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CONTENTS August 2020 Animal Technology and Welfare Vol 19 No 2 August 2020 Editorial Jas Barley Chair of the Editorial Board ix Report of the 2019 RSPCA UFAW Rodent 101 Welfare Group meeting Chloe Stevens Emily Finnegan Jasmine Clarkson Charlotte Burns Sonia Bains Colin Gilbert Caroline Chadwick Samantha Izzard Charlotte Inman Penny Hawkins Secretary and Huw Golledge Reduction of the negative effects of methionine on bone parameters in broilers embryos by intra egg injection of Vitamin B12 Mohammad Naser Nazem Shima Tasharofi Negin Amiri and Sepideh Sabzekar The care of the Children s Python Antaresia children Alexander Hosking and Gary Martinic Feline assisted therapy a promising part of animal assisted therapy AAT Eliska Mic Mi kov and and Krityna Krityna Machova Machova 112 118 123 The care of Central and Pygmy Bearded Dragons Alexander Hosking and Gary Martinic 125 PAPER SUMMARY TRANSLATIONS 131 French German Italian Spanish LOOKING BACK Physical hazards in the laboratory animal house R T Charles 139 The incidence of a pathogenic strain of pseudomonas in a rabbit colony G R Alpen and K Maerz 143 TECH 2 TECH 145 Development of a sifting cage change method for rats to improve welfare Seonagh Henderson i

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CONTENTS Animal Technology and Welfare August 2020 POSTER PRESENTATIONS Assessing pain in models of Rheumatoid Arthritis Samuel Singleton Meriam Nefla Ngaire Dennison Simon Arthur and Tim Hales 149 Refinements to health monitoring Hannah Jones and Rebecca King 153 Biosecurity risks and the pre implantation embryo lessons from the mouse Jean Cozzi Mendy Verrier and Jimmy Mancip 155 Environmental enrichment for a small colony of rats Nick Blackburn Gemma Cronshaw and Mike Mitchell 158 Oestrus checking increasing productivity and embracing the 3Rs Samantha Hoskins and Jack Brown 159 Using habituation to reduce stress for rats being transported short distances Sarah Taylor 162 Shining a light on rearing pigmentless Zebrafish Jacqueline Glover Thom Berriman Dimitra Mantzorou William Havelange Sam Berry and Bruno Correia da Silva The jacket with pulling power a novel approach to early stage evaluation of magnetic nanoparticles Alison Ritchie James Dixon Phil Clarke and Anna Grabowska 164 166 Index to Advertisers ABPI x xi AS ET OBC Datesand Ltd IFC Institute of Animal Technology vii IPS Product Supplies Ltd IBC ii LBS ii Somni Scientific iv Special Diets Services viii Tecniplast UK Ltd xii

