15 days before the trial began (mean of 10 days) except from one mouse which had the headplate installed 307 days before the trial (mean of 27 days inclusive). The mice were singly housed immediately following surgery and no surgical complications were reported. The animals in this study were already assigned to experiments that required head plate installation surgery. Conducting the study in parallel with ongoing research projects has the ethical advantage of investigating potential refinements and welfare improvements for head plate mice without exposing additional mice to head plate surgery. The quantity, age, strain, sex and period of time available for the animals was therefore dependent upon the availability of head plate mice already singly housed for experimental purposes within the animal facility.Housing and husbandryAll mice were housed in Tecniplast™ GM500 IVC cages with ad libitum access to food (2018 Harlan Teklad) and water (Tecniplast™ bottle) and lined with Aspen bedding (Eco-Pure™). As standard, the mice had been provided with Nestlets (Datesand Group), a GLP mini Fun Tunnel (LBS Biotechnology), a red plastic handling tunnel (Datesand Group) and a small Aspen Brick (Datesand Group) from birth. All cages were cleaned prior to the start of the study and were not changed for the duration of the experiment (10 days). The animal room housing the mice for this study was an average of 20.0°C and 35.8% RH. The room maintained a 12:12hr light cycle which began at 07:00.MaterialsThis study assessed the apparent nestability (ability to construct a nest) and safety of the following types of nesting material: Pure Comfort White (LBS Biotechnology), Nestlets (Datesand Group) and a combination/composite of both. Pure Comfort White was selected as it has previously been shown to be safe for head plate mice and produces good quality nests.9 Nestlets were selected as a control group as they are standard head plate mouse enrichment in the author’s own facility and there is currently no published information on its safety for head plate animals. Experimental protocolThis study replicated the protocol described in Windsor & Bate 2019 where this was feasible and amendments were made where deemed necessary.9 Animals were randomly assigned to test groups using the RAND function in Excel.Approximately 1hr before the dark cycle began, any shreddable environmental enrichment was removed from the cage. The trial nesting material was then added to the rear right quadrant of each cage (5 cages per trial group). The quantity of each material provided was based on perceived amount required to construct a high-quality nest: 10g of Pure Comfort White (in the PCW only group), 1 Nestlet (Nestlet only group) and 5g of Pure Comfort White and 1 Nestlet (in the Composite group). It was deemed that providing 10g and a whole nestlet would be excessive, therefore the amount of PCW was halved given that it is more practical to provide a smaller amount of PCW than half a Nestlet.The visual assessment of nest quality made use of a scoring system to improve the reproducibility of scores between animals, however as this is still a subjective process, only one observer recorded observations in this trial. Due to the visual nature of the study, blinding was not possible.Refinements in head plate mouse nesting: using composite nests to enhance welfare

137August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareStage 1: Assessing the safety of each test group The 15 mice were tested in 2 cohorts (n=5 and n=10) for 10 days each approximately 4 weeks apart due to availability of mice. The random group allocation within cohort 1 was as follows: Nestlet n=1, PCW n=1 and Composite n=3. Within cohort 2, the groups were randomly allocated as follows: Nestlet n=4, PCW n=4 and Composite n=2.The safety observation was performed once on day 1 of the trial. To allow the mice to construct their nests, the observation period began at 8:30am the day after the nesting material was provided to each cage and lasted for a period of 10 days. During the observational period, the observer recorded observations about the apparent safety of the nesting material as the mouse interacted with it (see Table 1). Observations were made during the early stages of the light phase (which began at 7:00am) as the mice would normally be asleep during this time and therefore be highly motivated to return to the nest after being disturbed. A prompt return to the nest site would increase the opportunities to record observations about the mouse’s interactions with each given nesting material.For the 5-minute observation period animals were scored on the frequency of tangling/snagging observed and for each tangling/snagging, the severity of the degree of the tangle (see Table 1). Severity ScoreCriteriaMildSome nesting material is caught in head plate but mouse can free itself with relative ease.ModerateMouse can free itself but some nesting material still attached to head plate.SevereMouse is unable to free itself, nest is dragged with the mouse when moving around the cage.Table 1. Severity score for each tangle/snagging observed in the observational period (Windsor 2019).Stage 2: Assessing nest qualityOn day 1, the undisturbed nests produced in each cage were assessed on a rating scale 1-5 (see Table 2). The nest quality assessment was repeated daily for 10 days.Data HandlingThe scores for each animal were averaged over the 10-day period and a one-way ANOVA test was used to test for differences between each test group. A two-way ANOVA was run to test for an effect of time on nest scores.ResultsIn Stage 1, no tangling was observed in any of the groups during the daily observation period. In Stage 2, the Nestlet group produced the lowest scores averaged over the 10-day period and the Composite group produced the highest scores (Composite= 4.62±0.739, 9 increase the opportunities to record observations about the mouse’s interactions with each given nesting material.For the 5-minute observation period animals were scored on the frequency of tangling/snagging observed and, for each tangling/snagging, the severity of the degree of the tangle (see Table 1). Severity ScoreCriteriaMildSome nesting material is caught in head plate but mouse can free itself with relative easeModerateMouse can free itself but some nesting material still attached to head plateSevereMouse is unable to free itself, nest is dragged with the mouse when 9 increase the opportunities to record observations about the mouse’s interactions with each given nesting material.For the 5-minute observation period animals were scored on the frequency of tangling/snagging observed and, for each tangling/snagging, the severity of the degree of the tangle (see Table 1). Severity ScoreCriteriaMildSome nesting material is caught in head plate but mouse can free itself with relative easeModerateMouse can free itself but some nesting material still attached to head plateSevereMouse is unable to free itself, nest is dragged with the mouse when 9 increase the opportunities to record observations about the mouse’s interactions with each given nesting material.For the 5-minute observation period animals were scored on the frequency of tangling/snagging observed and, for each tangling/snagging, the severity of the degree of the tangle (see Table 1). Severity ScoreCriteriaMildSome nesting material is caught in head plate but mouse can free itself with relative easeModerateMouse can free itself but some nesting material still attached to head plateSevereMouse is unable to free itself, nest is dragged with the mouse when Table 2.Assessment scale for nesting building phase (Windsor 2019).9Score Criteria1 Nesting material not noticeably touched (more than 90% left unmoved).2 Nesting material partially manipulated or shredded (50-90% remaining intact).3 Nesting material mostly manipulated or shredded but no identifi able nest site (nesting material is torn but the material is not gathered into a nest within one quarter of the area of the cage fl oor and is spread around the cage).4 An identifi able but fl at nest (nesting material is torn and gathered into a nest within one quarter of the area of the cage fl oor but is fl at and walls of the crater are no higher than the height of the mouse’s body).5 A well assembled crater (nesting material is torn and gathered into one quarter of the area of the cage fl oor. The nest is a hollow with walls higher than the mouse).6 A partially enclosed crater (nesting material is torn and gathered into one quarter of the area of the cage fl oor. The nest is a hollow with walls almost enclosing the mouse but with an opening still visible at the top of the nest).7 A full dome (nesting material is torn and gathered into one quarter of the area of the cage fl oor. The nest is a hollow with walls completely enclosing the mouse).Refi nements in head plate mouse nesting: using composite nests to enhance welfare

138Animal Technology and Welfare August 2020PCW=4.46±0.288 and Nestlet=3.8±0.316). A one-way ANOVA test indicated there is no statistically signifi cant difference between the 3 test groups (p= 0.05009), using the average score per animal over the trial. However, as can be seen in Figure 1, there is a trend towards a lower nest score in the Nestlet group, compared to the PCW and the Composite group. In the raw data, it was observed that over time in the Composite nest group each individual maintained a higher stable pattern of scoring than individuals in the Nestlet and PCW only groups.A two-way ANOVA test was used to investigate if there was an effect of time on nest scores and indicated that there was no signifi cant interaction (p=0.95192) between the two, therefore nest quality is not affected over time (Figure 2). All nests were constructed within the fi rst 24hrs and tended to maintain the initial quality throughout the experiment, never deviating more than one score above or below the day 1 score.DiscussionIn Stage 1, the cages were observed for 5 minutes each day to monitor for incidences of mice becoming tangled in the nesting material. No tangling or snagging on the head plate was observed during the observation period or throughout the 10-day trial period suggesting that all three test groups are safe for use with head plate mice. This is consistent with previous trials on PCW, which found it did not snag, or cause tangling or entrapment in head plate mice,9 currently there is only anecdotal information on the safety of Nestlets, however this trial confi rms that tangling does not present a problem with this nesting material. Lastly, a combination of both Nestlet and PCW provided together also did not result in incidences of tangling, therefore pairing the two did not alter their composition in a way that increased the risk of harm to the mouse. In Stage 2, the Nestlet group produced the lowest scores 13Figure 1: Using the average per animal over the 10 day period, the one-way ANOVA indicated no significant difference between the three test groups.A two-way ANOVA test was used to investigate if there was an effect of time on nest scores and indicated that there was no significant interaction (p=0.95192) between the two, therefore nest quality is not affected over time (Figure 2). All nests were constructed within the first 24hrs and tended to maintain the initial quality throughout the experiment, never deviating more than one score above or below the day 1 score.Figure 1. Using the average per animal over the 10 day period, the one-way ANOVA indicated no signifi cant difference between the three test groups.Nesting score range14Figure 2: A two-way ANOVA using the average score per group indicated that nest scores were not significantly affected over time (p=0.95192).DiscussionIn Stage 1, the cages were observed for 5 minutes each day to monitor for incidences of mice becoming tangled in the nesting material. No tangling or snagging on the head plate was observedduring the observation period or throughout the 10-day trial period suggesting that all three test groups are safe for use with head plate Figure 2.A two-way ANOVA using the average score per group indicated that nest scores were not signifi cantly affected over time (p=0.95192).Nesting over timeand the Composite group produced the highest scores. Results indicated that there was no statistically signifi cant difference between the 3 test groups. However, as seen in Figure 1, there is a trend towards a lower nest score in the Nestlet group (Figure 3), compared to the PCW and the Composite groups. The PCW group and the Composite group both scored highly throughout the trial (Figures 4, 5a and 5b). Windsor & Bate (2019) found that nests built of PCW frequently and quickly achieved a good quality score, indicating that PCW has qualities that are attractive to mice for nest building and allows them to construct a nest with relative ease.9Whilst there was no signifi cant difference between the PCW and Composite group, the Composite group achieved and maintained the highest scores, including scores of 6 which are characterised by the walls beginning to enclose the crater with only a small opening on top. This type of structure was not observed in the PCW group, potentially indicating that multiple materials are required to increase nest quality. Further personal observations indicated that nests in the Composite group were structurally sounder than nests in the PCW group, despite being of a similar quality score. It was observed that mice leaving the PCW nests often trailed nesting material with them resulting in a partial destruction to the nest, however the Composite nests held together more fi rmly and showed a tendency to stay intact as the mouse moved around the cage. A gentle nudge with the hand confi rmed that the Composite nests maintained their structure more easily than the PCW nests.Refi nements in head plate mouse nesting: using composite nests to enhance welfare

139August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAs it has been noted that compacted nesting materials such as Nestlets are generally used to create a border or structure,4 it is likely that the Nestlet and PCW complement each other in a way that allows for a higher quality nest: the Nestlet forming a sturdy frame (even partially shredded) and the PCW filling out the bulk of the nest for volume and comfort. The larger pieces of Nestlet interwoven with the small PCW pieces appears to allow for a structure which begins to enclose the mouse on top without caving in and has the added benefit of maintaining its structural integrity as the mouse enters and exits the nest. Composite nests in the literature have been shown to be of higher quality than nests of one sole material,3 and sturdy nests are biologically relevant as in the wild as mice require a safe shelter to hide from predators and to shield themselves form the environment.7Figure 3 - 4. Examples of nests produced in the Nestlet (top) and PCW group (bottom).16 to enclose the crater with only a small opening on top. This type of structure was not observed in the PCW group, potentially indicating that multiple materials are required to increase nest quality. Further personal observations indicated that nests in the Composite group were structurally sounder than nests in the PCW group, despite being of a similar quality score. It was observed that mice leaving the PCW nests often trailed nesting material with them resulting in a partial destruction to the nest, however the Composite nests held together more firmly and showed a tendency to stay intact as the mouse moved around the cage. A gentle nudge with the hand confirmed that the Composite nests maintained their structure more easily than the PCW nests. Figures 3 - 4: Examples of nests produced in the Nestlet (left) and PCW group (right) As it has been noted that compacted nesting materials such as Nestlets are generally used to create a border or structure,4 it is likely 16 to enclose the crater with only a small opening on top. This type of structure was not observed in the PCW group, potentially indicating that multiple materials are required to increase nest quality. Further personal observations indicated that nests in the Composite group were structurally sounder than nests in the PCW group, despite being of a similar quality score. It was observed that mice leaving the PCW nests often trailed nesting material with them resulting in a partial destruction to the nest, however the Composite nests held together more firmly and showed a tendency to stay intact as the mouse moved around the cage. A gentle nudge with the hand confirmed that the Composite nests maintained their structure more easily than the PCW nests. Figures 3 - 4: Examples of nests produced in the Nestlet (left) and PCW group (right) As it has been noted that compacted nesting materials such as Nestlets are generally used to create a border or structure,4 it is likely Figure 5a - 5b. Examples of nest produced in the Composite group.17 that the Nestlet and PCW complement each other in a way that allows for a higher quality nest: the Nestlet forming a sturdy frame (even partially shredded) and the PCW filling out the bulk of the nest for volume and comfort. The larger pieces of Nestlet interwoven with the small PCW pieces appears to allow for a structure which begins to enclose the mouse on top without caving in and has the added benefit of maintaining its structural integrity as the mouse enters and exits the nest. Composite nests in the literature have been shown to be of higher quality than nests of one sole material,3 and sturdy nests are biologically relevant as in the wild as mice require a safe shelter to hide from predators and to shield themselves form the environment.7 Figures 5a- 5b. Examples of nest produced in the Composite group 17 that the Nestlet and PCW complement each other in a way that allows for a higher quality nest: the Nestlet forming a sturdy frame (even partially shredded) and the PCW filling out the bulk of the nest for volume and comfort. The larger pieces of Nestlet interwoven with the small PCW pieces appears to allow for a structure which begins to enclose the mouse on top without caving in and has the added benefit of maintaining its structural integrity as the mouse enters and exits the nest. Composite nests in the literature have been shown to be of higher quality than nests of one sole material,3 and sturdy nests are biologically relevant as in the wild as mice require a safe shelter to hide from predators and to shield themselves form the environment.7 Figures 5a- 5b. Examples of nest produced in the Composite group There was no significant difference in nest quality over time (p=0.95192), indicating that in all groups, nests were constructed quickly and maintained a similar quality throughout the 10 days of the trial. As the lowest scoring group, Nestlets produced the lowest quality nests which remained unchanged throughout the trial. This is consistent with observations of compacted nesting material commonly being used to create a border or structure of the nest, however, these are frequently never fully shredded to fill out the bulk of it.4,9 This could be the reason why the scores remained low, as the mice may not have perceived the Nestlet to be suitably nestable. An alternative explanation is that Nestlets require a longer period of time than 10 days to be shredded and built into a high-quality nest. Windsor & Bate (2019) found that Rodent Rolls, a different variety of compacted nesting material also Refinements in head plate mouse nesting: using composite nests to enhance welfare

140Animal Technology and Welfare August 2020produced low nest quality scores over a period of 14 days, indicating that mice may not perceive them to be nestable or they simply take a lot of work over a longer period to shred. PCW has the benefit of being short fibre or pre-shredded and does not require much manipulation by the mouse before it can be used to build a nest. Aside from contributing to the higher scores observed in the PCW and Composite groups throughout the trial period, this could have also advantageous implications for post-operative animals as anaesthesia impairs nest building, indicating that the reduction in wellbeing and condition associated with surgical procedures inhibits mice’s ability and motivation to construct high quality nests.16 Therefore we can expect that during the post-operative period mice will not construct the same quality nest as they would in their pre-operative state. Facilitating quick nest building opportunities by providing short fibre materials such as PCW in combination with a Nestlet fulfils the mouse’s biological need to construct composite nests but also has positive welfare implications.Conclusions No tangling was observed in any of the groups, therefore Nestlets, Pure Comfort White and a combination of both (Composite) are all safe for use with head plate mice.The Pure Comfort White group produced the second highest and most stable scores throughout the trial. This is consistent with previous findings.The Nestlet group produced more variable, lower scores which did not improve significantly over time as the trial went on.Mice in the Composite group produced the highest scoring nests of the three groups. The quality of these nests remained consistent on an individual basis throughout the trial. The most structurally sound nests were recorded in this group. Head plate mice are able to safely construct higher quality nests when given both a Nestlet and Pure Comfort White in conjunction with each other. The provision of more than one nesting material fulfils the mouse’s biological motivation to create composite nests and serves as an enhancement to welfare.AcknowledgementsSpecial thanks to Dr. Ellen Forty for her advice and support throughout the study and to Irene Lopez for her contribution to the statistical analysis. I’d also like to thank Dr. Mikail Weston for kindly providing the mice for the study and finally, all the staff at Institute of Neurology BSU who leant their time and efforts to help with this trial.References1 Gaskill, BN., Karas, AZ., Garner, JP. & Pritchett-Corning, KR. (2013). Nest building as an indicator of health and welfare. JOVE Article e51012. http://www.jove.com/video/51012/nest-building-as-an-indicator-of-health-and-welfare-in-laboratory-mice.2 Olsson, A & Dahlborn, K. (2002). Improving housing conditions for laboratory mice: a review of ‘environmental enrichment’. Laboratory Animals 36: 243270.3 Van de Weerd H., Van Loo P.L.P., Van Zutphen L., Koolhaas J. and Baumans V. (1997). Preferences for nesting material as environmental enrichment for laboratory mice. Laboratory Animals 31: 133-14.4 Hess, S., Rohr, S., Dufour, B., Gaskill, B., Pajor, E. & Garner, J. (2008). Home improvement: C57BL/6J mice given more naturalistic nesting materials build better nests. J. Am. Assoc. Lab. Anim. Sci. 47 (6): 25-31.5 Sherwin, C.M. (1997). Observations on the prevalence of nest-building in non-breeding TO strain mice and their use of two nesting materials. Laboratory Animals 31 (2): 125-132.6 Gaskill, BN., Gordon, CJ., Davis, JK., Lucas, JR., Pajor, EA., and Garner, JP. 2013. Impact of nesting material on mouse body temperature and physiology.Physiol. Behav. (110): 87-95.7 Latham N., Mason G. (2004). From house mouse to mouse house: the behavioural biology of free-living Mus musculus and its implications in the laboratory. Appl. Anim. Behav. Sci. 86: 261-289.8 Garner, J.P. (2005). Stereotypies and other abnormal repetitive behaviors: potential impact on validity, reliability, and replicability of scientific outcomes. ILAR J 46 (2): 106-117.9 Windsor, Z. & Bate, S. (2019). Assessing the safety and suitability of nesting material for singly housed mice with surgically fitted head plates. Heliyon 5 (7).10 Hawkins, P., Morton, D.B., Bevan, R., Heath, K., Kirkwood J., Pearce P., Scott, L., Whelan, G., Webb, A. (2004). Husbandry refinements for rats, mice, dogs and non-human primates used in telemetry procedures. Laboratory Animals (38): 1-10.11 Würbel, H., Garner, J.P. (2007). Refinement of Rodent Research through Environmental Enrichment and Systematic Randomization. Accessed 10 23, 2020. https://www.nc3rs.org.uk/sites/default/files/ documents/Refinementenvironmentalenrichmentand systematicrandomization.pdf12 Gaskill, B.N., Lucas, J.R., Pajor, E.A., Garner, J.P. (2011). Working with what you’ve got: changes in thermal preference and behavior in mice with or without nesting material. J. Therm. Biol (36): 193-199.13 Pham, T.M., Hagman, B., Codita, A., Van Loo, P.L., Strömmer, L., Baumans, V. (2010). Housing environment influences the need for pain relief during post-operative recovery in mice. Behaviour & Physiology 99 (5): 663-668.Refinements in head plate mouse nesting: using composite nests to enhance welfare

141August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfare14 Consolidated version of ASPA 1986 – GOV.UK https://www.gov.uk/government/publications/consolidated-version-of-aspa-198615 Guidance on the operation of the Animals (Scientific Procedures) Act 1986 https://www.gov.uk/guidance/guidance-on-the-operation-of-the-animals16 Jirkof, P., Fleischmann, T., Cesarovic, N., Rettich, A., Vogel, J., Arras, M. (2013). Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring. Laboratory Animals 47 (3): 153–161.Refinements in head plate mouse nesting: using composite nests to enhance welfare

142Animal Technology and Welfare August 2020The 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 ABPIANDREW BLAKETRIBUTE AWARDDON’T KEEP YOUR GOOD IDEA TO YOURSELF!WE WANT TO HEAR ABOUT IT FOR THE 2022 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 INCLUDES - CONGRESS 2022 FREE ATTENDANCE next March WHICH WILL INCLUDE PRESENTING YOUR WORK - AN ENGRAVED GLASS PLAQUE - AND £250 CASH AWARDCLOSING DATE FRIDAY 29th OCTOBER 2021 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 ENTERSUBMISSIONS SHOULD CONTAIN AS MUCH AS POSSIBLE OF THE FOLLOWING HEADINGS AND YOU CAN INCLUDE PHOTOGRAPHS/IMAGES (THESE SHOULD BE SUPPLIED AS ATTACHMENTS):CRITERIA – The topic of work that you describe in your application may be undertaken as part of a project and presented EITHER as a POSTER / an ESSAY / a PROJECT / a SCIENTIFIC PAPER.The submission which should contain the content below must be submitted online via this link https://www.iat.org.uk/abta where you will see the Submission form for completion:- 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. - Acknowledgements & References. - Brief CV to include your overall contribution to the work. - Please list your supervisors or PPL holder if applicable for the work.To allow others to be able to replicate the work, please consult the ARRIVE guidelines: https://www.nc3rs.org.uk/arrive-guidelines