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August 2020 OFFICERS President Dr Robin Lovell Badge CBE FRS Immediate Past President Professor Sir Richard Gardner MA PhD FRSB FIAT Hon FRS Vice Presidents Senga Allan MIAT RAnTech David Anderson MRCVS Stephen Barnett BA MSc FIAT Hon CBiol FRSB RAnTech Miles Carroll PhD Brian Cass CBE Paul Flecknell MA Vet MB PhD DLAS DipLECVA MRCVS FIAT Hon Penny Hawkins PhD BSc Wendy Jarrett MA Judy MacArthur Clark CBE BVMS DLAS FRSB DVMS h c DipECLAM FRAgS DipACLAM MRCVS Fiona McEwen BSc BVM S MSc MRCVS Tim Morris BVetMed PhD DipACLAM DipECLAM CBiol FRSB CertLAS MRCVS Clive Page OBE PhD BSc Jan Bas Prins PhD MSc Vicky Robinson CBE BSc PhD Paul Sanders MIAT RAnTech David Spillane FIAT Gail Thompson RLATG Robert Weichbrod PhD RLATG Life Members Ken Applebee OBE FIAT CBiol FRSB RAnTech Charlie Chambers MIAT RAnTech Roger Francis MSc FIAT RAnTech Pete Gerson MSc FIAT RAnTech Cathy Godfrey FIAT RAnTech John Gregory BSc Hons FIAT CBiol FRSB RAnTech Patrick Hayes FIAT DipBA RAnTech Robert Kemp FIAT Hon RAnTech Phil Ruddock MIAT RAnTech Ted Wills FIAT Hon RAnTech Honorary Members Mark Gardiner MIAT RAnTech Sarah Lane MSc FIAT Sue McHugh BSc FIAT Norman Mortell BA Hons MIAT RAnTech Wendy Steel BSc Hons FIAT Members of Council Matthew Bilton Kally Booth Steven Cubitt Simon Cumming Haley Daniels Glyn Fisher Nicky Gent Alan Graham Nathan Hill Linda Horan Sam Jameson Elaine Kirkum Adele Kitching Theresa Langford Sylvie Mehigan Steve Owen Alan Palmer Allan Thornhill John Waters Lynda Westall Carole Wilson Adrian Woodhouse IAT REPRESENTATIVES Animal Technology and Welfare Council Officers Chair Linda Horan BSc Hons MIAT RAnTech Vice Chair Glyn Fisher FIAT RAnTech Honorary Secretary Simon Cumming BSc FIAT RAnTech Treasurer Glyn Fisher FIAT RAnTech Chair of Board of Educational Policy Steven Cubitt MSc FIAT RAnTech Chair Registration Accreditation Board Glyn Fisher FIAT RAnTech ATW Editor Jas Barley MSc FIAT RAnTech Bulletin Editor Carole Wilson BSc MIAT ATW Bulletin Editorial Board Jas Barley Chair Matthew Bilton Nicky Gent Patrick Hayes Elaine Kirkum Carole Wilson Lynda Westall Branch Liaison Officer Kally Booth MIAT RAnTech EFAT Representatives Glyn Fisher Alan Palmer Website Coordinator Allan Thornhill FIAT RAnTech Animal Welfare Officers and LABA Representatives Matthew Bilton Chair Kally Booth Lois Byrom Simon Cumming Nicky Gent Sylvie Mehigan John Waters Board of Educational Policy Steven Cubitt Chair Adele Kitching Secretary Communications Group Adrian Woodhouse Chair Nathan Hill Elaine Kirkum Teresa Langford Sylvie Mehigan Allan Thornhill Lynda Westall v

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Animal Technology and Welfare CPD Officer Alan Palmer MIAT RAnTech Registration and Accreditation Board Glyn Fisher Chair John Gregory Cathy Godfrey Kathy Ryder Home Office Stuart Stevenson Observer Ngaire Dennison LAVA Congress Committee Alan Graham Chair Haley Daniels Adele Kitching Allan Thornhill John Waters Diversity Officer Haley Daniels MBA MSc MIAT RAnTech CIPD UK Biosciences ASG Representative Home Office Alan Palmer MIAT RAnTech August 2020 IAT OFFICERS MAY BE CONTACTED VIA IAT Administrator admin iat org uk OR VIA THE IAT WEBSITE AT www iat org uk OR THE REGISTERED OFFICE 5 South Parade Summertown Oxford OX2 7JL Advertisement Managers PRC Associates Ltd Email mail prcassoc co uk BRANCH SECRETARIES 2020 Cambridge Edinburgh Huntingdon Suffolk Norfolk Ireland London Midlands North East England North West Oxford Surrey Hampshire Sussex West Middlesex Wales West West of Scotland Tony Davidge Kery Anne Lavin Thomson Jo Martin Lisa Watson Rebecca Towns Ian Fielding Zoe Smith and John Bland Nicky Windows Adam Truby Francesca Whitmore Josefine Woodley Rhys Perry Joanne King cambridgebranch iat org uk edinburghbranch iat org uk hssbranch iat org uk irelandbranch iat org uk londonbranch iat org uk midlandsbranch iat org uk northeastbranch iat org uk cheshirebranch iat org uk oxfordbranch iat org uk shsbranch iat org uk westmiddxbranch iat org uk waleswestbranch iat org uk westscotlandbranch iat org uk Although every effort is made to ensure that no inaccurate or misleading data opinion or statement appear in the journal 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 such inaccurate or misleading data opinion statement or advertisement being published Furthermore the opinions expressed in the journal do not necessarily reflect those of the Editor or the Institute of Animal Technology 2020 Institute of Animal Technology All rights reserved No part of this publication may be reproduced without permission from the publisher vi

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August 2020 2020 Animal Technology and Welfare THE INSTITUTE OF ANIMAL TECHNOLOGY 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 Editorial Jas Barley Chair of the Editorial Board Looking back over issues of the Journal through its various identities one thing is apparent and that is the contribution that overseas 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 been constant 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 when reproduced 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 different from what we use in the UK This issue welcomes contributions from Australia the Czech Republic and Iran as well of course from the UK Since ATW became an Open Access publication and is being published electronically it is enjoying a wider audience and is attracting more contributions than usual Not all are relevant to our profession but knowledge is transferable so what seems off beat today may become useful in the future However as Editor I will always strive to maintain the quality of our publications 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 have organised focussed on sentience positive welfare and psychological well being The report contains contributions from 11 presenters as well as notes on the interactive discussion session on sentience that closed the meeting A paper from Iran a first as far as I can see for the Journal on reducing the negative effects of methionine on bone parameters in broilers embryos may seem of little relevance but it offers a better understanding of how methionine affects bone structure which is important to most species Similarly Feline Assisted Therapy as described by the team at the University of Life Sciences Prague does not appear to fall into the realms of Animal Technology but it gives us a better understanding of how animals can have a positive effect on some people which in the current situation may be of significant benefit to a wider population Our final paper from the team at Western Sydney University details the care of the Children Python and two species of Bearded Dragons Not perhaps the run of the mill laboratory animals but just as important to many Animal Technologists globally as mice and rats If you keep reptiles at home or know of someone who is contemplating one as a pet these papers make useful reference documents We also offer two papers from previous issues of the Journal which were very different in appearance and content than today s Journal of Animal Technology and Welfare and not only because of the change of title Issues were printed in black and white and in the very early days were produced by hand The paper from France on Physical Hazards in the laboratory animal house will bring back many memories for some of the older technicians myself included but not necessarily good ones The use of ether as an anaesthetic which I know is still used in some countries where resources are limited for human surgery presented a very real danger to both animals and staff Disease in laboratory animal units was often a recurring problem bacterial infections such as Pseudomonas as described in the reprint of the article were still presenting Animal Technologists with problems as late as the end of the 1980s When importing animals and tissues from overseas it is important to realise that they may be carrying disease not seen in the UK for several decades In recent times Ectromelia was introduced into a unit in the USA via antibodies produced overseas Precautions must 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 on a novel technique of cage cleaning which has a positive effect on the welfare of laboratory rats Finally we included several posters prepared for AST2020 but sadly at the moment remain unpresented SDS Thanks again to all of our authors past and present both internationally and here in the UK There would not have been 70 years of the Journal without you Here is to the next seven decades and beyond ix

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Animal Technology and Welfare August 2020 ANDREW BLAKE TRIBUTE AWARD DON T KEEP YOUR GOOD IDEA TO YOURSELF WE WANT TO HEAR ABOUT IT FOR THE 2021 AWARD 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 ENTER SUBMISSIONS 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 x

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August 2020 Animal Technology and Welfare The Andrew Blake Tribute Award commemorates the work and life of Andrew Blake who suffered from Friedrich s ataxia a hereditary condition described as one of the worst of neurological diseases Andrew died in May 2002 aged 39 Andrew was passionate about the need to support scientists in their work and his commitment to speaking out against animal rights activists took up much of the last ten years of his life He died shortly before he was to collect his MBE DETAILS 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 2021 FREE ATTENDANCE next March WHICH WILL INCLUDE PRESENTING YOUR WORK AN ENGRAVED GLASS PLAQUE AND 250 CASH AWARD CLOSING DATE FRIDAY 13th NOVEMBER 2020 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 ANDREW BLAKE TRIBUTE AWARD SPONSORED BY THE ABPI xi