143August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe 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 ABPIANDREW BLAKETRIBUTE AWARDDON’T KEEP YOUR GOOD IDEA TO YOURSELF!WE WANT TO HEAR ABOUT IT FOR THE 2022 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 INCLUDES - CONGRESS 2022 FREE ATTENDANCE next March WHICH WILL INCLUDE PRESENTING YOUR WORK - AN ENGRAVED GLASS PLAQUE - AND £250 CASH AWARDCLOSING DATE FRIDAY 29th OCTOBER 2021 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 ENTERSUBMISSIONS SHOULD CONTAIN AS MUCH AS POSSIBLE OF THE FOLLOWING HEADINGS AND YOU CAN INCLUDE PHOTOGRAPHS/IMAGES (THESE SHOULD BE SUPPLIED AS ATTACHMENTS):CRITERIA – The topic of work that you describe in your application may be undertaken as part of a project and presented EITHER as a POSTER / an ESSAY / a PROJECT / a SCIENTIFIC PAPER.The submission which should contain the content below must be submitted online via this link https://www.iat.org.uk/abta where you will see the Submission form for completion:- 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. - Acknowledgements & References. - Brief CV to include your overall contribution to the work. - Please list your supervisors or PPL holder if applicable for the work.To allow others to be able to replicate the work, please consult the ARRIVE guidelines: https://www.nc3rs.org.uk/arrive-guidelines

144Animal Technology and Welfare August 202049Haven’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 article written for the National Centre for the 3RsApril 2020 Animal Technology and WelfareTECH-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 ischanged 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 electromagnetic 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 husbandrypractices to set our criteria, balancing hygiene 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 basechange. 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 Veteri narySurgeon (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 49Animal Technologists – the key workers for medical researchCALL FOR WORKSHOPSl take an active part in the leading annual meeting for Animal Technologistsldo 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 - 3 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 October 2021Contact: congress@iat.org.ukCongress2022CONGRESS Invitation to Participate29th March – 1st AprilSITUATIONS VACANTVisit the Careers pages on the IAT Website for the latest vacancieshttps://www.iat.org.uk/vacanciesRecruitment advertising details from mail@prcassoc.co.uk

145August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIntroduction Water quality is an important and yet relatively neglected area of laboratory animal science. There are no set water quality standards for rodent facilities other than to provide clean potable water. The Code of Practice for Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes1 limits the guidance to ‘Uncontaminated drinking water shall always be available to all animals’. It goes on to say ‘Water is a vital resource to all animals. However, water is also a potential vehicle for microorganisms, and due consideration should be given to arranging the supply so that the contamination risk is minimised’.1 How contamination of water in laboratory animal facilities is defined therefore, is questionable. Water is not naturally sterile; it contains var ying amounts of microorganisms depending on the geographical location and season. The only true guarantee of a sterile supply is achieved by autoclaving although many facilities have now opted for less burdensome filter systems, chlorination and acidification that reduce the microbial load of water to a tolerable level. Determining what constitutes a tolerable level is not defined, given that no standard currently exists for laboratory animal facilities. Health monitoring laboratories providing a water monitoring service should provide support in interpreting results of water analysis to enable facilities to better understand contamination and how these results can impact the health of laboratory animals and their potential research implications.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 article written for the National Centre for the 3RsApril 2020 Animal Technology and WelfareTECH-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 electromagnetic 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 basechangebased 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 husbandrypractices to set our criteria, balancing hygiene 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 Veteri narySurgeon (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 49Interpreting water monitoring results in laboratory rodent facilitiesLORNA CLEVERLEY Animal Health Monitoring Laboratories, Fera Science Ltd, York Biotech Campus, Sand Hutton, York YO41 1LZ UKCorrespondence: lorna.cleverley@fera.co.ukTECH-2-TECHAugust 2021 Animal Technology and Welfare

146Animal Technology and Welfare August 2020Why do rodent research facilities need to monitor water?Microbial contaminants in the water supplied to laboratory animals pose a threat to the health and welfare of the animals and consequently, the reliability of experimental data. Monitoring water quality should therefore be an important part of any robust biosecurity programme. Waterborne transmission of organisms such as Pseudomonas aeruginosa in animal facilities inevitably enter through poorly designed or poorly maintained watering systems. Organisms such as Pseudomonas can have substantial effects on the health of research colonies. Experimentally induced lung infection studies have demonstrated that some rodent strains are susceptible to Pseudomonas aeruginosa. C57BL/6, A/J mice and DBA/2 mice are all susceptible with DBA/2 mice demonstrating high mortality due to Pseudomonas infection.2 Natural Pseudomonas infections of immunocompromised mice including athymic nude mice and severe combined immunodeficient mice (SCIDS) present with clinical signs that include hunchback posture, ruffled coat, apathy, shortness of breath, oblique head posture, circus movement and emaciation.3 Spread of Pseudomonas to the organs causes tissue lesions, necrotic foci, abscess formation and suppuration of the liver, lung and kidney.4Pseudomonas is not the only microbiological contaminant that may enter a facility through the water supply. Cryptosporidia species, faecal coliforms including Klebsiella species and enteric viruses are all organisms that can be transmitted via water sources. Pseudomonas aeruginosa are ubiquitous bacteria that adhere to organic and inorganic materials when nutrient conditions are favourable producing slime to create biofilms. Most bacteria found in water detach from biofilms rather than existing as free-floating cells. Multiple organisms attach to biofilms creating communities of microorganisms that attach to the inside surfaces of pipes. They live in close proximity enabling them to support each other by exchanging nutrients and removing toxic end products. The structure of biofilm communities can protect the bacteria within them from standard disinfection processes and are therefore very difficult to remove once established. Pseudomonas can survive very easily by attaching to the surface of pipes even in waters with low nutrients such as Reverse osmosis (Ro) water.5The highest risk of contamination from biofilms is associated with facilities with poorly designed plumbing systems containing dead legs and outlets that may permit back contamination and biofilm formation. How is water monitored?For meaningful results, water samples should be representative and collected aseptically in sterile containers. Outlets should be thoroughly decontaminated before collection and water should be run for a period before collection to remove any residual disinfectant. The use of containers with the addition of sodium thiosulpate can be used to neutralise the antibacterial effect of chlorine during transit to the laboratory. Water is analysed in the laboratory using culture methods although there are some more rapid methods now available such as ATP Bioluminescence, light scattering and counting fluorescently labelled cells.6 The standard culture approach for assessing microbial levels in water systems involves passing water through a membrane filter to capture any colonies on the membrane. Organisms are then cultured by the addition of liquid culture media or by placing the filter onto an agar plate. For small volumes of water, the water is placed directly in the culture media (Pour Plate method). After incubation, colonies are counted to provide an estimation of the number of organisms in the water sample. For pure waters standard culture media is not recommended for use. Reasoner’s 2A agar is used instead to facilitate the growth of microorganisms that survive in low nutrient waters such as reverse osmosis water. Standard water microbiology reports provide a total viable count, a count for E. coli, Coliforms and a count for Pseudomonas aeruginosa. An example of a table of results is shown in Table 1.RESULTS Total Viable CountE.coli/Coliforms Pseudomonas aeruginosaIncubation 72 hours @ 22°C 72 hours @ 37°C 24 hours @ 37°C 24 hours @ 37°CVolume CFU/ml CFU/ml CFU/100ml CFU/100mlSample A 18 9 Not present Not PresentSample B 303 138 Not present Not presentSample C 37 29 Not present Not presentSample D 73 116 Not present Not presentSample E 724 513 7 Coliforms E.coli not present Not PresentSample F 63 45 Not Present Not PresentSample G 50 12 Not present Not presentTable 1. Example results from a Reverse Osmosis filtered water system in an SOPF rodent facility. Tech-2-Tech

147August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe microbial count from water is reported as a total viable count (TVC) and it is measured in colony forming units (CFUs). The total viable count is the number of living bacteria, yeasts and fungi in a given volume of water. The colony forming unit is the number of colonies on an agar plate. In the example, for sample A the total viable count at 37°C is 18 cfu/ml and at 22°C the TVC is 9 cfu/ml. The total number of living bacteria, yeasts and fungi are given using two growth temperatures. Two temperatures are used for incubation to allow growth of both environmental organisms (22°C) and organisms that may colonise/infect animals and humans (37°C). In addition, results provide the total viable count of Coliforms/E.coli and Pseudomonas aeruginosa per 100mls of water. This is achieved using culture media selective for these organisms after filtration. Interpreting water monitoring results? As there are no standards relating to water quality in rodent facilities it is difficult to determine if the water supply in the example in Table 1 is of an acceptable quality. However, there are standards which can provide some guidance. There are national drinking water/potable water standards relating to water quality. The Drinking Water Inspectorate (DWI) is the relevant body that regulates and monitors the quality of drinking water in England and Wales. Standards are provided in schedule 1 of The Water Supply (Water Quality) Regulations 2016.7 Water from the mains supply to any home or business is monitored by water authorities and must meet a standard which means it is ‘wholesome’ and fit for human consumption. There are no standards given for total viable counts, instead the standard is given as 0 E. coli cfu/100mls and 0 Enterococci cfu/100mls of potable water.7 E. coli and Enterococci are chosen as indicator organisms of human and animal sewage contamination. This standard is not entirely useful in laboratory animal facilities because it may not provide enough evidence of suitable water quality for rodents. This is because many strains of rodents may be immunosuppressed or immune-vague leaving them susceptible to organisms that humans would otherwise tolerate.Water used in the Pharmaceutical industry as a raw material, ingredients or final product must meet standards described in the European Pharmacopeia. Pharmaceutical manufacturers are expected to establish their own quantitative microbial specification suited to their water uses. However, these values should not exceed a total viable count of 100 cfu/mL for Purified Water or 10 cfu/100 mL for Water for Injection because these values represent the highest microbial levels for pharmaceutical water that are still suitable for manufacturing use.8 The purified water standard of <100 cfu/ml could be used as a standard for laboratory animal facilities particularly in facilities supplied by systems providing pure water such as RO systems.An alternative approach and more suitable approach for laboratory animal facilities who do not have pure water systems is to routinely monitor TVCs to provide a baseline specification. If the TVC rapidly increases above the baseline this may then indicate an issue in the water system which needs to be investigated. To set a specification, water monitoring should be carried out regularly. Testing TVCs regularly, allows facilities to understand their baseline contamination levels and identify any significant deviation from the baseline. Rather than setting a specific standard, assessing water systems for fluctuations is more appropriate and are most likely to inform about biofilms. In addition, inclusion of cultures for coliforms and Pseudomonas is useful because this can also provide information on the presence of biofilms and the requirement to initiate a decontamination process. In summary, taking a proactive approach to understand the microbiological integrity of facility water systems by regular water testing, can prevent serious outcomes in terms of rodent losses and negative research outcomes. Monitoring water using the European pharmacopeia purified water standard and/or establishing a quantitative microbial specification for individual facilities may be the most effective means of assessing water quality in laboratory rodent facilities. In addition, health monitoring laboratories should provide support to interpret meaningful water quality results. References1 The Code of Practice for Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes. December 2014.17-18,44,77. https://www.gov.uk/government/publications/code-of-practice-for-the...2 Stotland, P.K., Radzioch, D., Stevenson, M.M. (2000). Mouse models of chronic lung infection with Pseudomonas aeruginosa: models for the study of cysticfibrosis(review). Pediatr. Pulmonol. 30, 413–424.3 Gaydos, J.M., Carrick Jr., L., Berk, R.S., (1975). Experimental studies on mice challenged subcutaneously with Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 149,908–914.4 Gotoa,M., Shimadab,K., Satoc,A., et al., (2010). Rapid detection of Pseudomonas aeruginosa in mouse faeces by colorimetric loop-mediated isothermal amplification. Journal of Microbiological Methods 81 247–2525 Edstrom, E.K. and Curran, R. (2003). Quality Assurance of Animal Watering Systems. Laboratory Animals 32, No. 5 32-35Tech-2-Tech

148Animal Technology and Welfare August 20206 Sandle, T. (2017). Microbiological monitoring of water systems, European Pharmaceutical Review 22(2):25-2 7 The water supply (water quality) regulations. 2016 No 614 https://www.legislation.gov.uk/uksi/2016/614/contents/made8 European Pharmacopoeia, 9th edition, January 2017 https://www.pharmamicroresources.com/2017/ 01/european-pharmacopoeia-9 Tech-2-Tech

149August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIntroduction This article is a summary of a recent review paper by Stevens et al.Enrichment for laboratory ZebrafishCHLOE STEVENSAnimals in Science Department, RSPCA, Wilberforce Way, Southwater, West Sussex RH13 9RS UKCorrespondence: chloe.stevens@rspca.org.uk Introduction • This article is a summary of a recent review paper by Stevens et al. ‘ Figure 1. Wild Zebrafish ( Image by Peter Kunetssov from Pixaby) Environmental enrichment’ generally refers to modifications or additions which are made to the housing of a captive animal with the intention of improving that animal’s welfare. The welfare benefits of adding enrichment have been observed in a wide range of species, and may range from lower levels of stress hormones and changes in behaviours associated with boredom, stress or anxiety, to longer-term changes in the animal’s brain development and cognition, growth, reproductive rate, or immune system function. Environmental enrichment tends to be less-frequently used for laboratory zebrafish than for other laboratory species, and a commonly-cited reason for this is a belief that there is not yet enough evidence to support the position that zebrafish can benefit from enrichment, or to tell us what types of enrichment zebrafish may benefit from. However, a look at the literature shows that many studies have now been published on the effects of enrichment on zebrafish. We recently conducted and published a literature review 1 to better understand what the evidence says about the effects of enrichment on zebrafish, and I present a summary of this evidence here. Environmental enrichment’ generally refers to modifications or additions which are made to the housing of a captive animal with the intention of improving that animal’s welfare. The welfare benefits of adding enrichment have been observed in a wide range of species, and may range from lower levels of stress hormones and changes in behaviours associated with boredom, stress or anxiety, to longer-term changes in the animal’s brain development and cognition, growth, reproductive rate, or immune system function. Environmental enrichment tends to be less-frequently used for laboratory zebrafish than for other laboratory species, and a commonly-cited reason for this is a belief that there is not yet enough evidence to support the position that zebrafish can benefit from enrichment, or to tell us what types of enrichment zebrafish may benefit from. However, a look at the literature shows that many studies have now been published on the effects of enrichment on zebrafish. We recently Figure 1. Wild Zebrafish (Image by Peter Kunetssov from Pixaby).The term environmental enrichment generally refers to modifications or additions which are made to the housing of a captive animal with the intention of improving that animal’s welfare. The welfare benefits of adding enrichment have been observed in a wide range of species, and may range from lower levels of stress hormones and changes in behaviours associated with boredom, stress or anxiety, to longer-term changes in the animal’s brain development and cognition, growth, reproductive rate, or immune system function. Environmental enrichment tends to be less-frequently used for laboratory Zebrafish than for other laboratory species, and a commonly-cited reason for this is a belief that there is not yet enough evidence to support the position that Zebrafish can benefit from enrichment or to tell us what types of enrichment Zebrafish may benefit from. However, a look at the literature shows that many studies have now been published on the effects of enrichment on Zebrafish. We recently conducted and published a literature review1 to better understand what the evidence says about the effects of enrichment on Zebrafish, and I present a summary of this evidence here.August 2021 Animal Technology and Welfare

150Animal Technology and Welfare August 2020The natural habitat of the wild Zebrafish is both complex and dynamic. It may contain physical structures such as plants, rocks and debris. There may be sand, mud, silt or rocks at the bottom of the water body, and there may be water currents to swim against.2–4 There are predators to avoid, sounds and smells to respond to, food to be foraged, prey to capture and the opportunity for varied social interactions. In contrast, a standard laboratory tank is usually a simple environment with little or no change or stimulation. It therefore seems likely that there is scope to use enrichment to improve the welfare of Zebrafish housed in these conditions by creating a more complex environment which more closely resembles the wild habitat. These enrichments are not limited to putting objects into the tanks (physical or structural enrichment) - there are several other categories of enrichment which include social context (social enrichment), dietary variety (nutritional enrichment), sensory inputs (sensory enrichment) and the opportunity to carry out a wider range of behaviours (occupational enrichment).5 A well planned enrichment strategy would ideally take elements from all of these categories.Physical EnrichmentWhen people think about enrichment for Zebrafish, they tend to think of physical forms of enrichment first – real or plastic plants, shelters, or substrates like gravel or sand. Indeed, most research into the effects of enrichment on Zebrafish has focussed on physical enrichment. It has been suggested that part of the reason for this focus is anthropomorphism – the assumption that, because humans prefer the look of tanks containing lots of these structures, Zebrafish will too.6 However, multiple studies have now found that Zebrafish will actively choose physically enriched environments over barren ones when given the choice7–10 – and if presented with environments with a little, or a lot of enrichment, will choose the more enriched environment.11 The importance of providing animals with what they want is a well-established principle in Animal Welfare science,12 so these results are a good indicator that enrichment is important for Zebrafish welfare but it is also important to establish the effects of enrichment by looking at other welfare indicators.One of the clearest improvements in welfare which has been seen for Zebrafish housed with enrichment was a study by Lee et al ,13 in which larvae were housed with gravel and plants or in bare tanks. They found that larvae housed in the enriched tanks had much higher survival rates (83%) than in the bare tanks (55%), which the authors suggested could be partly explained by smaller larvae having to expend less energy to avoid larger larvae as the enrichment allowed them to access hiding places. Physical enrichment has also been found to contribute to lower levels of anxiety13–17 and stress17,18 in Zebrafish, which has clear benefits for fish wellbeing. Physical enrichment may also help Zebrafish cope better with unpredictable stressors17 or even pain15 – both of which might be experienced by Zebrafish during their life in the laboratory, so these are particularly noteworthy findings. As well as the benefits mentioned above, a number of other changes which are likely to indicate better welfare have been seen in Zebrafish housed with physical enrichment. For example, Zebrafish housed with enrichment have been found to have higher fertility and fecundity19 – as chronic stress is associated with lower fertility and fecundity in many animals, this might suggest that fish housed with physical enrichment are less stressed. Physical enrichment has even been found to improve learning, memory and brain size in Zebrafish.16,20,21 These kinds of changes may make Zebrafish better prepared to cope with challenges and appraise stressors and therefore improve welfare.One concern which has been expressed over the introduction of physical enrichment is whether adding structures to the tank might increase territoriality and aggression by giving the fish something to fight over. Although there is some evidence that Zebrafish in enriched conditions may show more aggression,22 other studies have found that aggression does not increase with enrichment.23–26 The reasons for these differences in findings are unclear, but may be related to stocking density - as aggression is likely to reflect competition for high-value resources, having lower stocking densities of fish and higher numbers of enrichment items may avoid any increase in aggression. Social enrichmentSocial enrichment could be described simply as providing animals with access to other individuals. Wild Zebrafish are found living in groups,4,27,28 and express clear preferences for being near other Zebrafish in the laboratory,14,29,30 so it may therefore seem easy to say that group-housed Zebrafish are already socially enriched. However this picture may not be as simple as it might first appear. For one thing, given the strong tendencies of wild Zebrafish and the preferences of laboratory Zebrafish, it might be more appropriate to say that housing Zebrafish in groups represents a basic, baseline need. Individual housing should therefore be viewed as an impoverished environment, rather than considering the presence of other Zebrafish to be ‘enrichment’. Furthermore, the social interactions a Zebrafish might have with others can be complex – and reflecting this some varied results as to the effects of social contact on welfare parameters have been found.A number of studies have found that being housed in a group leads to lower stress and anxiety in Zebrafish,31–33 Enrichment for laboratory Zebrafish

151August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfareand can also lead to increased development of new neurons in the brain34 and decrease fear responses to new situations.35 However, some studies have found that group-housing does not affect anxiety or stress18,36 or can even increase anxiety and stress.37,38 These mixed results might be because the social environment of the Zebrafish can be very complex, with a variety of both positive and negative interactions. It is therefore likely that, to create a tank which can be considered socially enriched, more consideration will need to be given to factors like how familiar the Zebrafish in the group are with each other, the overall sex ratio, the stocking density and the availability of other forms of enrichment.Dietary enrichmentMany laboratory Zebrafish will be mostly fed on a diet of commercially available dry food, often supplemented with some form of live food such as Artemia, rotifers, or Daphnia. Adding this live food is often considered enrichment because it provides some variety for the Zebrafish, and allows Zebrafish to express some of their natural hunting behaviour. This is likely to be true. However to our knowledge, no published studies have yet examined the welfare impacts of live food supplementary feeding on Zebrafish. Many Animal Technologists have observed that providing live food seems to benefit Zebrafish, so it seems advisable to continue giving this unless there is a good reason to stop. However, this presents an interesting question for those interested in Zebrafish enrichment, as some may show more hesitancy over using physical enrichment for their Zebrafish than live food, despite there being significantly more evidence for the use of physical enrichment. Occupational enrichmentCreating some kind of activity with which Zebrafish can occupy their time may present a challenge but one option is flowing water. This gives the Zebrafish something to swim against, which provides exercise – this kind of enrichment can have physical benefits such as better muscle and bone development,39,40 but can also have psychological benefits, including lower anxiety and better learning ability.41,42 However, Zebrafish may not want access to flowing water all the time – a preference test which gave Zebrafish a choice between tank compartments containing water flow, plastic plants, a combination of water flow and plastic plants, or a bare tank found that the fish avoided the flowing water condition but most strongly preferred the combined condition.9 This might have been because the presence of the plants made this most enriched condition more interesting to the fish, or could have been because the presence of the plants gave fish a choice over whether to interact with the water flow or not. Providing an animal with choice and control within their environment is key for promoting good welfare. This study also highlights that it is important to trial different types of enrichment together to see if the fish respond differently to a combination of conditions than to a type of enrichment on its own. Sensory enrichmentChanges to environments which provide stimulation through visual cues, sounds, smells, touch or taste can be considered sensory enrichments, and some of these may be possible for laboratory Zebrafish. For example, it may be possible to provide fish with visual enrichment by changing the colour of tanks or adding pictures to the outside of tanks to make them more interesting. Zebrafish have been found to express preferences for some colours,43–45 but different studies have found different results, so it may be too early to use this information to design enrichments. However, Zebrafish were found to express a strong preference for a tank with a gravel image affixed to the base, over a barren tank – and this preference was almost as strong as for the presence of real gravel in the tank.8 This kind of modification to laboratory tanks is straightforward to introduce, so is recommended and provides some exciting possibilities for trialling other similar types of enrichment.Another form of sensory enrichment which may benefit Zebrafish is auditory enrichment – for example, playing music in the laboratory. Playing classical music might lead to lower levels of anxiety in Zebrafish,46,47 although it may be too early to say whether this is because of some feature of the music itself, or because it simply drowns out other noises from the lab. However, it does show that Zebrafish are sensitive to auditory stimuli outside the tank and this is therefore an area that needs further investigation.A form of possible sensory enrichment which could be used in laboratories, but has not been directly investigated, is the use of dawn and dusk phases in between the light and dark phases of lighting cycles. Zebrafish show higher levels of anxiety in bright light,48 and are likely to startle and therefore experience stress if exposed to a bright light switching on each day. DiscussionAlthough there is still much to be done to better understand exactly what laboratory conditions would provide the optimal welfare for Zebrafish, it is clear that, on the basis of the available evidence, enrichment does improve Zebrafish welfare and it is therefore possible to make improvements to the standard conditions currently seen in many laboratories. For example, numerous studies now indicate that physical enrichments can have positive effects on a wide variety Enrichment for laboratory Zebrafish