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August 2020 2020 Animal Technology and Welfare Report of the 2019 RSPCA UFAW Rodent Welfare meeting CHLOE STEVENS 1 EMILY FINNEGAN 2 JASMINE CLARKSON 3 4 CHARLOTTE BURNS 5 SONIA BAINS 6 COLIN GILBERT 7 CAROLINE CHADWICK SAMANTHA IZZARD 9 CHARLOTTE INMAN 10 PENNY HAWKINS1 and HUW GOLLEDGE11 1 2 3 4 5 6 7 8 9 10 11 Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS UK Bristol Veterinary School University of Bristol Dolberry Building Langford House Bristol BS40 5DU UK Centre for Behaviour and Evolution Biosciences Institute Newcastle University Newcastle NE2 4HH UK Institute for Biodiversity Animal Health and Comparative Medicine University of Glasgow Glasgow G61 1QH UK The Royal Veterinary College Hawkshead Lane North Mymms Hatfield Hertfordshire AL9 7TA UK MRC Harwell Institute Harwell Science and Innovation Centre Oxford OX11 0RD UK The Babraham Institute Babraham Cambridge CB22 3AT UK Biomedical Services Unit University of Birmingham Vincent Drive Birmingham B15 2TT UK GSK Medicines Research Centre Gunnels Wood Road Stevenage Hertfordshire SG1 2NY UK Home Office Animals in Science Regulation Unit 14th Floor Lunar House 40 Wellesley Road Croydon CR9 2BY UK UFAW The Old School Brewhouse Hill Wheathampstead Hertfordshire AL4 8AN UK Introduction The RSPCA UFAW Rodent Welfare Group has held a one day meeting every autumn for the last 26 years so that its members can discuss current welfare research exchange views on welfare issues and share experiences of the implementation of the 3Rs of replacement reduction and refinement with respect to rodent use A key aim of the Group is to encourage people to think about the whole lifetime experience of laborator y rodents ensuring that ever y potential negative impact on their wellbeing is reviewed and minimised The 26th meeting was held in November 2019 at the University of Birmingham and was attended by over 60 delegates from across the UK The theme of this year s meeting was sentience positive welfare and psychological well being The meeting opened with a talk on animal sentience and what it means for animal welfare and this topic was returned to in the discussion session at the end of the day with delegates offering their own views on animal sentience Some talks related to various refinements including enrichment refined handling methods and clicker training and how these refinements affect behaviour and the affective emotional state of animals Other talks discussed new ways of monitoring animals for better welfare assessment addressing neonatal mor tality in laboratory mice and how small changes can have a cumulative positive effect on the lives of laboratory animals The day ended with an update from the Home Office Animals in Science Regulation Unit ASRU on their themed inspections relating to lack of food and water refined handling of mice and needle re use This report summarises the meeting and ends with a list of action points for readers to consider raising at their own establishments Sentience what does it mean Huw Golledge Universities Federation for Animal Welfare Sentience has been a topic of much debate recently It can roughly be defined as the capacity to have feelings in other words to have positive and negative 101

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Report of the 2019and RSPCA UFAW Rodent Welfare Welfare Group Group meeting meeting Animal Technology Welfare Rodent RSPCA UFAW experiences such as pleasure pain or distress However definitions vary some argue that sentience simply means an ability to experience the world whereas others suggest that it means having feelings that actually matter to the animal or being aware of one s own self awareness It is difficult to know for sure whether or not an animal is sentient but there are behavioural indicators we can look for such as avoiding painful stimuli seeking out pleasure and empathy We can also look to see if an animal has the physiology to support sentience such as a complex nervous system sophisticated enough to take in and process sensory inputs to create a conscious experience and mechanisms which would be necessary for pain sensation or which might underlie emotional states If some animals are sentient there is a moral responsibility to care about their welfare which is why so many people are keen to have sentience enshrined in law Although the Animals Scientific Procedures Act 1986 ASPA regulates the care and use of sentient animals it does not explicitly mention sentience ASPA does however recognise that animals can be harmed and defines harm as the pain suffering distress or lasting harm likely to be experienced by animals during the course of the procedures within a project after applying all appropriate refinement techniques By acknowledging that protected animals may have negative experiences like pain suffering and distress this definition implies that the animals protected by ASPA are sentient So what does this mean for rodents Even though ASPA does not explicitly say so it seems highly likely that rodents are sentient They have the necessary neural structures there is more extensive evidence for pain perception in rats and mice than in any other nonhuman species thanks to their extensive use in pharmaceutical research to identify analgesics they display complex learning memor y and other sophisticated cognitive abilities and may experience affective emotional states and empathy 1 This is why it is so important to avoid or minimise pain suffering distress and lasting harm to rodents and it shows that their use should be regulated by ASPA However sentience in rodents gives rise to two important questions regarding their use in research The first is are some animals more sentient than others By only regulating vertebrate and cephalopod use ASPA tacitly assumes that all other animals are not sentient although emerging research into the sentience of animals such as decapod crustaceans such as crabs may eventually lead to the addition of new species ASPA also assumes that some animals can suffer more than others as it requires that regulated procedures should involve animals with the 102 August 2020 lowest degree of neurophysiological sensitivity This has important implications for the use of animals for example should we assume that mice are less sentient than rats or rats less sentient than primates 2 Do some animals suffer more or does a more sophisticated brain allow some animals to cope better with challenges than others It is unclear at the moment whether these are reasonable assumptions but we might gain a better understanding of how best to choose species for research by going beyond neurophysiological complexity alone Gaining a scientific understanding of sentience and to what degree it varies between species might allow us to better predict which species are likely to suffer the least in various situations The second question is when does sentience develop Early stage fetuses are widely thought not to be sentient and some mammals such as rats and mice are thought to be born developmentally immature and therefore also insentient 3 Understanding the development of sentience might help us to understand at what stage of development animal use should be regulated for each species However in the meantime we can apply the precautionar y principle which suggests that we should treat animals as though they are sentient until proven otherwise As Marion Dawkins puts it animal welfare is far too important to be made to wait until the hard problem of consciousness has been solved 4 In summary although not explicitly acknowledged by ASPA almost everyone assumes that rodents are sentient and strives to improve their welfare accordingly As science continues to provide evidence that animals can have feelings that matter to them and as sentience science develops further we may be able to make more informed decisions about which animal models we should use However we shouldn t wait for hard proof of sentience to reduce suffering and improve welfare in those species we reasonably think are sentient Inactive but awake behaviour as a potential indicator of depression E Finnegan M Mendl 1 A Trevarthen 1 E Paul 1 A Resasco 3 C Fureix 2 1 2 3 Bristol Vet School University of Bristol Dolberry Building Langford House Bristol BS40 5DU UK School of Biological Marine Science A415A Portland Square Drake Circus Plymouth PL4 8AA UK Laboratorio de Animales de Experimentaci n Facultad de Cs Veterinarias UNLP Argentina Assessing an animal s affective emotional state is a challenging but important goal in animal welfare science Humans can experience chronic negative