152Animal Technology and Welfare August 2020of welfare parameters, so this should be included where possible. However some facilities may find that there are practical or technical challenges associated with introducing physical enrichment into adult stock or experimental tanks – in this case, it may still be possible to add enrichment for larvae, which may result in long-term benefits to Zebrafish welfare even when they are moved into bare tanks, or for fish which have to be housed in temporary isolation (e.g. for genotyping).Opportunities for introducing other forms of enrichment should be explored as well, especially where there are limitations which completely prevent physical enrichment from being used. For example, a gravel image underneath tanks is preferred by Zebrafish, and can be a low-cost, low-maintenance way to add some more enrichment to tanks. Other forms of enrichment which have not yet been properly investigated but either have some anecdotal evidence supporting them, or are likely to be worth giving the benefit of the doubt include providing fish with live food and using dawn and dusk lighting cycles, although ideally these will be appropriately empirically tested in the near future. Action points:– Share this summary paper with others in the animal unit, your Named Information Officer and the Animal Welfare and Ethical Review Body (AWERB).– Ask for a discussion about enrichment for Zebrafish in your facility. Which of the enrichments described in this paper are already in place, and which could you trial or implement? Are there any obstacles to implementation, and if so how could you overcome these?– Consider trialling enrichments i.e. adding gravel images under tanks.– If enrichment cannot be added to all stock or experimental tanks, consider trialling enrichment such as plastic plants in the tanks of fish which have to be temporarily singly housed. Also ensure that these fish remain in visual contact with other fish at all times. – Share the findings of any enrichment trials by publishing or presenting at conferences.To read the full review paper, please visit https://tinyurl.com/EnrichmentReviewReferences1 Stevens C.H., Reed B.T. & Hawkins P. (2021). Enrichment for Laboratory Zebrafish – A Review of the Evidence and the Challenges. Animals, Vol. 11, 698.2 Engeszer R.E., Patterson L.B., Rao A.A. & Parichy D.M. (2007). Zebrafish in The Wild: A Review of Natural History And New Notes from The Field. Zebrafish, Vol. 4, 21–40.3 Spence R. (2011). Zebrafish Ecology and Behaviour. In Zebrafish Models in Neurobehavioral Research (Kalueff A.V. & Cachat J.M., eds.), pp. 1–46. Humana Press, Totowa, NJ.4 Sundin J., Morgan R., Finnøen M.H. et al. (2019). On the Observation of Wild Zebrafish (Danio rerio) in India. Zebrafish, 546–553.5 Bloomsmith M.A., Brent L.Y. & Schapiro S.J. (1991). Guidelines for developing and managing an environmental enrichment program for nonhuman primates. Laboratory Animal Science, Vol. 41, 372–377.6 Message R. & Greenhough B. (2019). “But It’s Just a Fish”: Understanding the Challenges of Applying the 3Rs in Laboratory Aquariums in the UK. Animals, Vol. 9, 1075.7 Kistler C., Hegglin D., Würbel H. & König B. (2011).Preference for structured environment in Zebrafish (Danio rerio) and checker barbs (Puntius oligolepis). Applied Animal Behaviour Science, Vol. 135, 318–327.8 Schroeder P., Jones S., Young I.S. & Sneddon L.U. (2014). What do Zebrafish want? Impact of social grouping, dominance and gender on preference for enrichment. Laboratory Animals, Vol. 48, 328– 337.9 DePasquale C., Fettrow S., Sturgill J. & Braithwaite V.A. (2019). The impact of flow and physical enrichment on preferences in Zebrafish. Applied Animal Behaviour Science, Vol. 215, 77–81.10 Tan S.L.T., Handasyde K.A., Rault J.-L. & Mendl M. (2020). Insensitivity to reward shifts in Zebrafish (Danio rerio) and implications for assessing affective states. Animal Cognition, Vol. 23, 87–100.11 Lavery M., Braithwaite V., Miller N. & Mason G. (2019). Identifying enriched housing conditions for Zebrafish (Danio rerio) that vary along a scale of preference. In Proceedings of the 53rd Congress of the ISAE: Animals Lives Worth Living (Newberry, RC and Braastad, BO, ed.), p. 388. Wageningen Academic Publishers.12 Dawkins M.S. (2003). Behaviour as a tool in the assessment of Animal Welfare. Zoology, Vol. 106, 383–387.13 Lee C.J., Paull G.C. & Tyler C.R. (2018). Effects of environmental enrichment on survivorship, growth, sex ratio and behaviour in laboratory maintained Zebrafish Danio rerio. Journal of Fish Biology, Vol. 94, 86–95.14 Collymore C., Tolwani R.J. & Rasmussen S. (2015). The Behavioral Effects of Single Housing and Environmental Enrichment on Adult Zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science, Vol. 54, 280–285.15 Manuel R., Gorissen M., Stokkermans M. et al. (2015). The effects of environmental enrichment and age-related differences on inhibitory avoidance in Zebrafish (Danio rerio Hamilton). Zebrafish, Vol. 12, 152–165.Enrichment for laboratory Zebrafish

153August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfare16 DePasquale C., Neuberger T., Hirrlinger A.M. & Braithwaite V.A. (2016). The influence of complex and threatening environments in early life on brain size and behaviour. Proceedings of the Royal Society B: Biological Sciences, Vol. 283, 20152564.17 Marcon M., Mocelin R., Benvenutti R. et al. (2018) Environmental enrichment modulates the response to chronic stress in Zebrafish. Journal of Experimental Biology, Vol. 221, jeb176735.18 Giacomini A.C.V.V., Abreu M.S., Zanandrea R. et al. (2016). Environmental and Pharmacological Manipulations Blunt the Stress Response of Zebrafish in a Similar Manner. Scientific Reports, Vol. 6, 28986.19 Wafer L.N., Jensen V.B., Whitney J.C. et al. (2016). Effects of Environmental Enrichment on the Fertility and Fecundity of Zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science, Vol. 55, 291–294.20 Spence R., Magurran A.E. & Smith C. (2011). Spatial cognition in Zebrafish: the role of strain and rearing environment. Animal Cognition, Vol. 14, 607–612.21 Roy T. & Bhat A. (2016). Learning and memory in juvenile Zebrafish: What makes the difference--population or rearing environment? Ethology, Vol. 122, 308–318.22 Woodward M.A., Winder L.A. & Watt P.J. (2019). Enrichment Increases Aggression in Zebrafish. Fishes, Vol. 4, 22.23 Basquill S.P. & Grant J.W.A. (1998). An increase in habitat complexity reduces aggression and monopolization of food by Zebrafish (Danio rerio). Canadian Journal of Zoology, Vol. 76, 770–772.24 Carfagnini A.G., Rodd F.H., Jeffers K.B. & Bruce A.E.E. (2009). The effects of habitat complexity on aggression and fecundity in Zebrafish (Danio rerio). Environmental Biology of Fishes, Vol. 86, 403–409.25 Keck V.A., Edgerton D.S., Hajizadeh S. et al. (2015). Effects of Habitat Complexity on Pair-Housed Zebrafish. Journal of the American Association for Laboratory Animal Science, Vol. 54, 378–383.26 Czezyk A., Burn C. & Russell C. (2020). Does Providing Hiding Spaces for Zebrafish in Large Groups Reduce Aggressive Behaviour? Journal of young investigators, Vol. 38.27 Suriyampola P.S., Shelton D.S., Shukla R. et al. (2016). Zebrafish Social Behavior in the Wild. Zebrafish, Vol. 13, 1–8.28 Shelton D.S., Shelton S.G., Daniel D.K. et al. (2020). Collective Behavior in Wild Zebrafish. Zebrafish, Vol. 17, 243–252.29 Al-Imari L. & Gerlai R. (2008). Sight of conspecifics as reward in associative learning in Zebrafish (Danio rerio). Behavioural Brain Research, Vol. 189, 216–219.30 Saverino C. & Gerlai R. (2008). The social Zebrafish: behavioral responses to conspecific, heterospecific, and computer animated fish. Behavioural Brain Research, Vol. 191, 77–87.31 Pagnussat N., Piato A.L., Schaefer I.C. et al. (2013). One for all and all for one: the importance of shoaling on behavioral and stress responses in Zebrafish. Zebrafish, Vol. 10, 338–342.32 White L.J., Thomson J.S., Pounder K.C., Coleman R.C. & Sneddon L.U. (2017). The impact of social context on behaviour and the recovery from welfare challenges in Zebrafish, Danio rerio. Animal Behaviour, Vol. 132, 189–199.33 Tunbak H., Vazquez-Prada M., Ryan T.M., Kampff A.R. & Dreosti E. (2020). Whole-brain mapping of socially isolated Zebrafish reveals that lonely fish are not loners. eLife, Vol. 9.34 Lindsey B.W. & Tropepe V. (2014). Changes in the social environment induce neurogenic plasticity predominantly in niches residing in sensory structures of the Zebrafish brain independently of cortisol levels. Developmental Neurobiology, Vol. 74, 1053–1077.35 Kareklas K., Elwood R.W. & Holland R.A. (2018). Grouping promotes risk-taking in unfamiliar settings. Behavioural Processes, Vol. 148, 41–45.36 Forsatkar M.N., Safari O. & Boiti C. (2017). Effects of social isolation on growth, stress response, and immunity of Zebrafish. Acta Ethologica, Vol. 20, 255–261.37 Shams S., Chatterjee D. & Gerlai R. (2015). Chronic social isolation affects thigmotaxis and whole-brain serotonin levels in adult Zebrafish. Behavioural Brain Research, Vol. 292, 283–287.38 Shams S., Seguin D., Facciol A., Chatterjee D. & Gerlai R. (2017). Effect of social isolation on anxiety-related behaviors, cortisol, and monoamines in adult Zebrafish. Behavioral Neuroscience, Vol. 131, 492–504.39 Hasumura T. & Meguro S. (2016). Exercise quantity-dependent muscle hypertrophy in adult Zebrafish (Danio rerio). Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology, Vol. 186, 603–614.40 Suniaga S., Rolvien T., vom Scheidt A. et al. (2018). Increased mechanical loading through controlled swimming exercise induces bone formation and mineralization in adult Zebrafish. Scientific Reports, Vol. 8, 3646.41 Luchiari A.C. & Chacon D.M.M. (2013). Physical exercise improves learning in Zebrafish, Danio rerio. Behavioural Processes, Vol. 100, 44–47.42 DePasquale C. & Leri J. (2018). The influence of exercise on anxiety-like behavior in Zebrafish (Danio rerio). Behavioural Processes, Vol. 157, 638–644.43 Oliveira J., Silveira M., Chacon D. & Luchiari A. (2015). The Zebrafish world of colors and shapes: preference and discrimination. Zebrafish, Vol. 12, 166–173.44 Avdesh A., Martin-Iverson M.T., Mondal A. et al. (2012). Evaluation of color preference in Zebrafish for learning and memory. Journal of Alzheimer’s Disease, Vol. 28, 459–469.Enrichment for laboratory Zebrafish

154Animal Technology and Welfare August 202045 Roy T., Suriyampola P.S., Flores J. et al. (2019). Color preferences affect learning in Zebrafish, Danio rerio. Scientific Reports, Vol. 9, 14531.46 Barcellos H.H.A., Koakoski G., Chaulet F. et al. (2018). The effects of auditory enrichment on Zebrafish behavior and physiology. PeerJ, Vol. 6, e5162.47 Marchetto L., Barcellos L.J.G., Koakoski G. et al. (2021). Auditory environmental enrichment prevents anxiety-like behavior, but not cortisol responses, evoked by 24-h social isolation in Zebrafish. Behavioural brain research, Vol. 404, 113169.48 Sabet S.S., Van Dooren D. & Slabbekoorn H. (2016). Son et lumière: Sound and light effects on spatial distribution and swimming behavior in captive Zebrafish. Environmental Pollution, Vol. 212, 480–488.Enrichement for Laboratory Zebrafish

155August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAbstract Animal Welfare has developed into a science in its own right and, as a result, there is a growing amount of research into this subject. Now people of all age groups are coming forward to gain education about Animal Welfare as well as training in some of the practical aspects of the subject. Greater numbers of science graduates and scientists working in the field of laboratory animals are curious about this topic and want to be trained in this field. The National Institute of Animal Welfare (NIAW) examined a wide range of issues affecting the welfare of each animal species, including housing and husbandry systems, nutrition and feeding, transport and euthanasia methods. In the last five years, NIAW has played an important role in improving the welfare of animals anticipating the increase in public concern for Animal Welfare in India. In the year 2015-16, a total 674 persons were trained in Animal Welfare. For the year (2016-17) in the month of September this number rose to 300, all of which voluntarily participated in the training programmes. The majority of them were leading scientists with experience in areas such as veterinary sciences and animal behaviour, that wished to become of CPCSEA nominees for different institutions and provide scientific advice on the welfare of animals. Other than scientists, pharmacy and veterinary interns, Zookeepers, Honorary Animal Welfare Officers, Wild Life Crime Control Bureau Volunteers and Social Activists have come forward to be trained in Animal Welfare.Keywords: PCA Act, Animal Welfare, training programme, education IntroductionFor many years, Animal Welfare organisations have taken the lead in bringing about improvements in Animal Welfare, as well as helping to change attitudes towards animals. The Prevention of Cruelty to Animals Act, 1960 is an Act of the Parliament of India enacted to prevent the infliction of unnecessary pain or suffering on animals and to amend the laws relating to the prevention of cruelty to animals. India, despite having some of the best animal protection laws in the world, is still lacking in the way they are enforced. Unfortunately, in India the Animal Welfare organisations and professionals have often lagged behind in support for reform. It was vital therefore, that training programmes which include a good grounding in Animal Welfare education should be established for all interested persons. In 2009, the government of India decided to establish a National Institute of Animal Welfare (NIAW) to impart training and education and the syllabus stimulates focussed critical thinking on Animal Welfare issues.Some people use the terms Animal Welfare and animal rights interchangeably, suggesting that they represent the same concerns, principles, and practices. However the differences between the two are significant and irreconcilable. The National Institute of Animal Welfare (NIAW) was established in recognition of the need to promote awareness and disseminate information about Animal Welfare. NIAW has been conceptualised as an apex body in the field of Animal Welfare and its broad mandate covers the need to improve Animal Welfare through research, education and public outreach. Its objective is to create an enabling environment for fulfilment of the statutory requirements as laid down Scientific, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and education KUMUD KANT AWASTHI1,21 National Institute of Animal Welfare Ballabhgarh, Faridabad (Haryana) 121004 India2 Department of Life Sciences, Vivekananda Global University, Jaipur (Rajasthan) 303012 IndiaCorrespondence: awasthi.niaw@gmail.com, kumud.awasthi@vgu.ac.inAugust 2021 Animal Technology and Welfare

156Animal Technology and Welfare August 2020in the Prevention of Cruelty to Animals Act, 1960. This Institute is an imparting education in this discipline in a professional manner with a structured framework given the intrinsic value of animals and their contribution not only to the country’s economy but also regarding social and environmental issues. It is imparting training and education on diverse subjects in Animal Welfare including animal management, behaviour and ethics. This change in educational pattern will have to be tempered with a totally new concept of perceptions in the new era of education. It is felt that there is a need for trained personnel who are currently not available, to work in the Animal Welfare sector where over 70% of all agricultural households are involved with animals as a livelihood option.The necessity for establishing NIAW has to be viewed in the background of the requirements of the new century where new specialisations and specialties, based primarily on futuristic technologies, will form the primary core required for development in the general area of poverty alleviation. These will focus on the use of animals to provide employment opportunities and generation of wealth with a humane attitude for those who are below the poverty line in this country. Awareness, comprehension, skills, behaviours, and beliefs related to human intervention in the lives of animals are promoted through Animal Welfare education. It has the implications for animals’ ability to meet their basic needs, as well as human obligations as a result. It can result in positive improvements in how people handle animals, but it will not always result in long-term behavioural changes, especially if carried out in an instructional or piecemeal way.Animal care preparation must become an integral part of the career advancement of someone who plans to deal with animals or with animal policies, including, policy and enforcement officials, animal wardens, veterinarians, stockmen/women, slaughtermen/women and all other animal industries and researchers. Training modules at NIAW are summarised in Table 1. Training module of Animal Welfare in NIAWNIAW through its different training modules spreads the message of Animal Welfare for almost all types of persons in the country. Training courses are being organised for personnel who are leading scientists having experience in areas such as veterinary sciences and animal behaviour. Those wishing to become nominees for the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and (Institutional Animal Ethics Committee (IAEC) for different institutions and who Table 1. Training modules for Animal Welfare at NIAW.Sr. No.Module TopicsCore module1. Alternative to animal experiments Yes2. Animal behaviour Yes3. Animal Welfare during disasters Yes4. Animal Welfare introduction Yes5. Animal Welfare organisations Yes6. Animals in experiments Yes7. Animals used in entertainment No8. Anti-rabies programme Yes9. Behavioural indicators Yes10. Commercial exploitation of wildlife No11. CPCSEA and IAEC Yes12. Enforcement and political pressure No13. Euthanasia Yes14. Farm Animal Welfare assessment and issues Yes15. Farm, wild and laboratory animal transport Yes16. GLP and GMP Yes17. Group assessment and management of welfare Yes18. Handling of animals (No Practical) Yes19. Human-animal interactions No20. Humane education Yes21. Immune and production indicators of welfare No22. Influence of the marketplace No23. Interaction with other ethical concerns No24. Introduction to Animal Welfare ethics Yes25. Introduction of 3Rs to 5Fs concept Yes26. Other societies and organisation for Animal Welfare Yes27. Pain and stress management Yes28. PCA Act 1960 Yes29. Physiological indicators of welfare No30. Population control programmes (ABC) Yes31. Protection legislation Yes32. Rational use of animals in research Yes33. Record keeping for animal house Yes34. Religion and animals No35. Slaughter of farm animals Yes36. Social relationship in animals Yes37. Stray Animal Welfare Yes38. The role of the veterinarian Yes39. Visit of shelter house Yes40. Visit of small laboratory house Yes41. War and natural disasters Yes42. Welfare assessment and the Five Freedoms Yes43. Wild/ Farm/ Laboratory Animal management Yes44. Wildlife protection act Yes45. Zoonoses and anthroponosis YesScientific, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and education

157August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfareprovide scientific advice on the welfare of animals. Other than scientists, there is course for pharmacy and veterinary interns, science graduates from different institutes/universities from around the country. There is a separate module for zookeepers, wild life crime control bureau volunteers and social activists who are working for Animal Welfare through different animal shelter houses and non-governmental organisations. There is a separate course structure for persons who want to work in the society and are identified by Animal Welfare Board of India as Honorary Animal Welfare Officers (HAWO). Other separate courses are for Group ‘A’ Officers of State Animal Welfare Board (SAWB), an Interactive Session with CPCSEA scientists working with small laboratory animals and a special training programme on Animal Welfare (STPAW) is also organised for persons who do not have knowledge of science and Animal Welfare but as a part of society take keen interest for improvement of animal health. The training programme with training module and trainees organised from year 2012 to 2016 is summarised in Table 2. Trends in Animal Welfare education in IndiaSeveral countries have recently adopted constitutional provisions that provide a basis for the protection of animals, though none unambiguously establishes Animal Welfare principles. The first Asian country to constitutionally address Animal Welfare may be India. India’s Prevention of Cruelty to Animals Act (1960) was unique for its era in that it established an oversight body, the Animal Welfare Board of India, for the promotion of Animal Welfare generally and for the purpose of protecting animals from being subjected to unnecessary pain or suffering. The creation of such a Board to implement the anti-cruelty law led to the promulgation of a series of specific rules that are closer to the realm of Animal Welfare than anti-cruelty legislation. NIAW is doing magnificent and pioneering work spreading education of Animal Welfare amongst the public via conducting various training programmes aimed at increasing the understanding of welfare for job seekers and people Table 2. Types of trainees for Animal Welfare.S. No.Title2012-13 2013-14 2014-15 2015-16Trainings Trainees Trainings Trainees Trainings Trainees Trainings Trainees1. Interactive Session1 16 - - - - - -2. ZooKeepers Programme1 11 - - - - 1 123. CPCSEA Nominee Training Programme9 78 11 107 10 110 9 734. Internship Training Programme5 56 9 126 6 120 13 3915. Group ‘A’ Officers of SAWB- - 1 2 - - - -6. HAWO’s Training Programme2 28 6 50 2 32 4 657. Special Training Programme on Animal Welfare (STPAW)- - - - 4 56 8 1158. WCCB Volunteers- - - - - - 1 18Total 18 189 27 285 22 318 36 674Scientific, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and education

158Animal Technology and Welfare August 2020working in the fi eld. The data presented here for the last three years (2013-16) shows a tremendous growth of trainees in Animal Welfare. However from 2016, the academic responsibility of NIAW was handed over to other organisations. Philosophical bases of Animal WelfareUnderstanding of ‘Animal Welfare’ depends in part on values that differ between cultures and individuals. These differences lead people to emphasise different elements of Animal Welfare that can be summarised under three broad headings. The fi rst is a focus on animals’ physical wellbeing and biological functions. Disease, illness and malnutrition are almost universally treated as Animal Welfare issues since there is almost universal consensus that those elements are essential for Animal Welfare. The second is concern about the ‘affective states’ of animals, especially negative states such as pain, distress and hunger. These are common concerns in many cultures, but in some cases they are de-emphasised by certain people – often animal producers and veterinarians who may, for example, regard the short-term pain of castration as not important enough to warrant pain management interventions. The third is a belief that the welfare of animals depends on their ability to live in a reasonably ‘natural’ manner, either by being free to perform important elements of their natural behaviour or by having natural elements (daylight, fresh air) in their environment. This last belief arises especially in industrialised countries and is common in critiques of industrialised forms of animal production. Different facets of Animal Welfare would be assigned greater or lesser importance depending on the combination of these cost considerations and the moral bases for Animal Welfare most common in each culture.Animal Welfare in cultural practiceAnimal health has been an increasing issue in many countries around the world, impacting the acceptability of farming processes. For most people, an earlier 12increasing the understanding of welfare for job seekers and people working in the field. The data presented here for last three years (2013-16) shows a tremendous growth of trainees in animal welfare. However from 2016, the academic responsibility of NIAW was handed over to other organisations. Figure 1: Trends in animal welfare education at NIAW189285318674182722362012-13 2013-14 2014-15 2015-16Animal welfare Training Programme at NIAW No. of Trainees No. of TrainingsFigure 1. Trends in Animal Welfare education at NIAW.13CPCSEA Nominees11%HAWO9%Internship10%Pharmacy 48%STPAW 17%WCCB3%Zoo Keepers2%2015-16CPCSEA Nominees34%HAWO10%Internship12%Pharmacy 26%STPAW 18%2014-15Figure 2. Interest of Trainees for Animal Welfare Programme in last three years (2013-16).13CPCSEA Nominees11%HAWO9%Internship10%Pharmacy 48%STPAW 17%WCCB3%Zoo Keepers2%2015-16CPCSEA Nominees34%HAWO10%Internship12%Pharmacy 26%STPAW 18%2014-1514Figure 2: Interest of Trainees for Animal Welfare Programme in last three years (2013-16)Philosophical bases of animal welfareUnderstanding of "animal welfare" depends in part on values that differ between cultures and individuals. These differences lead people to emphasize different elements of animal welfare that can be summarised under three broad headings. The first is a focus on animals' physical well-being and biological functions. Disease, illness, and malnutrition are almost universally treated as animal welfare issues since there is almost universal consensus that those elements are essential for animal welfare. The second is concern about the "affective states" of animals, especially negative states such as pain, distress and hunger. These are CPCSEA Nominees37%HAWO17%Internship20%Pharmacy 25%SAWB1%2013-142015-162014-152013-14Scientifi c, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and education

159August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareJudeo-Christian reading of the Bible, stated dominion over animals meant some level of cruelty was permissible. This attitude has changed to mean that everyone is responsible for Animal Welfare. This view was evident in some ancient Greek writings and has parallels in Islamic teaching. A minority view of Christians, which is a widespread view of Jains, Buddhists and many Hindus, is that animals should not be used by humans as food or for other purposes. The most widely held philosophical views on how animals can be handled today are a mix of deontological and utilitarian perspectives. Most people think that extremes of poor welfare in animals are unacceptable and that those who keep animals should strive for good welfare. Hence Animal Welfare science, which allows the evaluation of welfare, has developed rapidly. Animals have long been respected and held a special role in Indian culture and tradition. Animals have always been treated with kindness and compassion in Hinduism, Buddhism, and Jainism. Each Hindu God or Goddess is seen with an animal. Lord Krishna was a shepherd and is seen with a cow, Lord Rama with the monkeys, Lord Vishnu with the eagle and the snake, Lord Shiva with a snake around his neck and the bull ‘Nandi’ at his feet, the Goddess Saraswati, goddess of wisdom and literacy is seen with a swan. Goddess Amba’s symbol of power is riding a tiger, Lord Dattatraya always has dogs at his feet and so on. The foundation of Buddhism and Jainism is ‘Ahimsa’ or ‘non-violence’, not only towards fellow humans and animals but also to every living creature, including an insect.There was no need for Animal Welfare organisations because each home was an Animal Welfare establishment in and of itself, thanks to such a rich community and history where goodness and love were the foundations of society. Every home had cattle in the back yard. The bullocks worked in the fields alongside the farmer, the cows and buffaloes provided milk to the family but only after the calves had had their fill. Dogs and cats were welcomed into the households as family members. The hen was content to remain in the front yard and give eggs to the family. Just dead animals’ skins were used to make leather. As a result, it was a lovely picture of animal and human coexistence.Animal Welfare is a ScienceThe World Organisation for Animal Health (OIE) is the primary international standard-setting organisation for veterinary matters and takes a strong science-based approach, beginning with its definition of Animal Welfare: ‘Animal Welfare’ means how an animal is coping with the conditions in which it lives. An animal is in a good state of welfare if (as indicated by scientific evidence) it is healthy, comfortable, well nourished, safe, able to express innate behaviour, and if it is not suffering from unpleasant states such as pain, fear, and distress.Many of the scientists that use laboratory animals are worried about their health. Their interest is humanitarian as well as scientific. They have little excuse to mistreat laboratory animals and numerous reasons to handle them properly, because using sick, nervous or scared animals decreases the reliability of study findings. Veterinary staff are responsible for ensuring that research animals are well fed, free of infections and other illnesses and are kept in an appropriate environment.Animal Welfare movementAs it aims to improve society’s view and handling of animals, the animal rights movement is clearly a social change movement. Animal rights activists also regard themselves as distinct from other social reform groups. Other social justice campaigns, on the other hand, can teach us a lot, as this course has demonstrated. Also, as with any similar movement, the Animal Welfare movement cannot be isolated from social change, politics, culture and economics. In fact, the expansion of the Animal Welfare movement is strongly connected to these areas. In different countries, the animal rights campaign is at various levels of growth. The position of animal rights and the level of the movement’s growth are influenced by culture and historical development. Culture and environment also have an effect on how the animal rights community will better carry out its activism. Religion can also impact upon attitudes towards Animal Welfare, hampering or advancing the cause.Our ethical roots (especially in the West) have evolved as a human-biased morality but a major shift has occurred in the last 30 years. The Animal Welfare and environmental campaigns have also changed their emphasis to encompass the non-human world. This viewpoint is not really novel. Native Indians and Aborigines have ancient yet living rituals that demonstrate a love and understanding for the natural world that incorporates environmental preservation with human animal treatment.Lacuna in Animal WelfareUntil recently, there have been no international legislation or policy initiatives around which the movement could unite. Animal Welfare is lacking in India because of the lack of urgency about the mission for Animal Welfare, professionalism and efficiency in this field. Deficiency of capacity, skills and expertise for dealing with Animal Welfare, this also includes low numbers of Animal Welfare-trained veterinarians. Other than these issues, dearth of funding, long-term and sustained campaigns, strategic advocacy and operational impact are also the key points which lag behind Animal Welfare movement. Failure to develop feasible alternatives to current Scientific, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and education

160Animal Technology and Welfare August 2020paradigms and orthodoxies is also a co-factor for poor Animal Welfare concern. Animal Welfare is considered as a luxury for the privileged class in developing countries. Last but not the least, institutional tendency towards competition, rather than genuine collaboration is also a big challenge for proper Animal Welfare. Conclusion and future prospectsAlthough Animal Welfare education is necessary and part of the community’s move towards harmonisation of professional qualifications, Animal Welfare education is underdeveloped in most of the places in India. In education, we believe it has not received the attention it deserves in our schools’ curriculum. However, there are increasing numbers of courses on Animal Welfare being implemented. This change in educational pattern will have to be tempered with a totally new concept of perceptions in the new era of education. There is a need for niche education for specific job requirements in the Animal Welfare sector.AcknowledgementI am thankful to all the trainers for the different training courses at NIAW, who have given their paramount time for Animal Welfare education to the trainees of different groups. I pay my sincere gratitude to Director Mr. S. Gowri Shankar, and Former Director Mr. Anil Bahuguna for their encouragement. Office staff also thankfully acknowledged for providing the previous records of training programmes at NIAW.Scientific, philosophical and cultural basis of Animal Welfare and its enhanced public concern via training and education

161August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIntroductionMice are removed from their home cage for several reasons, including health and welfare checks; being placed in a warming cabinet; being placed on weighing scales or for regulated procedures. For many years people used the tail as the initial capture method. This has changed because evidence has shown that tunnel handling is a non-aversive method which can improve the welfare of mice.1,2,3,4 In 2019 we successfully introduced the use of tunnel handling as our preferred method for catching mice. The key to non-aversive methods of handling lies in understanding what capture method creates the least anxiety in mice: be this tunnel or cupping or another method. It is important that we do not get so focussed on tunnel handling, we fail to notice that in some instances tunnel capture is an aversive experience. One of the benefits we observed with tunnel handling is that it enables mice to show us this handling method may be aversive to them. For example, we observed that some mice refused to enter the tunnel by aiming their noses away from it or pushing against it. If they avoid the tunnel we know it is likely due to the increased anxiety the mouse is feeling. Rather than label the mouse as aggressive or grumpy we try to find the method that best suits the individual mouse. Some of the alternative methods to tail handling we developed are outlined below.We also adopted a hierarchy of capture methods, starting with the tunnel, followed by the cupping method and finally asking staff to use non-aversive alternatives if either of the methods were not appropriate. Having a variety of methods has drawbacks, as consistency in handling is important because mice learn to expect to be picked up in a certain way.3 If they are tunnel handled, they will usually enter the tunnel and if they are offered a lid of a cage they will readily walk on it. If the same approach is used by all handlers it is likely to further reduce the anxiety of the mice. Sometimes handlers can feel under pressure to get tasks completed quickly and the use of tunnel handling will remove the impact of this on the mouse. It is important to closely observe the mice we are working with because they can react to the demeanour of the handler. If the handler gets more stressed and agitated this could be reflected in the reaction of the mouse. We should always keep in mind that regardless of what we are doing there should be no sense of urgency when it comes to handling the mouse. Ethical statementAll animal work was carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals.Some alternative methods:Grid cage lidWe noticed our hairless mice occasionally had abrasions which were likely due to the plastic tunnels (they tended to get sticky with use after a few days) we kept in the cages for tunnel handling. For these mice we bury a clean cage grid into the sawdust and guide the mice onto it, they seem quite relaxed about staying on the lid as we lift it up. This may be because mice climb on to the grid so they are used to the feel of it underfoot, they can then be picked up from the lid. Please note mice in the picture are hairy albino females and the pink patches at the base of their tails is the fur dye we use for identification purposes (see Figure 1). Thinking outside of the tunnel for non-aversive mouse handling JOANNA MOORE and MARK WICKERT GlaxoSmithKline, Laboratory Animal Medicine, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY UK Figure 1. Left image; mice walking onto lid, right image; mice stepping from lid onto the handler’s hand. 5 Figure 1. Left image; mice walking onto lid, right image; mice stepping from lid onto the handler’s hand. Mouse wheels If mice are already on an activity wheel, which can be removed from the cage, we lift the wheel and either move them to the hand or transfer them to another cage (see Figure 2). Figure 2. Left image; mice on running wheel, right image; mice being lifted using the wheel. 5 Figure 1. Left image; mice walking onto lid, right image; mice stepping from lid onto the handler’s hand. Mouse wheels If mice are already on an activity wheel, which can be removed from the cage, we lift the wheel and either move them to the hand or transfer them to another cage (see Figure 2). Figure 2. Left image; mice on running wheel, right image; mice being lifted using the wheel. August 2021 Animal Technology and Welfare

162Animal Technology and Welfare August 2020Mouse wheelsIf mice are already on an activity wheel, which can be removed from the cage, we lift the wheel and either move them to the hand or transfer them to another cage (see Figure 2).Nervous handlersSome strains avoid being caught and make handlers feel uneasy about picking the mice up, even with a tunnel, due to the perceived concerns around being bitten by the mice during the guiding process. One method we devised to reduce the anxiety for the handler was to use a cage card to guide the mice. This protects the handler’s fi ngers and they feel calmer, in turn this keeps the mice calmer, so they are unlikely to bite when transferred from tunnel to hand (see Figure 3).Figure 2. Left image; mice on running wheel, right image; mice being lifted using the wheel. 5 Figure 1. Left image; mice walking onto lid, right image; mice stepping from lid onto the handler’s hand. Mouse wheels If mice are already on an activity wheel, which can be removed from the cage, we lift the wheel and either move them to the hand or transfer them to another cage (see Figure 2). Figure 2. Left image; mice on running wheel, right image; mice being lifted using the wheel. 5 Figure 1. Left image; mice walking onto lid, right image; mice stepping from lid onto the handler’s hand. Mouse wheels If mice are already on an activity wheel, which can be removed from the cage, we lift the wheel and either move them to the hand or transfer them to another cage (see Figure 2). Figure 2. Left image; mice on running wheel, right image; mice being lifted using the wheel. Figure 3. Top left; Label is placed in the cage and fi ngers can be tucked behind the label, top right; label is used to gently guide the mouse towards the tunnel. Lower left; guiding into tunnel; Lower right; if the label is laid fl at on the base of a cage, mice often step on the label to be lifted and moved to the hand. 6 Nervous handlersSome strains avoid being caught and make handlers feel uneasy about picking the mice up, even with a tunnel, due to the perceivedconcerns around being bitten by the mice during the guiding process.One method we devised to reduce the anxiety for the handler was to use a cage card to guide the mice. This protects the handler’s fingers and they feel calmer, in turn this keeps the mice calmer, so they are unlikely to bite when transferred from tunnel to hand (see Figure 3). Figure 3. Top left; Label is placed in the cage and fingers can be tucked behind the label, top right; label is used to gently guide the mouse towards the tunnel. Lower left; guiding into tunnel; Lower6 Nervous handlersSome strains avoid being caught and make handlers feel uneasy about picking the mice up, even with a tunnel, due to the perceivedconcerns around being bitten by the mice during the guiding process.One method we devised to reduce the anxiety for the handler was to use a cage card to guide the mice. This protects the handler’s fingers and they feel calmer, in turn this keeps the mice calmer, so they are unlikely to bite when transferred from tunnel to hand (see Figure 3). Figure 3. Top left; Label is placed in the cage and fingers can be tucked behind the label, top right; label is used to gently guide the mouse towards the tunnel. Lower left; guiding into tunnel; Lower6 Nervous handlersSome strains avoid being caught and make handlers feel uneasy about picking the mice up, even with a tunnel, due to the perceivedconcerns around being bitten by the mice during the guiding process.One method we devised to reduce the anxiety for the handler was to use a cage card to guide the mice. This protects the handler’s fingers and they feel calmer, in turn this keeps the mice calmer, so they are unlikely to bite when transferred from tunnel to hand (see Figure 3). Figure 3. Top left; Label is placed in the cage and fingers can be tucked behind the label, top right; label is used to gently guide the mouse towards the tunnel. Lower left; guiding into tunnel; Lower6 Nervous handlersSome strains avoid being caught and make handlers feel uneasy about picking the mice up, even with a tunnel, due to the perceivedconcerns around being bitten by the mice during the guiding process.One method we devised to reduce the anxiety for the handler was to use a cage card to guide the mice. This protects the handler’s fingers and they feel calmer, in turn this keeps the mice calmer, so they are unlikely to bite when transferred from tunnel to hand (see Figure 3). Figure 3. Top left; Label is placed in the cage and fingers can be tucked behind the label, top right; label is used to gently guide the mouse towards the tunnel. Lower left; guiding into tunnel; LowerMouse iglooOne strain of mice actively avoided the tunnels. As each of our cages include an igloo retreat for mice, we turn the igloo over and herd them in the same way we would using a tunnel, then lift them up with the igloo and move them to the hand (see Figure 4).Figure 4. Left image; mice being guided onto igloo, right image; mice in igloo. 7 right; if the label is laid flat on the base of a cage, mice often step on the label to be lifted and moved to the hand. Mouse igloo One strain of mice actively avoided the tunnels. As each of our cages include an igloo retreat for mice, we turn the igloo over and herd them in the same way we would using a tunnel, then lift them up with the igloo and move them to the hand (see Figure 4). Figure 4. Left image; mice being guided onto igloo, right image; mice in igloo. Handling by hand There are three options for just using our hands, we can cup the mouse, or it is possible with some mice to let them simply walk onto the hand, either with the hand on the base or the edge of the cage (See Figure 5). 7 right; if the label is laid flat on the base of a cage, mice often step on the label to be lifted and moved to the hand. Mouse igloo One strain of mice actively avoided the tunnels. As each of our cages include an igloo retreat for mice, we turn the igloo over and herd them in the same way we would using a tunnel, then lift them up with the igloo and move them to the hand (see Figure 4). Figure 4. Left image; mice being guided onto igloo, right image; mice in igloo. Handling by hand There are three options for just using our hands, we can cup the mouse, or it is possible with some mice to let them simply walk onto the hand, either with the hand on the base or the edge of the cage (See Figure 5). Handling by handThere are three options for just using our hands, we can cup the mouse, or it is possible with some mice to let them simply walk onto the hand, either with the hand on the base or the edge of the cage (See Figure 5).Figure 5. Left image; mouse jumping on the side of a cage to walk onto an open hand; right image; mouse stepping onto a hand; Lower image; cupping method. 8 Figure 5. Left image; mouse jumping on the side of a cage to walk onto an open hand; right image; mouse stepping onto a hand; Lower image; cupping method. Tunnel handlingWe remain committed to selecting tunnel handling as the initial method either using the cardboard tunnel (see Figure 6) or a clear tunnel (see Figure 7). When staff are trained and comfortable, using the tunnel technique outlined on the NC3Rs website, mice generally remain calm and often voluntarily enter the tunnel. Our cages usually include mouse handling tunnels as part of the enrichment and we often find the mice enter the tunnel as soon as the cage lid is removed. This could be an indication that (for some 8 Figure 5. Left image; mouse jumping on the side of a cage to walk onto an open hand; right image; mouse stepping onto a hand; Lower image; cupping method. Tunnel handlingWe remain committed to selecting tunnel handling as the initial method either using the cardboard tunnel (see Figure 6) or a clear tunnel (see Figure 7). When staff are trained and comfortable, using the tunnel technique outlined on the NC3Rs website, mice generally remain calm and often voluntarily enter the tunnel. Our cages usually include mouse handling tunnels as part of the enrichment and we often find the mice enter the tunnel as soon as the cage lid is removed. This could be an indication that (for some 8 Figure 5. Left image; mouse jumping on the side of a cage to walk onto an open hand; right image; mouse stepping onto a hand; Lower image; cupping method. Tunnel handlingWe remain committed to selecting tunnel handling as the initial method either using the cardboard tunnel (see Figure 6) or a clear tunnel (see Figure 7). When staff are trained and comfortable, using the tunnel technique outlined on the NC3Rs website, mice generally remain calm and often voluntarily enter the tunnel. Our cages usually include mouse handling tunnels as part of the enrichment and we often find the mice enter the tunnel as soon as the cage lid is removed. This could be an indication that (for some Thinking outside of the tunnel for non-aversive mouse handling

163August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareTunnel handlingWe remain committed to selecting tunnel handling as the initial method either using the cardboard tunnel (see Figure 6) or a clear tunnel (see Figure 7). When staff are trained and comfortable, using the tunnel technique outlined on the NC3Rs website, mice generally remain calm and often voluntarily enter the tunnel. Our cages usually include mouse handling tunnels as part of the enrichment and we often fi nd the mice enter the tunnel as soon as the cage lid is removed. This could be an indication that (for some mice) the removal of the lid may act as a visual cue for the mice to prepare for being removed from the cage. Often three or four mice will be in the plastic handling tunnel. Recently we have started to include a suspended tunnel in our cages as well as the tunnel on the cage fl oor, mice do not tend to enter the suspended tunnel for handling. When removing the cage lid for handling it is better to ensure the mice are all on the base of the cage and not in the suspended tunnel.There may be a temptation to use the tail method when removing them from a warming cabinet. When removing mice from a cabinet the handling tunnel should be used to initially catch the mouse in the same way as we would from the home cage. Figure 6. Left image, three mice around the cardboard tunnel. Right image: two mice being picked up via cardboard tunnel. 9 mice) the removal of the lid may act as a visual cue for the mice to prepare for being removed from the cage. Often three or four mice will be in the plastic handling tunnel. Recently we have started to include a suspended tunnel in our cages as well as the tunnel on the cage floor, mice do not tend to enter the suspended tunnel for handling. When removing the cage lid for handling it is better to ensure the mice are all on the base of the cage and not in the suspended tunnel. Figure 6. left image, three mice around the cardboard tunnel. Right image: two mice being picked up via cardboard tunnel. 9 mice) the removal of the lid may act as a visual cue for the mice to prepare for being removed from the cage. Often three or four mice will be in the plastic handling tunnel. Recently we have started to include a suspended tunnel in our cages as well as the tunnel on the cage floor, mice do not tend to enter the suspended tunnel for handling. When removing the cage lid for handling it is better to ensure the mice are all on the base of the cage and not in the suspended tunnel. Figure 6. left image, three mice around the cardboard tunnel. Right image: two mice being picked up via cardboard tunnel. Figure 7. Left image, two mice in plastic tunnel and one mouse in a cardboard tunnel waiting to be picked up. Right image; mouse in handling tunnel. 9 mice) the removal of the lid may act as a visual cue for the mice to prepare for being removed from the cage. Often three or four mice will be in the plastic handling tunnel. Recently we have started to include a suspended tunnel in our cages as well as the tunnel on the cage floor, mice do not tend to enter the suspended tunnel for handling. When removing the cage lid for handling it is better to ensure the mice are all on the base of the cage and not in the suspended tunnel. Figure 6. left image, three mice around the cardboard tunnel. Right image: two mice being picked up via cardboard tunnel. 9 mice) the removal of the lid may act as a visual cue for the mice to prepare for being removed from the cage. Often three or four mice will be in the plastic handling tunnel. Recently we have started to include a suspended tunnel in our cages as well as the tunnel on the cage floor, mice do not tend to enter the suspended tunnel for handling. When removing the cage lid for handling it is better to ensure the mice are all on the base of the cage and not in the suspended tunnel. Figure 6. left image, three mice around the cardboard tunnel. Right image: two mice being picked up via cardboard tunnel. In conclusion, non-aversive handling of mice is a great refi nement in mouse care, it is important we continue to observe and understand the behavioural cues from the animals in our care. Having a range of non-aversive options for handling as alternatives to tail methods for the initial pick up of the mouse is not just a great refi nement in handling, it can give the mice an opportunity to let the handler know their capture preference. By encouraging handlers to have alternative methods we may help further improve the wellbeing of laboratory mice.References1 Clarkson, J.M., Dwyer, D.M., Flecknell, P.A., Leach, M.C. and Rowe, C. (2018). Handling method alters the hedonic value of reward in laboratory mice. Scientifi c Reports. 2018: 8, 448. Available from: https://doi.org/10.1038/s41598-018-20716-32 Gouveia, K., Hurst, J.L. (2013). Reducing mouse anxiety during handling: Effect of experience with handling tunnels. PloS ONE 8. 2013: 6, e66401. Available from: https://doi.org/10.1371/journal.pone.00664013 Gouveia, K., Hurst, J.L. (2017). Optimising reliability of mouse performance in behavioural testing: themajor role of non-aversive handling. Scientifi c Reports.2017: 7: 44999. Available from: https://doi.org/ 10.1038/srep449994 Hurst, J.L., West, R.S. (2010). Taming anxiety in laboratory mice. Nature Methods. 2010: 7, 825-826. Available from: https://doi.org/10.1038/nmeth.1500Thinking outside of the tunnel for non-aversive mouse handling

164Animal Technology and Welfare August 2020Animal Technologists – the key workers for medical researchCALL FOR POSTERSCongress2022CONGRESS Invitation to Participate29th March – 1st Aprill take an active part in the leading annual meetingfor Animal Technologistsl submit a poster – final date Friday 4th February 2022*l choose to do an oral presentation of your posterand receive a discount – closing date Friday 17th December 2021*l send your ideas today on the Submission form available from www.iat.org.uk (*posters will notbe accepted for display at Congress unless theyhave been properly submitted and approved bythe Congress Committee)l two best posters will receive a prize based on the criteria: environmental enrichment / scientific basisl plus claim up to 10 CPD points – details given on acceptance of submissionContact: congress@iat.org.uk PET PORTRAITS Watercolour painting from your chosen photograph, provided framed Dimensions approximately 35cm x 25cm £120 All profits going to AS-ET For more details contact wendy.steel1@outlook.com Registered Charity Number 1133119 Bulletin16 •July 2020AS-ET NewsThe Trustees of AS-ET would like to send our best wishes to all those of you whoare having to work in difficult circumstances, ensuring your animals are cared forand essential science carries on. It is an amazing achievement to have continuedto get to work while public transport has been lacking and while avoiding beinginfected yourselves. Congratulations to all of you for demonstrating your cultureof care in a really practical way in these dangerous times. Please make sure tostill stay safe now that the restrictions have been lifted slightly – the incidence ofinfections has slowed down but the virus is still around.As you can imagine this has been a quiet time for AS-ET but we are ready forwhen circumstances allow people to get back to enrolling on courses. Our plansfor events to mark our tenth anniversary will remain on hold until the countrybecomes more settled, however remember the Congress Bursary Competition isstill running so start writing. Congress Bursary CompetitionIf you are studying for the IAT level 2 qualification or you passed it in 2018, 2019or 2020 and you are employed as a laboratory animal technician in the UK or theRepublic of Ireland you can enter the competition. This year’s topic is —‘What challenges and rewards have you experienced as an animal technician?’ Your 1,000 word essay should be submitted by 20th October 2020. Full details areon our website (www.as-et.org.uk).Patron: Professor Lord Naren Patel KTChair of Trustees: Professor Sir Richard Gardner FIAT (Hon) FRSSecretary to the Trust: Ken Applebee OBE FIAT FRSBTrustees: Stephen Barnett MSc (Hon) FRSB, Jasmine Barley MSc FIAT,Karen J Gardner, Wendy Steel BSc (Hons) FIATRegistered Charity Number 113319Registered Office: 5 South Parade, Summertown, Oxford OX2 7JLJul20:IATB NEW 10/7/20 10:02 Page 16

165August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareDevelopment of a feeding device to reduce reliance on fi eld trials to test novel poultry red mite controlsFRANCESCA NUNN, KATHRYN BARTLEY and ALASDAIR NISBETMoredun Research Institute, Edinburgh EH26 0PZ UKCorrespondence: francesca.nunn@moredun.ac.ukWinner of the Best Poster exhibited at IAT Virtual Congress 2021POSTER PRESENTATIONSOriginally presented at:IAT Congress 2021IntroductionDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5 m M e s h% to t a l m ite s re c o v e re d / p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m it e s re c o v e r e d / p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8 m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m it e s re c o v e r e d / p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m it e s re c o v e r e d / p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra te1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro to n y m p h fe e d in g r a te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionAugust 2021 Animal Technology and WelfarePoultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production.2Testing of novel control methods uses mites in laborator y-based tests followed by fi eld trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periods of time.3This strategy has 2 major drawbacks:• the data from the in vitro feeding devices (Figure 1) are highly variable leading to misleading results• it requires invasive sampling of hensDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to t a l m ite s re c o v e re d /p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra te1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro t o n y m p h fe e d in g ra te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionSponsored byDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m ite s re c o v e re d /p o u c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m ite s re c o v e re d /p o u c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m ite s re c o v e re d /p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra te1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro t o n y m p h f e e d in g ra te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 5  m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 0 5  m M e s h% to t a l m ite s re c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 0  m M e s h% to t a l m ite s re c o v e re d /p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra te1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro t o n y m p h fe e d in g ra te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. Introduction

166Animal Technology and Welfare August 2020Prototype in vivo feeding deviceTo replace the in vitro feeding device we developed a prototype in vivo feeding device for adult mites that can be attached to the hens’ thighs for short periods. Consistent feeding rates of 50% and a low background mite mortality were demonstrated.Development of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5 m M e s h% to t a l m ite s re c o v e re d / p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m it e s re c o v e r e d / p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8 m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m it e s re c o v e r e d / p o u c hA d u lt sD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m it e s re c o v e r e d / p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra te1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro to n y m p h fe e d in g r a te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionFigure 2. Graphs showing mite feeding rates associated with different mesh aperture sizes.5Poster PresentationsFigure 1. In vitro feeding device.4 An important innovation in Reduction and Refi nement – Reduction: of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large fi eld trials.– Refi nement (improved welfare): reduces the frequency and duration of exposure to mites in fi eld trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. – For optimal use, the device needed further development for uses against all stages of blood feeding PRM life stages. Device optimisationMeshes of different aperture size, thickness and materialswere tested to optimise use for all haematophagous PRM life stages (Fig. 2). Feeding rates were signifi cantlyhigher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mites prior to feeding.4 Development of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m ite s r e c o v e re d /p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra t e1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro to n y m p h fe e d in g r a te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m ite s r e c o v e re d /p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra t e1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro to n y m p h fe e d in g r a te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to ta l m ite s r e c o v e r e d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m ite s r e c o v e re d /p o u c hA d u lt sD e u t o n y m p h sP r o t o n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m ite s r e c o v e re d /p o u c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g ra t e1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p ro to n y m p h fe e d in g r a te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. IntroductionDevelopment of a feeding device to reduce reliance onfield trials to test novel poultry red mite controlsFrancesca Nunn1, Kathryn Bartley & Alasdair Nisbet1 Moredun Research Institute, Edinburgh EH26 0PZwww.moredun.org.ukMoredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland. Francesca.Nunn@moredun.ac.uk1) The data from the in vitrofeeding devices (Fig. 1) arehighly variable leading tomisleading results2) Requires invasive sampling ofhensThis strategy has 2 major drawbacks:To replace the in vitro feeding device we developed a prototype in vivofeeding device for adult mites that can be attached to the hens’ thighs forshort periods. Consistent feeding rates of 50% and a low background mitemortality were demonstrated.Prototype in vivo feeding device Reduction: Use of the in vivo feeding device to pre-screen vaccines leads to a decreased number of hens per study (384 ‘v’ 4 per group) and less testing of suboptimal products in large field trials Refinement (improved welfare): reduces the frequency and duration of exposure to mites in field trials (50-100 mites for 3 hours/time point ‘v’ tens of thousands mites for several weeks) and less invasive blood sampling of hens. For optimal use, the device needed further development for use against all blood feeding PRM life stagesAn important innovation in Reduction and RefinementFig. 1. In vitro feeding device4Device optimisationMeshes of different aperture size, thickness and materials were tested tooptimise use for all haematophagous PRM life stages (Fig. 2). Feeding rateswere significantly higher for protonymphs using a 105µm aperture,polyester mesh and this was chosen to then study conditioning of the mitesprior to feeding4.A d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 07 5  m M e s h% to ta l m it e s r e c o v e r e d /p o u c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 5 m M e s h% to ta l m it e s re c o v e r e d/p o u c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 06 8  m M e s h% to ta l m it e s r e c o v e r e d /p o uc hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 0 5 m M e s h% to ta l m it e s re c o v e r e d/p ou c hA d u ltsD e u to n y m p h sP r o to n y m p h s02 04 06 08 01 0 01 2 0 m M e s h% to ta l m it e s re c o v e r e d/ p ou c hFig. 2. Graphs showing mite feeding rates associated with different mesh aperture sizes5.To determine the best treatment of mites prior to feeding assays, fourprotocols were tested: Each mite preparation was stored at roomtemperature (RT) for 1 week and thereafter at 4oC for up to 3 weeks prior tofeeding on hens.Mite conditioningFig. 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c)502 04 06 08 01 0 0% a d u lt fe e d in g r a t e1 w e e k R T1 w e e k R T , 1 w e e k 4oC1 w e e k R T , 2 w e e k 4oC1 w e e k R T , 3 w e e k 4oC02 04 06 08 01 0 0% d e u to n y m p h fe e d in g ra te02 04 06 08 01 0 0% p r o to n y m p h fe e d in g r a te• Protonymph feeding rates were significantly higher following starvation for 1wk RT 3 wks 4oC• Mortality for adult mites was significantly lower after 1wk RT 3 wks 4oC conditioning than at one week RT. • Mortality for deutonymphs was not significantly different across the different conditioning points and no protonymph mortality was demonstrated. Summary Device optimised to allow all hematophagous PRM life stages to feed in vivo Compared to the in vitro device and initial in vivo prototype: Improved mite feeding rates Reduced background mortality High welfare for the hens Easily used by trained individualsReferences:1. Chauve, C. Vet. Para. 79 (1998) 239-2452. Flochlay, S., Thomas, E. Sparagano, O. Parasites and Vectors (2017) 10:3573. Bartley et al.. Vet Para. 244 (2017) 25-354. Bartley et al,. Int J Parasitol. 45 (2015) :819-8305. Nunn et al,. Vet. Para. 267 (2019) 42-46.Poultry red mites (PRM) are small, mobile ectoparasites that feed on the blood of hens. They are considered to be the most important ectoparasites in laying hens and are found in all housing systems worldwide. Infestation can cause an increase in mortality and stress behaviours1and a decrease in egg production2.Testing of novel control methods uses mites in laboratory-based tests followed by field trials. Field trials use large numbers of hens (~400) per experimental group, which are then exposed to high numbers of mites for extended periodsof time3. Introduction75µm Mesh 125µm Mesh 68µm Mesh 105µm Mesh 120µm Mesh75µm Mesh 125µm Mesh68µm Mesh105µm Mesh 120µm Mesh75µm Mesh 125µm Mesh 68µm Mesh 105µm Mesh 120µm Mesh

167August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareFigure 3. Percentages of mites fed following different conditioning periods: adult (a) deutonymph (b) protonymph feeding rates (c).5Mite conditioning To determine the best treatment of mites prior to feeding assays, four protocols were tested: each mite preparation was stored at room temperature (RT) for 1 week and thereafter at 4°C for up to 3 weeks prior to feeding on hens. Poster PresentationsSummary– Device optimised to allow all hematophagous PRM life stages to feed in vivo: – Compared to the in vitro device and initial in vivo protype: Improved mite feeding rates. Reduced background mortality. High welfare for the hens. Easily used by trained individuals. • Protonymph feeding rates were signifi cantly higher following starvation for 1wk RT 3 wks 4°C• Mortality for adult mites was signifi cantly lower after 1wk RT 3 wks 4°C conditioning than at one week RT.• Mortality for deutonymphs was not signifi cantly different across the different conditioning points and no protonymph mortality wasReferences1 Chauve, C. (1998). Activation of the poultry red mite, Dermanyssus gallinea current situation and future prospects for capital. Vet. Para. 79 (1998) 239-245. 2 Flochlay, S., Thomas, E. Sparagano, O. (2017). Poultry red mite (Dermanyssus gallinea) infestation: a broad impact parasitological disease that still remains a signifi cant challenge for the egg-laying industry in Europe. Parasites and Vectors (2017) 10:357. 3 Bartley, et al. (2017). Field evaluation of poultry red mite (Dermanyssus gallinea) native and recombinant prototype vaccines. Vet Para. 244 (2017) 25-35. 4 Bartley, et al. (2015). Identifi cation and evaluation of vaccine consolidate aligns from the poultry red mite (Dermanyssus gallinea). Int J Parasitol. 45 (2015): 819-830. 5 Nunn, et al. An improved method for the in vitro feeding of adult female (Dermanyssus gallinea) (poultry red mite) using Badenoch membrane (goldheiden’s stan). Vet. Para. 267 (2019). 42-46.

168Animal Technology and Welfare August 2020The present debate about the so-called ‘reproducibility crisis’ within preclinical studies has been dominated by eloquent papers focussing on the very real, but more ‘mathematical’ issues. These are only part of the problem.The solution to this crisis must involve greater attention to the animal-related issues and the facilities in which they are kept. These issues may be less obvious to those who have not worked in an animal unit but they constitute by far the greatest potential source of variation in preclinical studies.To make matters worse, it is frequently stated that the solution lies in better reporting of animal studies. An experiment cannot be improved by describing it. Our fi rm belief, after many years of working within animal facilities, is that the clue lies in closer collaboration between all parties from day one of planning. In this way, specialists in laboratory animal science, statisticians and scientists can attend to the many factors which will determine the outcome of the study: both its validity and the animals’ welfare.The PREPARE Guidelines have been written to solve this challenge. They are based on experiences over the last 30 years in conducting and supervising preclinical research.PREPARE for better science:guidelines for animal researchADRIAN J SMITH,1 R EDDIE,2 ELLIOT LILLEY,3 KRISTINE EA HANSEN4andTROND BRATTELID51 Norecopa, Oslo, Norway2 Royal (Dick) School of Veterinary Studies, Easter Bush, Midlothian EH25 9RG UK3 Research Animals Department, Science Group, RSPCA, Wilberforce Way, Southwater, Horsham, West Sussex RH13 9RS UK 4 Section of Experimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, PO Box 8146 Dep., 0033 Oslo, Norway5 Division for Research Management and External Funding, Western Norway University of Applied Sciences, 5020 Bergen, NorwayCorrespondence: adrian.smith@norecopa.noAdrian J Smith1, R Eddie Clutton2, Elliot Lilley3, Kristine EA Hansen4& Trond Brattelid51Norecopa, Oslo, Norway (adrian.smith@norecopa.no);2Royal (Dick) School of Veterinary Studies, Easter Bush, Midlothian, EH25 9RG, U.K.;3Research Animals Department, Science Group, RSPCA, Wilberforce Way, Southwater, Horsham, West Sussex, RH13 9RS, U.K. ;4Section ofExperimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of LifeSciences, P.O. Box 8146 Dep., 0033 Oslo, Norway;5Division for Research Management and External Funding, Western Norway University ofApplied Sciences, 5020 Bergen, Norway.PREPARE for better Science: guidelines for animal researchThe PREPARE Guidelines have been writtento solve this challenge. They are based onexperiences over the last 30 years inconducting and supervising preclinicalresearch.PREPARE consists of a 2-page checklist, in 20languages, and, importantly, webpages withguidance on each of the 15 topics on thechecklist. The PREPARE webpages are partof the Norecopa website, which containsover 8,500 pages of resources designed toattend to the 3Rs: Replacement, Reductionand Refinement of animal research.norecopa.no/PREPARENorecopa gratefully acknowledges financial support from the Norwegian Ministry of Agriculture and Food, Finn Rahn's legacy, Dag Stiansen’s Foundation, Laboratory Animals Ltd., the Nordic Society against Painful Animal Experiments (NSMSD), the Norwegian Society for Protection of Animals, Novo Nordisk, the Scottish Accreditation Board, the US Department of Agriculture and the Universities Federation for Animal Welfare (UFAW). Stock photos: colourbox.comGroup sizePowerBiasHARKingp-hackingThe present debate about the so-called"reproducibility crisis" within preclinical studieshas been dominated by eloquent papers focusingon the very real, but more "mathematical"issues. These are only part of the problem.To make matters worse, it is frequently stated that the solution lies in better reporting of animal studies. Anexperiment cannot be improved by describing it. Our firm belief, after many years of working within animalfacilities, is that the clue lies in closer collaboration between all parties from day one of planning. In this way,specialists in laboratory animal science, statisticians and scientists can attend to the many factors which willdetermine the outcome of the study: both its validity and the animals' welfare.We have produced a 3-minute cartoon film to illustrate this,using the aviation industry as an analogy with its impressiveability to ensure quality and reproducibility, despite variabletraffic conditions and the risk of human errors.https://norecopa.no/PREPARE/filmThe solution to this crisis must involve greater attention to theanimal-related issues and the facilities in which they are kept. Theseissues may be less obvious to those who have not worked in ananimal unit, but they constitute by far the greatest potential sourceof variation in preclinical studies.PREPAREThe PREPARE Guidelines ChecklistPlanning Research and Experimental Procedures on Animals: Recommendations for ExcellenceAdrian J. Smitha, R. Eddie Cluttonb, Elliot Lilleyc, Kristine E. Aa. Hansend & Trond BrattelideaNorecopa, c/o Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway; bRoyal (Dick) School of Veterinary Studies, Easter Bush,Midlothian, EH25 9RG, U.K.; cResearch Animals Department, Science Group, RSPCA, Wilberforce Way, Southwater, Horsham, West Sussex, RH13 9RS, U.K.; dSection of Experimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, P.O. Box 8146 Dep., 0033 Oslo, Norway; eDivision for Research Management and External Funding, Western Norway University of Applied Sciences, 5020 Bergen, Norway.PREPARE1 consists of planning guidelines which are complementary to reporting guidelines such as ARRIVE2.PREPARE covers the three broad areas which determine the quality of the preparation for animal studies:1. Formulation of the study2. Dialogue between scientists and the animal facility3. Quality control of the components in the studyThe topics will not always be addressed in the order in which they are presented here, and some topics overlap. The PREPARE checklist can be adapted to meet special needs, such as field studies. PREPARE includes guidance on the management of animal facilities, since in-house experiments are dependent upon their quality. The full version of the guidelines is available on the Norecopa website, with links to global resources, at https://norecopa.no/PREPARE. The PREPARE guidelines are a dynamic set which will evolve as more species- and situation-specific guidelines are produced, and as best practice within Laboratory Animal Science progresses.Topic Recommendation(A) Formulation of the study2. Legal issues Consider how the research is affected by relevant legislation for animal research and other areas, e.g. animal transport, occupational health and safety.Locate relevant guidance documents (e.g. EU guidance on project evaluation).3. Ethical issues, harm-benefit assessment and humane endpointsConstruct a lay summary.In dialogue with ethics committees, consider whether statements about this type of research have already been produced.Address the 3Rs (replacement, reduction, refinement) and the 3Ss (good science, good sense, good sensibilities). Consider pre-registration and the publication of negative results.Perform a harm-benefit assessment and justify any likely animal harm.Discuss the learning objectives, if the animal use is for educational or training purposes.Allocate a severity classification to the project.Define objective, easily measurable and unequivocal humane endpoints.Discuss the justification, if any, for death as an end-point.1. Literature searchesForm a clear hypothesis, with primary and secondary outcomes.Consider the use of systematic reviews.Decide upon databases and information specialists to be consulted, and construct search terms.Assess the relevance of the species to be used, its biology and suitability to answer the experimental questions with the least suffering, and its welfare needs.Assess the reproducibility and translatability of the project.4. Experimental design and statistical analysisConsider pilot studies, statistical power and significance levels.Define the experimental unit and decide upon animal numbers.Choose methods of randomisation, prevent observer bias, and decide upon inclusion and exclusion criteria.5. Objectives and timescale, funding and division of labour7. Education and trainingArrange meetings with all relevant staff when early plans for the project exist.Construct an approximate timescale for the project, indicating the need for assistance with preparation, animal care, procedures and waste disposal/decontamination.Discuss and disclose all expected and potential costs.Construct a detailed plan for division of labour and expenses at all stages of the study.Assess the current competence of staff members and the need for further education or training prior to the study.Topic Recommendation(B) Dialogue between scientists and the animal facility(C) Quality control of the components in the study11. Quarantine and health monitoringDiscuss the animals’ likely health status, any needs for transport, quarantine and isolation, health monitoring and consequences for the personnel.15. Necropsy Construct a systema tic plan for all stages of necropsy, including location, and identification of all animals and samples.13. Experimental proceduresDevelop refined procedures for capture, immobilisation, marking, and release or rehoming.Develop refined procedures for substance administration, sampling, sedation and anaesthesia, surgery and other techniques.14. Humane killing, release, reuse or rehomingConsult relevant legislation and guidelines well in advance of the study.Define primary and emergency methods for humane killing.Assess the competence of those who may have to perform these tasks.12. Housing and husbandryAttend to the animals’ specific instincts and needs, in collaboration with expert staff.Discuss acclimatization, optimal housing conditions and procedures, environmental factors and any experimental limitations on these (e.g. food deprivation, solitary housing).9. Test substances and proceduresProvide as much information as possible about test substances.Consider the feasibility and validity of test procedures and the skills needed to perform them.10. Experimental animalsDecide upon the characteristics of the animals that are essential for the study and for reporting.Avoid generation of surplus animals.6. Facility evaluationConduct a physical inspection of the facilities, to evaluate building and equipment standards and needs.Discuss staffing levels at times of extra risk. 8. Health risks, waste disposal and decontaminationPerform a risk assessment, in collaboration with the animal facility, for all persons and animals affected directly or indirectly by the study.Assess, and if necessary produce, specific guidance for all stages of the project.Discuss means for containment, decontamination, and disposal of all items in the study.References1. Smith AJ, Clutton RE, Lilley E, Hansen KEA & Brattelid T. PREPARE:Guidelines for Planning Animal Research and Testing. Laboratory Animals, 2017, DOI: 10.1177/0023677217724823.2. Kilkenny C, Browne WJ, Cuthill IC et al. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PloS Biology, 2010; DOI: 10.1371/journal.pbio.1000412. Further informationhttps://norecopa.no/PREPARE | post@norecopa.no | @norecopaGenotypeMicrobiomeTransportSocial hierarchyAcclimationEnvironmentProceduresContingent sufferingProcedural sufferingPoor welfareVariability of responseUnreliable and invalid dataPoor replicabilityGenotypeMicrobiomeTransportContingentsufferingresponseVariabilityofresponseContingentresponseAdrian J Smith1, R Eddie Clutton2, Elliot Lilley3, Kristine EA Hansen4& Trond Brattelid51Norecopa, Oslo, Norway (adrian.smith@norecopa.no);2Royal (Dick) School of Veterinary Studies, Easter Bush, Midlothian, EH25 9RG, U.K.;3Research Animals Department, Science Group, RSPCA, Wilberforce Way, Southwater, Horsham, West Sussex, RH13 9RS, U.K. ;4Section ofExperimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of LifeSciences, P.O. Box 8146 Dep., 0033 Oslo, Norway;5Division for Research Management and External Funding, Western Norway University ofApplied Sciences, 5020 Bergen, Norway.PREPARE for better Science: guidelines for animal researchThe PREPARE Guidelines have been writtento solve this challenge. They are based onexperiences over the last 30 years inconducting and supervising preclinicalresearch.PREPARE consists of a 2-page checklist, in 20languages, and, importantly, webpages withguidance on each of the 15 topics on thechecklist. The PREPARE webpages are partof the Norecopa website, which containsover 8,500 pages of resources designed toattend to the 3Rs: Replacement, Reductionand Refinement of animal research.norecopa.no/PREPARENorecopa gratefully acknowledges financial support from the Norwegian Ministry of Agriculture and Food, Finn Rahn's legacy, Dag Stiansen’s Foundation, Laboratory Animals Ltd., the Nordic Society against Painful Animal Experiments (NSMSD), the Norwegian Society for Protection of Animals, Novo Nordisk, the Scottish Accreditation Board, the US Department of Agriculture and the Universities Federation for Animal Welfare (UFAW). Stock photos: colourbox.comGroup sizePowerBiasHARKingp-hackingThe present debate about the so-called"reproducibility crisis" within preclinical studieshas been dominated by eloquent papers focusingon the very real, but more "mathematical"issues. These are only part of the problem.To make matters worse, it is frequently stated that the solution lies in better reporting of animal studies. Anexperiment cannot be improved by describing it. Our firm belief, after many years of working within animalfacilities, is that the clue lies in closer collaboration between all parties from day one of planning. In this way,specialists in laboratory animal science, statisticians and scientists can attend to the many factors which willdetermine the outcome of the study: both its validity and the animals' welfare.We have produced a 3-minute cartoon film to illustrate this,using the aviation industry as an analogy with its impressiveability to ensure quality and reproducibility, despite variabletraffic conditions and the risk of human errors.https://norecopa.no/PREPARE/filmThe solution to this crisis must involve greater attention to theanimal-related issues and the facilities in which they are kept. Theseissues may be less obvious to those who have not worked in ananimal unit, but they constitute by far the greatest potential sourceof variation in preclinical studies.PREPAREThe PREPARE Guidelines ChecklistPlanning Research and Experimental Procedures on Animals: Recommendations for ExcellenceAdrian J. Smitha, R. Eddie Cluttonb, Elliot Lilleyc, Kristine E. Aa. Hansend & Trond BrattelideaNorecopa, c/o Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway; bRoyal (Dick) School of Veterinary Studies, Easter Bush,Midlothian, EH25 9RG, U.K.; cResearch Animals Department, Science Group, RSPCA, Wilberforce Way, Southwater, Horsham, West Sussex, RH13 9RS, U.K.; dSection of Experimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, P.O. Box 8146 Dep., 0033 Oslo, Norway; eDivision for Research Management and External Funding, Western Norway University of Applied Sciences, 5020 Bergen, Norway.PREPARE1 consists of planning guidelines which are complementary to reporting guidelines such as ARRIVE2.PREPARE covers the three broad areas which determine the quality of the preparation for animal studies:1. Formulation of the study2. Dialogue between scientists and the animal facility3. Quality control of the components in the studyThe topics will not always be addressed in the order in which they are presented here, and some topics overlap. The PREPARE checklist can be adapted to meet special needs, such as field studies. PREPARE includes guidance on the management of animal facilities, since in-house experiments are dependent upon their quality. The full version of the guidelines is available on the Norecopa website, with links to global resources, at https://norecopa.no/PREPARE. The PREPARE guidelines are a dynamic set which will evolve as more species- and situation-specific guidelines are produced, and as best practice within Laboratory Animal Science progresses.Topic Recommendation(A) Formulation of the study2. Legal issues Consider how the research is affected by relevant legislation for animal research and other areas, e.g. animal transport, occupational health and safety.Locate relevant guidance documents (e.g. EU guidance on project evaluation).3. Ethical issues, harm-benefit assessment and humane endpointsConstruct a lay summary.In dialogue with ethics committees, consider whether statements about this type of research have already been produced.Address the 3Rs (replacement, reduction, refinement) and the 3Ss (good science, good sense, good sensibilities). Consider pre-registration and the publication of negative results.Perform a harm-benefit assessment and justify any likely animal harm.Discuss the learning objectives, if the animal use is for educational or training purposes.Allocate a severity classification to the project.Define objective, easily measurable and unequivocal humane endpoints.Discuss the justification, if any, for death as an end-point.1. Literature searchesForm a clear hypothesis, with primary and secondary outcomes.Consider the use of systematic reviews.Decide upon databases and information specialists to be consulted, and construct search terms.Assess the relevance of the species to be used, its biology and suitability to answer the experimental questions with the least suffering, and its welfare needs.Assess the reproducibility and translatability of the project.4. Experimental design and statistical analysisConsider pilot studies, statistical power and significance levels.Define the experimental unit and decide upon animal numbers.Choose methods of randomisation, prevent observer bias, and decide upon inclusion and exclusion criteria.5. Objectives and timescale, funding and division of labour7. Education and trainingArrange meetings with all relevant staff when early plans for the project exist.Construct an approximate timescale for the project, indicating the need for assistance with preparation, animal care, procedures and waste disposal/decontamination.Discuss and disclose all expected and potential costs.Construct a detailed plan for division of labour and expenses at all stages of the study.Assess the current competence of staff members and the need for further education or training prior to the study.Topic Recommendation(B) Dialogue between scientists and the animal facility(C) Quality control of the components in the study11. Quarantine and health monitoringDiscuss the animals’ likely health status, any needs for transport, quarantine and isolation, health monitoring and consequences for the personnel.15. Necropsy Construct a systematic plan for all stages of necropsy, including location, and identification of all animals and samples.13. Experimental proceduresDevelop refined procedures for capture, immobilisation, marking, and release or rehoming.Develop refined procedures for substance administration, sampling, sedation and anaesthesia, surgery and other techniques.14. Humane killing, release, reuse or rehomingConsult relevant legislation and guidelines well in advance of the study.Define primary and emergency methods for humane killing.Assess the competence of those who may have to perform these tasks.12. Housing and husbandryAttend to the animals’ specific instincts and needs, in collaboration with expert staff.Discuss acclimatization, optimal housing conditions and procedures, environmental factors and any experimental limitations on these (e.g. food deprivation, solitary housing).9. Test substances and proceduresProvide as much information as possible about test substances.Consider the feasibility and validity of test procedures and the skills needed to perform them.10. Experimental animalsDecide upon the characteristics of the animals that are essential for the study and for reporting.Avoid generation of surplus animals.6. Facility evaluationConduct a physical inspection of the facilities, to evaluate building and equipment standards and needs.Discuss staffing levels at times of extra risk. 8. Health risks, waste disposal and decontaminationPerform a risk assessment, in collaboration with the animal facility, for all persons and animals affected directly or indirectly by the study.Assess, and if necessary produce, specific guidance for all stages of the project.Discuss means for containment, decontamination, and disposal of all items in the study.References1. Smith AJ, Clutton RE, Lilley E, Hansen KEA & Brattelid T. PREPARE:Guidelines for Planning Animal Research and Testing. Laboratory Animals, 2017, DOI: 10.1177/0023677217724823.2. Kilkenny C, Browne WJ, Cuthill IC et al. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PloS Biology, 2010; DOI: 10.1371/journal.pbio.1000412. Further informationhttps://norecopa.no/PREPARE | post@norecopa.no | @norecopaGenotypeMicrobiomeTransportSocial hierarchyAcclimationEnvironmentProceduresContingent sufferingProcedural sufferingPoor welfareVariability of responseUnreliable and invalid dataPoor replicabilityAnimal Technology and Welfare August 2021

169August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfarePREPARE consists of a 2-page checklist, in 20 languages and importantly, webpages with guidance on each of the 15 topics on the checklist. The PREPARE webpages are part of the Norecopa website, which contains over 8,500 pages of resources designed to attend to the 3Rs: Replacement, Reduction and Refi nement of animal research.norecopa.no/PREPARE4 collaboration between all parties from day one of planning. In this way, specialists in laboratory animal science, statisticians and scientists can attend to the many factors which will determine the outcome of the study: both its validity and the animals' welfare.The PREPARE Guidelines have been written to solve this challenge. They are based on experiences over the last 30 years in conducting and supervising preclinical research.PREPARE consists of a 2-page checklist, in 20 languages, and, importantly, webpages with guidance on each of the 15 topics on thechecklist. The PREPARE webpages are part of the Norecopa website, which contains over 8,500 pages of resources designed toattend to the 3Rs: Replacement, Reduction and Refinement of animal research.norecopa.no/PREPAREFigure 1. PREPARE checklist. We have produced a 3-minute cartoon fi lm to illustrate this, using the aviation industry as an analogy with its impressive ability to ensure quality and reproducibility, despite variable traffi c conditions and the risk of human errors.5 Figure 1. PREPARE checklist We have produced a 3-minute cartoon film to illustrate this, using the aviation industry as an analogy with its impressive ability to ensure quality and reproducibility, despite variable traffic conditions and the risk of human errors. https://norecopa.no/PREPARE/filmAcknowledgementsThe PREPARE checklist is published with permission from: Smith AJ, Clutton RE, Lilley E, Hansen KEA Andersona & Brattelid T (2018): PREPARE: Guidelines for planning animal research and testing. Laboratory Animals, 52(2): 135-141. doi: 10.1177/0023677217724823.https://norecopa.no/PREPARE/fi lmPoster PresentationsPREPAREThe PREPARE Guidelines ChecklistPlanning Research and Experimental Procedures on Animals: Recommendations for ExcellenceAdrian J. Smitha, R. Eddie Cluttonb, Elliot Lilleyc, Kristine E. Aa. Hansend & Trond BrattelideaNorecopa, c/o Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway; bRoyal (Dick) School of Veterinary Studies, Easter Bush, Midlothian, EH25 9RG, U.K.; cResearch Animals Department, Science Group, RSPCA, Wilberforce Way, Southwater, Horsham, West Sussex, RH13 9RS, U.K.; dSection of Experimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, P.O. Box 8146 Dep., 0033 Oslo, Norway; eDivision for Research Management and External Funding, Western Norway University of Applied Sciences, 5020 Bergen, Norway.PREPARE1 consists of planning guidelines which are complementary to reporting guidelines such as ARRIVE2.PREPARE covers the three broad areas which determine the quality of the preparation for animal studies: 1. Formulation of the study 2. Dialogue between scientists and the animal facility 3. Quality control of the components in the studyThe topics will not always be addressed in the order in which they are presented here, and some topics overlap. The PREPARE checklist can be adapted to meet special needs, such as field studies. PREPARE includes guidance on the management of animal facilities, since in-house experiments are dependent upon their quality. The full version of the guidelines is available on the Norecopa website, with links to global resources, at https://norecopa.no/PREPARE. The PREPARE guidelines are a dynamic set which will evolve as more species- and situation-specific guidelines are produced, and as best practice within Laboratory Animal Science progresses.Topic Recommendation(A) Formulation of the study2. Legal issues Consider how the research is affected by relevant legislation for animal research and other areas, e.g. animal transport, occupational health and safety.Locate relevant guidance documents (e.g. EU guidance on project evaluation).3. Ethical issues, harm-benefit assessment and humane endpointsConstruct a lay summary.In dialogue with ethics committees, consider whether statements about this type of research have already been produced.Address the 3Rs (replacement, reduction, refinement) and the 3Ss (good science, good sense, good sensibilities). Consider pre-registration and the publication of negative results.Perform a harm-benefit assessment and justify any likely animal harm.Discuss the learning objectives, if the animal use is for educational or training purposes.Allocate a severity classification to the project.Define objective, easily measurable and unequivocal humane endpoints.Discuss the justification, if any, for death as an end-point.1. Literature searchesForm a clear hypothesis, with primary and secondary outcomes.Consider the use of systematic reviews.Decide upon databases and information specialists to be consulted, and construct search terms.Assess the relevance of the species to be used, its biology and suitability to answer the experimental questions with the least suffering, and its welfare needs.Assess the reproducibility and translatability of the project.4. Experimental design and statistical analysisConsider pilot studies, statistical power and significance levels.Define the experimental unit and decide upon animal numbers.Choose methods of randomisation, prevent observer bias, and decide upon inclusion and exclusion criteria.5. Objectives and timescale, funding and division of labour7. Education and trainingArrange meetings with all relevant staff when early plans for the project exist.Construct an approximate timescale for the project, indicating the need for assistance with preparation, animal care, procedures and waste disposal/decontamination.Discuss and disclose all expected and potential costs.Construct a detailed plan for division of labour and expenses at all stages of the study.Assess the current competence of staff members and the need for further education or training prior to the study.Topic Recommendation(B) Dialogue between scientists and the animal facility(C) Quality control of the components in the study11. Quarantine and health monitoringDiscuss the animals’ likely health status, any needs for transport, quarantine and isolation, health monitoring and consequences for the personnel.15. Necropsy Construct a systematic plan for all stages of necropsy, including location, and identification of all animals and samples.13. Experimental proceduresDevelop refined procedures for capture, immobilisation, marking, and release or rehoming.Develop refined procedures for substance administration, sampling, sedation and anaesthesia, surgery and other techniques.14. Humane killing, release, reuse or rehomingConsult relevant legislation and guidelines well in advance of the study.Define primary and emergency methods for humane killing.Assess the competence of those who may have to perform these tasks.12. Housing and husbandryAttend to the animals’ specific instincts and needs, in collaboration with expert staff.Discuss acclimatization, optimal housing conditions and procedures, environmental factors and any experimental limitations on these (e.g. food deprivation, solitary housing).9. Test substances and proceduresProvide as much information as possible about test substances.Consider the feasibility and validity of test procedures and the skills needed to perform them.10. Experimental animalsDecide upon the characteristics of the animals that are essential for the study and for reporting.Avoid generation of surplus animals.6. Facility evaluationConduct a physical inspection of the facilities, to evaluate building and equipment standards and needs.Discuss staffing levels at times of extra risk. 8. Health risks, waste disposal and decontaminationPerform a risk assessment, in collaboration with the animal facility, for all persons and animals affected directly or indirectly by the study.Assess, and if necessary produce, specific guidance for all stages of the project.Discuss means for containment, decontamination, and disposal of all items in the study.References1. Smith AJ, Clutton RE, Lilley E, Hansen KEA & Brattelid T. PREPARE:Guidelines for Planning Animal Research and Testing. Laboratory Animals, 2017, DOI: 10.1177/0023677217724823.2. Kilkenny C, Browne WJ, Cuthill IC et al. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PloS Biology, 2010; DOI: 10.1371/journal.pbio.1000412. Further informationhttps://norecopa.no/PREPARE | post@norecopa.no | @norecopa

170Animal Technology and Welfare August 2020College of LaboratoryAnimal Science & TechnologyAnimal Law &WelfareExplore the legislation and ethical principles governing the use of animals in science, essential knowledge for all existing and prospective NACWOs.Biological ScienceExplore the principles of animal anatomy and physiology, and learn how to apply this knowledge to improve animal welfare and scientic research.Disease Recognition& ControlDiscover how the mammalian body defends itself against disease and how to utilise those defences for experimental and husbandry purposes.Genetic AlterationTechnologiesAll you need to know about breeding. Maintaining and using GA animals, including the maintenance and development of specic animal models.Physiology ofPain & StressConsider the biological basis of pathological change and animal behaviour with particular reference to pain and stress.ToxicologyAn introduction to the theory, methods and regulations governing the assessment of biochemical toxicology and the role of the study director.CONTINUAL PROFESSIONAL DEVELOPMENT UNITSCLAST oers 11 dierent subjects to tailor your education to meet your specic needsAnimal FacilityManagement & DesignLearn about the process of animal facility design, construction and modication, and develop your ability to reect on management theories and strategies.SupervisoryManagement SkillsAn introduction to supervisory management within an animal facility, focusing on the legislative responsibilities and management principles needed in the workplace.ExperimentalDesignAn introduction to the principles of good experimental design and reporting. Develop your skills in eective research, review and analysis.Applied Learning& DevelopmentDevelop your skills in reection, research and the creation of eective plans. Particularly useful for existing or prospective NACWOs, NTCOs and NIOs.Project Planning& ProjectResearch, review, analyse and debate current scientic theories, and learn how to manage a project eectively through your chosen research topic.ACADEMIC SKILLSMANAGEMENT SKILLSANIMAL HEALTH & WELFAREATW_CLAST_426x303.indd 1 29/06/2020 11:39

171August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareCollege of LaboratoryAnimal Science & TechnologyAnimal Law &WelfareExplore the legislation and ethical principles governing the use of animals in science, essential knowledge for all existing and prospective NACWOs.Biological ScienceExplore the principles of animal anatomy and physiology, and learn how to apply this knowledge to improve animal welfare and scientic research.Disease Recognition& ControlDiscover how the mammalian body defends itself against disease and how to utilise those defences for experimental and husbandry purposes.Genetic AlterationTechnologiesAll you need to know about breeding. Maintaining and using GA animals, including the maintenance and development of specic animal models.Physiology ofPain & StressConsider the biological basis of pathological change and animal behaviour with particular reference to pain and stress.ToxicologyAn introduction to the theory, methods and regulations governing the assessment of biochemical toxicology and the role of the study director.CONTINUAL PROFESSIONAL DEVELOPMENT UNITSCLAST oers 11 dierent subjects to tailor your education to meet your specic needsAnimal FacilityManagement & DesignLearn about the process of animal facility design, construction and modication, and develop your ability to reect on management theories and strategies.SupervisoryManagement SkillsAn introduction to supervisory management within an animal facility, focusing on the legislative responsibilities and management principles needed in the workplace.ExperimentalDesignAn introduction to the principles of good experimental design and reporting. Develop your skills in eective research, review and analysis.Applied Learning& DevelopmentDevelop your skills in reection, research and the creation of eective plans. Particularly useful for existing or prospective NACWOs, NTCOs and NIOs.Project Planning& ProjectResearch, review, analyse and debate current scientic theories, and learn how to manage a project eectively through your chosen research topic.ACADEMIC SKILLSMANAGEMENT SKILLSANIMAL HEALTH & WELFAREATW_CLAST_426x303.indd 1 29/06/2020 11:39

172Animal Technology and Welfare August 2020Introduction The assessment and understanding of Absorption, Distribution, Metabolism and Elimination (ADME) for new pharmaceuticals is required in regulatory submissions. Typically, ADME studies are conducted using metabolism cages designed for the single housing of animals to enable the quantitative collection of urine and faeces, normally over a 1week period. It is well documented that providing pair or group housing of social animals such as non-human primates has a signifi cant positive impact on the welfare of the animals.Charles River, Edinburgh have successfully performed many ADME studies with pair housed animals, with radioactivity recovery and plasma concentrations comparable to single housing.*As traditional single housing cages are relatively small, we designed new and larger cages with improved features. In addition to the welfare benefi ts for the animals with social housing, the refi nements enable more effi cient study conduct, dosing, blood sampling and faecal collections.This poster describes some of the advantages of the enhancements we have made.Cage design Our original metabolism cages were entirely stainless-steel construction. Whilst effective, metal is also unfortunately, cold to the touch. Trespa panels are warmer, refl ect light well and provide a better ambience to the caging. Following extensive testing we concluded that these could be used without detriment to the radioactive recovery, so we used these for the caging sides. Refi ning cages for social housing of non-human primates on ADME studiesWILLIAM ARCHIBALD and COLIN GLYNN Charles River, Elphinstone Research Centre, Tranent, East Lothian EH33 2NE UK Correspondence: william.archibald@crl.comPoster presented at IAT Virtual Congress 2021Figure 1. New metabolism cage.We made the cages as large as possible, whilst still manoeuvrable enough to go through the facility doors and fi t inside the cage-washer. The internal volume is 1.65m3 per cage.The squeeze back mechanism is incorporated into a central bar running the length of the cage, also providing an extra perch for the animals to sit on.Animal Technology and Welfare August 2021

173August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareA further, wide perch is situated at the rear of the cage. This slides through the back panel when using the squeeze back device. Both the fl oor and roof utilise bars running parallel to the sides, minimising the risk of fi nger, toes and tails getting pinched when using the squeeze back.Manipulanda is placed on the roof to provide additional stimulus for the animals. A Trespa® ‘crown’ is positioned to prevent these rolling off the cage. The crown is removable, to enable the cages to fi t in the cage washer.Refining Cages for Social Housing of Non-Human Primates on ADME StudiesWilliam Archibald, Colin Glynn3DOSING AND BLOOD SAMPLING2CAGE DESIGN1INTRODUCTION5ACKNOWLEGEMENTSThe assessment and understanding of Absorption, Distribution, Metabolism and Elimination (ADME) for new pharmaceuticals is required in regulatory submissions. Typically, ADME studies are conducted using metabolism cages designed for the single housing of animals to enable the quantitative collection of urine and faeces, normally over a 1 week period. It is well documented that providing pair or group housing of social animals such as nonhuman primates has a significant positive impact on the welfare of the animals.Charles River, Edinburgh have successfully performed many ADME studies with pair housed animals, with radioactivity recovery and plasma concentrations comparable to single housing.*As traditional single housing cages are relatively small, we designed new and larger cages with improved features. In addition to the welfare benefits for the animals with social housing, the refinements enable more efficient study conduct, dosing, blood sampling and faecal collections.This poster describes some of the advantages of the enhancements we have made.Our original metabolism cages were entirely stainless steel construction. Whilst effective, metal is also, unfortunately, cold to the touch. Trespa panels are warmer, reflect light well and provide a better ambience to the caging. Following extensive testing we concluded that these could be used without detriment to the radioactive recovery, so we used these for the caging sides. We made the cages as large as possible, whilst still manoeuvrable enough to go through the facility doors and fit inside the cage-washer. The internal volume is 1.65m3 per cage.The squeeze back mechanism is incorporated into a central bar running the length of the cage, also providing an extra perch for the animals to sit on.A further, wide perch is situated at the rear of the cage, this slides through the back panel when using the squeeze back device. Both the floor and roof utilise bars running parallel to the sides, minimise the risk of finger, toes and tails getting pinched when using the squeeze back.Manipulanda is placed on the roof to provide additional stimulus for the animals. A Trespa ‘crown’ is positioned to prevent these rolling off the cage. The crown is removable, to enable the cages to fit in the cage washer. The cage doors have a combination of vertical and horizontal bars. Using well established positive reinforcement training techniques, animals will present an arm through the vertical bars. This enables comfortable restraint on the door for dosing. Many intravenous, intramuscular and subcutaneous administrations can be performed without requiring removal from the cage.Blood samples can be taken from the cephalic veins. Alternatively, legs can be presented through the horizontal bars, enabling femoral vein sampling.*Assessment of Social Housing of Non-Human Primates on Excretion Mass Balance Studies: Charles River poster, Karen Stevenson, Colin GlynnNew cages designed and built in collaboration with UNO BV, Netherlands4FLEXIBILITYThe cages incorporate connecting hatches enabling several cages to be combined together. This modular approach facilitates a variety of uses in addition to ADME studies, for example; use as recovery housing following surgery.Figure 2. New metabolism cage showing perches. Figure 3. New metabolism showing cage roof manipulanda. Dosing and blood sampling The cage doors have a combination of vertical and horizontal bars. Using well-established positive reinforcement training techniques, animals will present an arm through the vertical bars. This enables comfortable restraint on the door for dosing. Many intravenous, intramuscular and subcutaneous administrations can be performed without requiring removal from the cage. Blood samples can be taken from the cephalic veins. Alternatively, legs can be presented through the horizontal bars, enabling femoral vein sampling.Figure 4. New metabolism cage showing cephalic sampling. Poster Presentations

174Animal Technology and Welfare August 2020Figure 5. New metabolism cage showing femoral sampling. Flexibility The cages incorporate connecting hatches enabling several cages to be combined together. This modular approach facilitates a variety of uses in addition to ADME studies, for example, use as recovery housing following surgery.Figure 6. New metabolism cage showing two cages combined. Acknowledgements *Assessment of Social Housing of Non-Human Primates on Excretion Mass Balance Studies: Charles River poster, Karen Stevenson, Colin Glynn.New cages designed and built in collaboration with UNO BV, Netherlands.Poster Presentations

175August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareBackground and additional informationRegulatory guidelines for relative humidity (RH) when housing laboratory rodents are 55±10%. These were set based on quite limited data and little is known on how fl uctuation in RH affects laboratory rodents or how high versus low RH, within these boundaries affects them. Studies through time have shown that RH has an impact on rodent health.Controlling humidity – improved breeding and validity of researchKAREN EKKELUND PETERSEN SCANBUR A/S, Silovej16-18, 2690 Karlslunde, Denmark Correspondence: kep@scanbur.comPoster displayed at IAT Virtual Congress 2021Figure 1. Relative humidity has been found to have effects on breeding, food consumption, behaviour and microorganisms.1-6We aim to investigate the effect of tighter control of RH on health and physiology of research animals. For example, by investigating the effect on breeding performance. Additionally, the potential of uncontrolled RH to affect outcomes of studies and thus validity of research, are part of our studies as well. We specifi cally look at RH controlled accurately at 55% or above.How relative humidity affects laboratory rodents is summarised below and in Figure 1.– Puberty is delayed in female mice housed under 15-30% RH, whereas fi rst oestrus was attained earlier when housed under a RH of 75%.1– At 35% RH compared to 75% rats consume 5% more food.2– Low RH has shown to increase activity in mice.2– RH impacts growth conditions for bacteria and fungus and transmission of virus3-6ConclusionThe literature and the results we have found support that RH affects study results, animal physiology and our results show that tighter control of RH can improve murine breeding performance signifi cantly. Further investigation is warranted on how different levels of stable RH versus variation in humidity affect the breeding performance.Studies are ongoing to further investigate the effect of RH on breeding performance, embryo transfer success rate, aggression of male mice, skin health, etc. The anecdotal feedback from existing installations is investigated further to accept or reject hypotheses of effects of RH on animals and research results.August 2021 Animal Technology and Welfare

176Animal Technology and Welfare August 2020References1 Drickamer, L.C. (1990). Environmental factors and age of puberty in female house mice. Developmental psychobiology 23,63-73, doi:10.1002/dev. 42023 0107 (1990)2 Clough, G. (1982). Environmental effects on animals used in biomedical research. Biological reviews of the Cambridge Philosophical Society 57, 487-523 (1982)3 Arundel, A.V., Sterling, E.M., Biggin, J.H., Sterling, T.D. (1986). Indirect health effects of relative humidity in indoor environments. Environ Health Perspect 65, 351-361, doi:10.1289/ehp.8665351 (1986)4 Alsmo, T., Alsmo, C. (2014). Ventilation and Relative Humidity in Swedish Buildings. Journal of Environmental Protection 05,1022-1036, doi: 10.4236/ jep.2014.511102 (2014)5 Lowen, A.C., Mubareka, S., Steel, J. Palese, P. (2007). Influenza virus transmission is dependent on relative humidity and temperature. PLoS pathogens 3,1470-1476,doi:10.1371/journal.ppat.0030151 (2007).6 Van Der Veen, J., Poort, Y., Birchfield, D.J. (1972). Effect of Relative Humidity on Experimental Transmission of Sendai Virus in Mice. Proceedings of the Society for Experimental Biology and Medicine 140,1437-1440,doi:10.3181/00379727-140-36691 (1972).Results from studies with control of RH above 55%Complete studies: Preliminary findings:– Less male aggressive behaviour.– An indication of higher success rate of embryo transfer.– Significantly lower water intake. Find a detailed description here.– Significantly higher number of pups per birth and lower number of total litters lost. Find a detailed description here.Poster Presentations

177August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareIntroductionThere are several items we often use in animal facilities that when fi nished with are thrown away. However many of these items come in packaging that can be recycled as enrichment for our animals, particularly facilities housing primates.The use of recycled packaging as enrichment for primatesis especially useful here in the UK because there are alimited number of suppliers of laboratory primate enrichment with a small range of variation in the primateenrichment available. In addition, the recycling of materials such as plastic and cardboard for use as enrichment, along with supplier bought products, is a great way of enriching the lives of the monkeys we work with.The ideas we have found to be most successful are when enrichment is used in novel and more challenging ways of presenting the monkeys with food.In this presentation these are a number of enrichment ideas using a range of common materials found in animal facilities, that we at Cambridge have made and used over the previous year or so with our macaques and we hope may provide other facilities with ideas on how to recycle such materials for use as enrichment.Plastic containers Plastic containers of various sizes and shapes can be used to hide forage hidden amongst substrate and/or hung from the cage top. The primates can then reach into the containers through the openings at the top, or alternatively holes of differing sizes and shapes can be made in the sides or bottoms of the containers, ensuring that any edges are fi led down to remove any sharp areas which may be potentially dangerous.We use empty disinfectant or ethanol containers, ensuringthat they are thoroughly rinsed out and left to dry prior to being used.Laboratory primate enrichment ideasJOE PEPLOE and CHRIS MACAULAY University of Cambridge, UBS – Joint Animal Facility (Anatomy Building), Downing Site, Cambridge CB2 3DY UKCorrespondence: Jp725@cam.ac.uk Based on a poster displayed at the IAT Virtual Congress 2021Figure 1. Typical examples of redundant plastic containers.3 Plastic containersFigure 1. Typical examples of redundant plastic containers.Plastic containers of various sizes and shapes can be used to hide forage hidden amongst substrate and/or hung from the cage top. The primates can then reach into the containers through the openings at the top, or alternatively holes of differing sizes and shapes can be made in the sides or bottoms of the containers, ensuring that any edges are filed down to remove any sharp areas which may be potentially dangerous.We use empty disinfectant or ethanol containers, ensuring that they are thoroughly rinsed out and left to dry prior to being used.Figure 2. Monkeys investigating enrichment.4 Figure 2. Monkeys investigating enrichment. 4 Figure 2. Monkeys investigating enrichment. August 2021 Animal Technology and Welfare

178Animal Technology and Welfare August 2020As well as simply being hung from the cage top as they are, plastic containers can be used to make more complex enrichment. Below are a few ideas for enrichment using plastic containers that we have found to be successful.Spin the bottleA container attached to a broken trolley piece, (or any similarly shaped item) and hung from the cage top with padlocks. Forage is placed inside the container. The monkeys need to spin the container on the trolley piece so that the forage falls out and onto the cage fl oor when the container is spun upside down. Once spun, the container will return to its upright position when the monkey lets go.Figure 3. Bottle adapted for ‘spin the bottle’.Below you can see Yaa peering into the container to see if the forage inside is worth spinning the bottle for.5 As well as simply being hung from the cage top as they are, plastic containers can be used to make more complex enrichment. Below are a few ideas for enrichment using plastic containers that we have found to be successful.Spin the bottleA container attached to a broken trolley piece, (or any similarly shaped item) and hung from the cage top with padlocks. Forage is placed inside the container. The monkeys need to spin the container on the trolley piece so that the forage falls out and onto the cage floor when the container is spun upside down. Once spun, the container will return to its upright position when the monkey lets go.Figure 4. To spin or not, Yaa trying to decide. 6 Figure 3. Bottle adapted for ‘spin the bottle. ’ Below you can see Yaa peering into the container to see if the forage inside is worth spinning the bottle for.Figure 4. To spin or not, Yaa trying to decide. Barrel of laughsA plastic bottle with a hole cut out on one side and a smaller hole on the bottom for a metal rod to run through. It is hung from the cage top with a chain that runs through the metal rod. Forage is then placed inside the bottle where the monkey can rotate the bottle around the rod until the hole is facing downwards so the forage falls onto the cage fl oor.As you can see, Athos is having a barrel of laughs trying to get his hands on some peanuts.Figure 5. Plastic bottle adapted for ‘barrel of laughs’ enrichment.7 Barrel of laughsFigure 5. Plastic bottle adapted for ‘barrel of laugh’ enrichment. A plastic bottle with a hole cut out on one side, and a smaller hole on the bottom for a metal rod to run through. It is hung from the cage top with a chain that runs through the metal rod. Forage is then placed inside the bottle, where the monkey can rotate the bottle around the rod until the hole is facing downwards so the forage falls onto the cage floor.As you can see, Athos is having a barrel of laughs trying to get his hands on some peanuts.Figure 6. Athos on a peanut hunt.8 Figure 6. Athos on a peanut hunt. KerplunkFigure 7. Plastic bottle ready for ‘Kerplunk’ KerplunkFigure 7. Plastic bottle ready for ‘Kerplunk’.8 Figure 6. Athos on a peanut hunt. KerplunkFigure 7. Plastic bottle ready for ‘Kerplunk’ Poster Presentations

179August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareA plastic bottle with slits cut out on both sides and a hole cut out of the bottom. Cardboard shelves are cut and inserted through the slits. Forage is then placed on the top shelf, and in order to get the food the monkeys need to remove the shelves by pulling them out so the forage gradually falls to the bottom of the bottle and through the hole to the cage fl oor.The RattleA plastic container cut in half, with a plastic ball with holes (available from most enrichment retailers) placed inside. Forage can be placed inside the ball and the monkeys can then shake the container to cause the forage to fall out of the ball, and then out of the open lid of the container. Alternatively the monkey can pull the container parts apart to access the ball.Figure 8. Wurzle is demonstrating the kerplunk by removing the shelves in order.9 A plastic bottle with slits cut out on both sides and a hole cut out of the bottom. Cardboard shelves are cut and inserted through the slits. Forage is then placed on the top shelf, and in order to get the food the monkeys need to remove the shelves by pulling them out so the forage gradually falls to the bottom of the bottle and through the hole to the cage floor.Figure 8. Wurzle is demonstrating the kerplunk by removing the shelves in order. Figure 9. Components of a rattle. 10The RattleA plastic container cut in half, with a plastic ball with holes (available from most enrichment retailers) placed inside. Forage can be placedinside the ball, and the monkeys can then shake the container to cause the forage to fall out of the ball, and then out of the open lid of the container. Alternatively the monkey can pull the container parts apart to access the ball. Figure 9. Components of a rattle. The HulkTwo plastic containers of different sizes but the same shape, both cut in half. The halves then fi tted together as in the image.Figure 10. Athos trying to work out the intricacies of the rattle.11Figure 10. Athos trying to work out the intricacies of the rattle.The HulkTwo plastic containers of different sizes, but the same shape, both cut in half. The halves then fitted together as in the image. Figure 11. Different sized container halves. Figure 11. Different sized container halves.11Figure 10. Athos trying to work out the intricacies of the rattle.The HulkTwo plastic containers of different sizes, but the same shape, both cut in half. The halves then fitted together as in the image. Figure 11. Different sized container halves. The smaller container is then placed inside the larger one and hung from the cage top with a chain running through both. Forage is placed inside the smaller container and the monkeys need to pull apart both containers to access the forage.Figure 12. Assembled ‘hulk’ enrichment. 12The smaller container is then placed inside the larger one and hung from the cage top with a chain running through both. Forage is placed inside the smaller container and the monkeys need to pull apart both containers to access the forage.Figure 12. Assembled ‘hulk’ enrichment. Figures 13 -14. Athos and Aragorn showing off their super-macaque strength.Poster Presentations

180Animal Technology and Welfare August 2020Concealed CapA plastic tube with the bottom cut off, with a container cap placed upside down inside, with a chain running through both the cap and tube.Figure 13-14. Athos and Aragorn showing off their super-macaque strength.12The smaller container is then placed inside the larger one and hung from the cage top with a chain running through both. Forage is placed inside the smaller container and the monkeys need to pull apart both containers to access the forage.Figure 12. Assembled ‘hulk’ enrichment. Figures 13 -14. Athos and Aragorn showing off their super-macaque strength.12The smaller container is then placed inside the larger one and hung from the cage top with a chain running through both. Forage is placed inside the smaller container and the monkeys need to pull apart both containers to access the forage.Figure 12. Assembled ‘hulk’ enrichment. Figures 13 -14. Athos and Aragorn showing off their super-macaque strength.A handful of forage can be hidden inside the cap, which when hung from the cage top is concealed by the tube. The monkey must then lift the tube to reveal the forage.Figure 15-16. The ‘concealed cap’ enrichment. 13Concealed CapA plastic tube with the bottom cut off, with a container cap placed upside down inside, with a chain running through both the cap and tube.Figures 15-16. The ‘concealed cap enrichment. A handful of forage can be hidden inside the cap, which when hung from the cage top is concealed by the tube. The monkey must then lift the tube to reveal the forage.13Concealed CapA plastic tube with the bottom cut off, with a container cap placed upside down inside, with a chain running through both the cap and tube.Figures 15-16. The ‘concealed cap enrichment. A handful of forage can be hidden inside the cap, which when hung from the cage top is concealed by the tube. The monkey must then lift the tube to reveal the forage.Figure 17. Ulysses is trying to fi gure out where his breakfast is.14Figure 17. Ulysses is trying to figure out where his breakfast is.The cap-tivatorThe caps from plastic containers are placed on a chain, through holes made in the centre of them. The caps should be positioned with one upside down on top of one facing upwards, so that when forage is placed in the bottom cap it is hidden by the one above. The monkeysthen need to slide the upper cap along the chain to reveal the forage.The cap-tivatorThe caps from plastic containers are placed on a chain, through holes made in the centre of them. The caps should be positioned with one upside down on top of one facing upwards, so that when forage is placed in the bottom cap it is hidden by the one above. The monkeys then need to slide the upper cap along the chain to reveal the forage.Figure 18. The ‘cap-tivator’. 15Figure 18. The cap-tivator Figure 19. Athos utterly captivated by the cap-tivator. Poster Presentations

181August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareThe hangoutThree plastic containers with holes cut out, each fi lled with substrate with forage hidden amongst it and hung from the cage top by a chain running through. Cardboard tubes are placed between the containers to stop them from moving along the chain.Figure 19. Athos utterly captivated by the ‘cap-tivator’.15Figure 18. The cap-tivator Figure 19. Athos utterly captivated by the cap-tivator. Figure 20. The hangout.16The hangoutThree plastic containers with holes cut out, each filled with substrate with forage hidden amongst it, and hung from the cage top by a chain running through. Cardboard tubes are placed between the containers to stop them from moving along the chain.Figure 20. The hangout This is particularly useful for group housed monkeys as it enables each monkey to feed at the same time and prevents the dominant monkey from being able to monopolise all of the forage.This is particularly useful for group housed monkeys as it enables each monkey to feed at the same time and prevents the dominant monkey from being able to monopolise all of the forage.Cardboard As well as plastic containers, cardboard is another material commonly found in the animal facility. Most products ordered are delivered in cardboard boxes including gloves and face masks. We also get our fruit delivered in a cardboard ‘tray’ like the one shown above. These boxes can be put into the enclosures as they are for the monkeys to destroy or they can be fi lled with forage hidden amongst substrate.Figure 21-22. Yaa and Athos can be seen hanging out.17Figure 21-22. Yaa and Athos can be seen hanging out. Cardboard As well as plastic containers, cardboard is another material commonly found in the animal facility. Most products ordered are delivered in cardboard boxes, including gloves and face masks. We also get our fruit delivered in a cardboard ‘tray’ like the one shownabove. These boxes can be put into the enclosures as they are for the monkeys to destroy or they can be filled with forage hidden amongst substrate.17Figure 21-22. Yaa and Athos can be seen hanging out. Cardboard As well as plastic containers, cardboard is another material commonly found in the animal facility. Most products ordered are delivered in cardboard boxes, including gloves and face masks. We also get our fruit delivered in a cardboard ‘tray’ like the one shownabove. These boxes can be put into the enclosures as they are for the monkeys to destroy or they can be filled with forage hidden amongst substrate.Figure 23. Examples of cardboard used as enrichment.18Figure 23. Examples of cardboard used as enrichment.Cardboard can also be used in different ways to provide slightly more complex and challenging enrichment for the monkeys. Cardboard boxes can also be hung from the cage top to allow the monkeys to sit or forage inside them. Figures 24 and 25demonstrate two of the ideas we have used.18Figure 23. Examples of cardboard used as enrichment.Cardboard can also be used in different ways to provide slightly more complex and challenging enrichment for the monkeys. Cardboard boxes can also be hung from the cage top to allow the monkeys to sit or forage inside them. Figures 24 and 25demonstrate two of the ideas we have used.Cardboard can also be used in different ways to provide slightly more complex and challenging enrichment for the monkeys. Cardboard boxes can also be hung from the cage top to allow the monkeys to sit or forage inside them. Figures 24 and 25 demonstrate two of the ideas we have used.Poster Presentations

182Animal Technology and Welfare August 2020Pass the parcelFigure 24-25. Macques using the enrichment.19Figures 24-25. Macques using the enrichment.Pass the parcel19Figures 24-25. Macques using the enrichment.Pass the parcelFigure 26. Examples of boxes used for pass the parcel.19Figures 24-25. Macques using the enrichment.Pass the parcelCardboard boxes of different sizes can be placed inside one another, with the forage hidden amongst substrate within the smallest box. The monkeys then need to work their way through each of the boxes in order to get to the smallest box where they can then access the forage.Figure 27-28. Tigger and Yaa not passing the parcel.20Figure 26. Examples of boxes used for pass the parcel. Cardboard boxes of different sizes can be placed inside one another, with the forage hidden amongst substrate within the smallest box. The monkeys then need to work their way through each of the boxes in order to get to the smallest box where they can then access the forage.Figures 27-28. Tigger and Yaa not passing the parcel. The cuppaIn a similar fashion to the cap-tivator, cardboard cups from water dispensers can be used, by cutting holes in the bottom and beingplaced on a chain hung from the cage top. Forage can then be 20Figure 26. Examples of boxes used for pass the parcel. Cardboard boxes of different sizes can be placed inside one another, with the forage hidden amongst substrate within the smallest box. The monkeys then need to work their way through each of the boxes in order to get to the smallest box where they can then access the forage.Figures 27-28. Tigger and Yaa not passing the parcel. The cuppaIn a similar fashion to the cap-tivator, cardboard cups from water dispensers can be used, by cutting holes in the bottom and beingplaced on a chain hung from the cage top. Forage can then be The ‘cuppa’In a similar fashion to the ‘cap-tivator’, cardboard cups from water dispensers can be used, by cutting holes in the bottom and being placed on a chain hung from the cage top. Forage can then be placed inside the cups which the monkeys need to manipulate along the chain in order to obtain the forage.Poster Presentations

183August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareConclusionBy recycling materials commonly found in the facility as enrichment, we have been able to provide our monkeys with novel and challenging ways of presenting food.We have found that by doing so, together with feeding a wider variety of forage types, the monkeys have been more active following feeding and have anticipated feeding times with more excitement.We have also found that the monkey’s interest is maintained for longer periods of time than when forage is scattered onto the cage fl oor, as it requires more effort to reach.In group housed monkeys, by using a number of different foraging enrichment spread throughout the enclosure, it has given the subordinate monkeys more chance of getting an equal share of food.Figure 29-30. The ‘cuppa’ and Wicket is enjoying a morningcuppa.21placed inside the cups which the monkeys need to manipulate the along the chain in order to obtain the forage. Figures 29-30. The cuppa and Wicket is enjoying a morning cuppa. ConclusionBy recycling materials commonly found in the facility as enrichment, we have been able to provide our monkeys with novel and challenging ways of presenting food.We have found that by doing so, together with feeding a wider variety of forage types, the monkeys have been more active following feeding and have anticipated feeding times with more excitement.We have also found that the monkey’s interest is maintained for longer periods of time than when forage is scattered onto the cage floor, as it requires more effort to reach.21placed inside the cups which the monkeys need to manipulate the along the chain in order to obtain the forage. Figures 29-30. The cuppa and Wicket is enjoying a morning cuppa. ConclusionBy recycling materials commonly found in the facility as enrichment, we have been able to provide our monkeys with novel and challenging ways of presenting food.We have found that by doing so, together with feeding a wider variety of forage types, the monkeys have been more active following feeding and have anticipated feeding times with more excitement.We have also found that the monkey’s interest is maintained for longer periods of time than when forage is scattered onto the cage floor, as it requires more effort to reach.AcknowledgementsWith many thanks to Aragorn, Athos, Tigger, Ulysses, Wicket, Wurzle, Yaa and Yum Yum. Poster Presentations

184Animal Technology and Welfare August 2020Home cage monitoring: investing in the future JOANNA MOORE1and HILARY LANCASTER21GSK, Laboratory Animal Medicine, Medicines Research Centre, Gunnels Wood Road, Stevenage,Hertfordshire SG1 2NY UK 2 GSK, Vivo Science and Delivery, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY UK Correspondence: joanna.l.moore@gsk.comAll animal studies were ethically reviewed and carried out in accordance with Animals (Scientifi c Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals.IntroductionHome cage monitoring is not a new technique, the idea of using technology to observe animals over a 24 hour period to help staff monitor welfare and health has been around for many years. Yet there is still a long way to go before the ideal system will be available. If indeed such a system could ever exist? Several criteria need to be considered and a system that fi ts all situations may not be feasible. There are a few ‘off the shelf’ cage monitoring options available, each working in a different way to the other. Selecting the best system for a facility is not as easy as choosing cages, and that is not simple.The concept of home cage monitoringAs a result of the increased desire to integrate technology into animal facilities, most animal areas now rely heavily on computer databases for daily routines and management. It was only a matter of time before our attention turned towards the animal’s cage to see how we could use technology to help us understand the animal more. The idea of having cameras peering into animal cages and giving us feedback on their health and welfare has often been considered as the ultimate goal for care we can offer the animals.Certainly, there are benefi ts to home cage monitoring:–Ability to observe nocturnal animal activity during theiractive period, giving us a greater insight into their behaviour.– Having an alarm to indicate when an animal may need closer observation due to a change in behaviour or reduction in activity. This will lead to more refi ned humane endpoints, as we are better able to ‘see’ the reactions to novel compounds and new models. – Develop a greater understanding of the animal’s behaviour, and their interactions with the home cage environment, which may indicate whether the cage furnishings are enabling a more species specifi c behaviour. When initially determining what system will work best in a given environment, the availability of the right information enables the three key areas of focus to be addressed.4 Figure 1. Key areas of focus. Algorithms The outputs from a home-cage monitoring software rely on the algorithms of code that have been programmed into a computer to give us the answers to complex questions. But how can we be certain the algorithm, which is essentially a set of instructions for a computer, really tells us what is occurring in the cage. Can we be WelfareIncrease our understanding of the animals reactions to eventsCareReduce times cages are disturbed, with increased oversight of micro environmentScienceIncreased objective data feeds into refined experimental resultsFigure 1. Key areas of focus. Animal Technology and Welfare August 2021’

185August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareAlgorithms The outputs from a home-cage monitoring software rely on the algorithms of code that have been programmed into a computer to give us the answers to complex questions. But how can we be certain the algorithm, which is essentially a set of instructions for a computer, really tells us what is occurring in the cage. Can we be confi dent a computer interprets behaviour with a good degree of accuracy? The answer is yes but there is a proviso - the instructions we give the computer must refl ect what is happening in the cage. If they do not then the data we get will be false. Avoid asking complicated questions, for example, if you want to know if the mouse in Figure 2 remains healthy over a period of time, use a key indicator as your instruction to the computer rather than lots of key indicators. Foundation for the algorithmAlgorithms are only as good as the instructions we give them and those instructions usually come in the form of a recipe, the key ingredient of behaviour monitoring for any species is a well-designed ethogram, which is a list of defi nitions describing the functions of a behaviour to such a degree that an untrained observer will recognise it (see Table 1).It is critical that behaviour and postures are properly defi ned and consistent, maybe even across the industry for the sake of reproducibility. Otherwise the algorithms may be fl awed. This is important when the use of 5 confident a computer interprets behaviour with a good degree of accuracy? The answer is yes, but there is a proviso - the instructions we give the computer must reflect what is happening in the cage. If they do not then the data we get will be false. Avoid asking complicated questions, for example, if you want to know if the mouse in Figure 2 remains healthy over a period of time, use a key indicator as your instruction to the computer rather than lots of key indicators. Figure 2. Mouse moving around a cage. Figure 2. Mouse moving around a cage. Figure 2. Examples of defi nitions of behaviour. *Example of ethogram defi nition *(N.B –see website for full defi nitions) Exploration – Goal-directed, typically occurs in the following sequence; search, attend, approach, investigate. Investigate – Typically multi-modal, and primarily involves olfactory and tactile senses such as olfactory (sniffi ng). Sniffi ng – Rapid twitching movements of the nose, either with the nose in contact with the stimulus, or held in an elevated position in the air.camera technology is a primary part of the monitoring system, especially when each movement of the animal has been defi ned and inputted into a computer.WelfareSome justifi cation of using home cage monitoring is that it improves Animal Welfare. However, this is not strictly accurate as the presence of a camera or any beams, does have a direct impact on the wellbeing of the cage occupants. In fact they may have a detrimental effect on welfare, which the animals may sometimes express by covering the cameras up (Figure 3).Poster Presentations

186Animal Technology and Welfare August 2020Figure 3. Schematic of mouse activity cameras, mouse covering the red hut shortly after camera placement. One important piece of information it does offer, is a greater understanding of their activity and behaviour which then enables us to make changes to their care, environment and study designs that can have a positive direct impact on their welfare. However, for this to occur and improve the welfare of the animals, we need to be sure that we have asked the most appropriate questions for our science and animal care programme. CareCaring for laboratory animals includes husbandry routines and doing everything we can to meet their needs and improve their wellbeing. Home cage monitoring can address this to a degree, especially if the animal is not the sole focus. This is only possible if we use a system that monitors the elements of care that can sometimes be missed, especially in a busy unit, such as ensuring every cage has access to water and food, and that every animal is behaving as expected. Even well enriched cages may have a drawback, for example, sometimes a well enriched cage can mask incidents of lameness. Routine husbandry can cause increased anxiety to animals, especially for mice where it is commonly agreed that a reduction in the frequency cages are cleaned may improve their welfare, How frequent this should be needs to be balanced against the need to have a routine that ensures mistakes such as missing cages, are not made. Therefore 9 Figure 4; Decision tree for home cage monitoring. Home cage monitoring decision treeCan animals be group housed?Can enrichment still be used?No YesSystem can be usedJustification requiredHas the behaviour and/or activty criteria been validated?Has the system been validated for studies?System needs validatingSystem can be usedNoYesCan the system recognise changes in activity and/or behaviour?Has the system been validated for studies?Has the system been validated for studies?System needs trainingSystem can be usedYesNoDoes it include data analysis tools?Is the system easy to navigate?Does the system aid husbandry and care?Does the system work with other animal management systems?Is this the right system for your work?System can be usedYesReview optionsNoNoYesDid the training work?YesNoWas the validation successful?NoYesJustification accepted?YesNo8 Figure 3. Schematic of mouse activity cameras, mouse covering its red hut shortly after camera placement. One important piece of information it does offer, is a greater understanding of their activity and behaviour which then enables us to make changes to their care, environment and study designs that can have a positive direct impact on their welfare. However, for this to occur and improve the welfare of the animals, we need to be sure that we have asked the most appropriate questions for our science and animal care program. 8 Figure 3. Schematic of mouse activity cameras, mouse covering its red hut shortly after camera placement. One important piece of information it does offer, is a greater understanding of their activity and behaviour which then enables us to make changes to their care, environment and study designs that can have a positive direct impact on their welfare. However, for this to occur and improve the welfare of the animals, we need to be sure that we have asked the most appropriate questions for our science and animal care program. Figure 4. Decision tree for home cage monitoring. Poster Presentations

187August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and Welfarethese factors are an important element of caring for the animals and a system that enables us to improve this will, indirectly, benefi t the welfare of the animals. Imagine a situation where you can view two cages. Cage 1has an activity monitor only and Cage 2 has a camera system. Each system alerts you that there is a problem in a cage. Which cage would you check? If the answer is both, then is a complex camera system necessary for your home cage monitoring? ScienceWhen we have a greater understanding of the behaviour of animals we can design protocols and studies that fi t around the animal to a greater degree than we are currently able to. A system that can give us an objective answer for activity patterns after cage disturbances such as husbandry procedures or a regulated procedure, or a reduction in activity due to an intervention or model design, may enable us to design studies where we can ascertain whether a refi nement to a procedure has been successful. We can use this type of data to determine shorter humane end points as we can clearly see when activity is reduced indicating a mouse which may need attention. Figure 6 shows there is high activity within this cage of female C57/BL6J mice when staff are present during the light phase.Without some behaviour data to back it up, subtle changes may be missed, therefore the use of cameras to validate a system is important to enable us to better understand the behaviour that has led to this activity (for example, increased activity may be due to fi ghting). No system will be fl exible enough for every situation we encounter and some hurdles may not be possible to overcome without an invasive procedure such as a subcutaneous microchip to enable us to identify individual animals in a cage, which in turn can cause problems with an MRI scanner. ConclusionsOn balance is the use of cameras that use complex algorithms, to inform researchers on every movement an animal makes, helping or in reality are they over-complicating things for people? Perhaps true objectivity and, in turn the ability to refi ne humane endpoints and improve the welfare of the animals is found in systems where cameras are not the primary source of data. After all these systems can give a more defi nite answer in terms of a disruption in activity which highlights a potential welfare concern in the cage. 11 check? If the answer is both, then is a complex camera system necessary for your home cage monitoring? Figure 5. Which cage would you check if both indicate a problem but only the occupants of one cage are visible? Science When we have a greater understanding of the behaviour of animals we can design protocols and studies that fit around the animal to a greater degree than we are currently able to. A system that can give us an objective answer for activity patterns after cage disturbances such as husbandry procedures or a regulated procedure, or a reduction in activity due to an intervention or model design, may enable us to design studies where we can ascertain whether a refinement to a procedure has been successful. We can use this type of data to determine shorter humane end points as we can clearly Figure 5. Which cage would you check if both indicate a problem but only the occupants of one cage are visible?Figure 6. Activity pattern of four female C57/BL6 mice over a four day period. 12 see when activity is reduced indicating a mouse which may need attention. Figure 6 shows there is high activity within this cage of female C57/BL6J mice when staff are present during the light phase. Figure 6. Activity pattern of four female C57/BL6 mice over a four day period. Without some behaviour data to back it up, subtle changes may be missed, therefore, the use of cameras to validate a system is important to enable us to better understand the behaviour that has led to this activity (for example, increased activity may be due to fighting). No system will be flexible enough for every situation we encounter, and some hurdles may not be possible to overcome without an invasive procedure such as a subcutaneous microchip to enable us 12 see when activity is reduced indicating a mouse which may need attention. Figure 6 shows there is high activity within this cage of female C57/BL6J mice when staff are present during the light phase. Figure 6. Activity pattern of four female C57/BL6 mice over a four day period. Without some behaviour data to back it up, subtle changes may be missed, therefore, the use of cameras to validate a system is important to enable us to better understand the behaviour that has led to this activity (for example, increased activity may be due to fighting). No system will be flexible enough for every situation we encounter, and some hurdles may not be possible to overcome without an invasive procedure such as a subcutaneous microchip to enable us Poster Presentations

188Animal Technology and Welfare August 2020CaseThis presentation concerns a purpose bred juvenile female Beagle dog presenting with unilateral hindlimb muscular atrophy and lameness since arrival at a research establishment.BackgroundTransient lameness in Beagle dogs is a familiar case in the research environment and can be easily treated with short term rest and medication however, chronic lameness resulting from a malformation or injury is much rarer and more challenging to treat. Management of the chronic condition represents a scientific and ethical challenge; the animal’s welfare and lifetime experience must be considered when determining the suitability of a treatment plan and its impacts on any cumulative severity, against the option of euthanasia as the best case for the animal. Consideration should also be given to the implications of premature removal or replacement of that animal on study (3Rs) and the impact that it may have on the sample size, scientific outcome and the welfare of the other animals on study.Objective A plan of action was prepared to test the hypothesis that a physiotherapy plan could be used successfully to improve welfare and quality of life of a Beagle dog presenting with chronic injury-related atrophy and lameness, thereby enabling its use in a scientific study, as authorised by the Animals (Scientific Procedures) Act 1986 and avoid the requirement for premature euthanasia.MethodologyA female juvenile Beagle dog, aged 10 months old, weighing 5.9kg, body score of 3/5, intended for use on a 13-week inhalation toxicity study for regulatory submission, presented after delivery with right hindlimb lameness with associated lateral lift during gait and advanced muscular atrophy of the affected limb. A series of diagnostic digital radiographs were conducted under general anaesthesia in-house (Cuattro Slate 6 Veterinary Digital X-ray) and radiographic evaluation revealed a small area of higher ossific density on the right femoral neck.It was hypothesised that both the atrophy and lameness were resultant of a historic injury, or congenital malformation and that management of the current condition could be achieved to a satisfactory level with a physiotherapy treatment plan to improve the animal’s welfare and as an alternative to euthanasia and potential use of another dog.Referencing techniques used in veterinary and human clinical practice, we devised a treatment plan using a mixture of passive and physical exercise methods that we thought were achievable and appropriate.We applied techniques on a daily basis (minimum of five of seven days per week) for a period of thirteen weeks (until end of study), starting with the easier tasks, then slowly introducing the more challenging tasks (Balance Using physiotherapy to successfully manage Chronic Atrophic hindlimb lameness in the Beagle dog – a case studySAMANTHA SHANKS Charles River, Veterinary Services, Elphinstone Research Centre, Tranent, East Lothian EH33 2NE UK Correspondence: Samantha.shanks@crl.comPoster from IAT Virtual Congress 2021Animal Technology and Welfare August 2021

189August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareTherapy, Cavalletti). Challenge progression was driven by the patient’s successful achievement of the foundation tasks. The patient demonstrated a strong desire for human contact time, which allowed us to utilise that as a reward for execution of the tasks. We introduced cardboard tubes for retrieval play during latter-stage cavalletti training, which proved successful.– Thermotherapy – using a heated oat bag for pain management, circulatory improvement;– Passive Range of Movement (PROM) – assisted exercises to improve integrity, range and function of joint and limb, and proprioception. Performed in lateral recumbency;– Figure of 8s (+/-stepover) on lead – improve joint fl exion, proprioception, core and spinal fl exibility;– Hindlimb lift – lift contralateral limb to encourage short-term use of affected limb;– Balance therapy – encourage use of hindlimbs by having forefeet on a raised surface, improve core muscle strength, muscle mass, proprioception;– Cavalletti – escalating in height/number through time.Encourages enthusiasm for treatment sessions. Improvements driven through gradually increasing theintensity of physical challenge (pace/height/ordination).Charted below are the breakdowns of the time per day spent per exercise, across the individual week. It depicts the progression from the basic exercises to more demanding plyometric exercise and the transition from fl oor-based cavalletti to the raised cavalletti, highlighting the improvement in functional movement and limb use.Figure 1-2. Beagle patient participating in cavellitti exercise.Poster Presentations

190Animal Technology and Welfare August 2020Shown below are photos taken at sequential timepointsdepicting progression through the treatment plan. Musclebulk can be observed to increase as the timepoints progress.Physiotherapy treatment commenced on the 20th May 2019. Figure 3. 2 weeks pre-treatment 6th May 2019.Figure 4. Week 9. 16th July 2019.Figure 5. Week 11. 2nd August 2019.Figure 6. Week 13. 15th August 2019.Poster Presentations

191August 2020 Animal Technology and WelfareAugust 2020 Animal Technology and WelfareFigure 7. Cavalletti – raised 15th August 2019. 13 Figure 7. Cavalletti –raised 15thAugust 2019. Discussion It is the aim of physiotherapy to promote mobility and to alleviate impairments. There are three main groups of treatments that can be implemented to achieve this; hands-on therapies, physiotherapeutic modalities and therapeutic exercises. Despite the lack of directly relevant evidence in support of these techniques, it is widely accepted that managing chronic pain using a physiotherapeutic approach is benefi cial and can signifi cantly improve mobility in most patients in both the short and long term.1,2 Improvements in proprioception, joint ROM (Range of Movement) and health, muscular strength and function can be accomplished by implementing a patient-specifi c strengthening and exercise regime.3A successful regime, such as the one designed, for strengthening hip muscles in the dog must include at least some controlled aerobic exercise (e.g. fi gure of 8s, cavalletti) and some targeted exercises designed to improve strength and joint ROM.4,5 In the early stages and throughout, the patient will also benefi t from thermotherapy, the therapeutic effects of heat include promoting increased blood fl ow; reducing joint stiffness, associated pains and/or muscle spasm; reducing infl ammation and oedema, and it aids in the post acute phase of healing.6 Application of heat treatment to collagenous tissue permits an increase in extensibility through stretch-based exercises. Plyometric exercise is used by athletes to enhance performance and as a form of physiotherapeutic exercise used to improve muscle performance once signifi cant healing and recovery of basic daily use has taken place. We used cavalletti for this purpose, the benefi ts gained through this exercise are core strengthening, improved strength and ROM in limbs, spinal fl exibility, proprioception and associated improvements to balance and coordination.7,8It can be used to improve muscle mass and joint strength,proprioception and neuromuscular effi ciency, and with the aim to correct any core imbalance acquired through previously limited use of the limb. Progress was patient-led but the physical challenge was gradually intensifi ed as physical capabilities improved. There should be no evidence of pain before, during or after exercise. Our results demonstrated a credible improvement in muscle mass gained on the affected limb and in overall improved locomotion. We observed growing enthusiasm in the patient’s approach to physical exercise and reduced avoidance of affected limb use. When considering the evidence and observations, it is reasonable to conclude that the life of the patient was improved as a result of the treatment and extended in the absence of euthanasia.From a practical perspective, this simple treatment plan was relatively low maintenance to manage, requiring only 15-30 minutes of the working day to set up, implement and pack down, and only 1 member of staff at any time. When considering the principles of the 3Rs, Replacement, Reduction, Refi nement, we can see the application of these pillars within this case. As well as refi ning the experience for the patient the physiotherapy plan allowed us to refi ne the experience of all animals on the study; by enabling the use of the patient and avoiding acquisition of an additional animal we avoided scheduling delays. Conclusions This clinical case study demonstrates that 10-20 minutes a day of progressively active physiotherapy treatment, under an ascending challenge plan, can facilitate reversal of severe leg muscle atrophy, improve joint mobility and limb use, in the laboratory Beagle dog. This can be achieved within a relatively short period of time provided that the dog is compliant and its interest in the treatment structure is maintained. Achievements may vary depending on origin and severity of injury however the exercises listed can be used interchangeably depending on the patient and over a variable period of time. Implementation of such a simple treatment plan could be used across the industry, where indicated, in order to reduce individual suffering and to reduce the requirement for use of replacement animals.Poster Presentations

192Animal Technology and Welfare August 2020Acknowledgements A special thank you to the following Animal Technologists for their supportive roles in delivering this treatment plan consistently for 13 weeks; Heather Turley, Claire Whitecross, Donna Livingstone, Ryan Cockerell and Alistair Colhoun.I would also like to acknowledge my veterinary colleagues,Rastislav Bator MRCVS, who supported my plan and provided the prescription for the treatment, and Bryony Few BVSC MSc MRCVS, who provided insight and help with constructing both the report and presentation.References1Fox, S.M. and Mills, D. (2010). Multimodal Management of Canine Osteoarthritis, 1st ed., Manson Publishing, London, 2010.2Millis, D. (2014). l therapy and rehabilitation in dogs. In GAYNOR JS and MUIR III WW (eds), Handbook of Veterinar y Pain Management, 3rd ed., Elsevier, St Louis, United States, 2014.3McGowan, C., Stubbs, N., and Goff., (2007). AnimalPhysiotherapy: Assessment, Treatment andRehabilitation of Animals, 1st ed., Blackwell Publishing Ltd, UK, 2007.4Edge-Hughs, L., (2007). Hip and Sacroiliac Disease: Selected Disorders and Their Management with Physical Therapy; Clin Tech Small Animal Practice, 2007; 22:183-194.5Starr, L., (2013). Rehabilitation for geriatric patients. In ZINK MC and VAN DYKE JB (eds), Canine Sports Medicine and Rehabilitation, 1st ed., Wiley-Blackwell Publishing, Chichester, UK, 2013. 6Prentice, W.E. and Arnhem, D.D., Arnheim’s Principles of Athletic Training: a Competency Based Approach, 13th ed., McGraw-Hill, United States, 2008.7Sayers, E. (2018). Emma Sayers Veterinar y Physiotherapy, [online] 2018, viewed on 06/01/2020. https://www.emmasayersvetphysio.com/post/polework-for-horses8Towson, S. (2016). Equine Mechanics, [online] 2016,viewed on 06/01/2020, https://equinemechanics.com/equinemechanics.com/post/133211502783/the-equine-back-part-3-ridden-exercises-to-buildPoster Presentations

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04Animal Technology and Welfare August 2020Congress 2022 makes a return to face-to-face meetings  Meet colleagues and friends in a relaxed atmosphere  More flexibility for attendance - book your own accommodation - select the days / meal plans / evening entertainment you wish to participate in - take full advantage of the EARLY BIRD DISCOUNT available to 31st December 2021  ‘Calls for’ PAPERS, WORKSHOPS and POSTERS announcements appear in this issue  Once you have registered, download the Congress 2022 App in order to make the most of your Congress experience INVITATION TO PARTICIPATE We’ve missed you – come and see us in 2022 ONLINE REGISTRATION OPENS 1st SEPTEMBER 2021 Venue North East England And don’t forget Ken’s Quiz makes a long-awaited return! All enquiries to congress@iat.org.uk Check for updates www.iat.org.uk