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August 2020 Report Rodent Technology Welfare Group and meeting Welfare Report of of the the 2019 2019 RSPCA UFAW RSPCA UFAW Animal affective states such as clinical depression which can be associated with reduced activity and a lack of engagement with the environment In animals inactive but awake IBA behaviour has been studied as a potential indicator of depression but its link to affective state is yet to be validated 5 6 However the link between these behaviours termed inactive but awake IBA behaviour and affective state in animals has not yet been validated We investigated the link between IBA behaviour and affective state in mice by identifying whether comparatively more stressful housing conditions are a risk factor for IBA behaviour and examining the effects of changing housing conditions We predicted that IBA behaviour would be observed more in non enriched cages than in enriched cages would increase if the amount of enrichment was reduced and would reduce if enrichment was added Based on the results of previous research 7 we also predicted that different strains of mice would show different amounts of IBA behaviour To test our hypotheses we housed 31 C57BL 6J mice and 31 DBA 2J mice in mixed strain pairs in either standard laboratory cages or large highly enriched cages for 3 weeks After this period half of the cages were swapped to the opposite housing condition for a further 3 weeks to examine the effects of a changed environment on IBA behaviour Over both phases we observed mice during the dark phase when they would usually be most active under red light to see how much IBA behaviour was displayed IBA behaviour was defined as the mouse being still with their eyes open for at least 15 consecutive seconds We found that as expected mice housed in nonenriched cages displayed significantly more IBA behaviour than those housed in enriched cages suggesting that IBA behaviour is an indicator of a negative affective state Moving mice from an enriched to a non enriched cage caused a bigger increase in IBA behaviour than any other condition this is in keeping with other studies which have found that losing enrichment can cause pessimism and therefore likely a negative affective state in several species 8 9 However we found that moving mice from a nonenriched cage to an enriched cage did not affect the amount of IBA behaviour suggesting that even though the housing conditions had improved it was not enough to reverse the negative effects of the nonenriched cage We did find differences in mouse strains in the amount of IBA behaviour but contrary to previous studies we found that DBA 2J mice showed more IBA behaviour than C57BL 6J mice This may be because of individual differences in the mice or differences in the experiences of the mice used in each study they may have differed in age early life conditions source or a combination of these factors Overall our results suggest that IBA behaviour may be a useful indicator of negative affective states in mice however more research is warranted to fully validate IBA behaviour as a welfare indicator Tail handling affects the capacity of laboratory mice to express disappointment and elation J M Clarkson 1 2 M C Leach 3 P A Flecknell 4 C Rowe1 1 2 3 4 Centre for Behaviour and Evolution Biosciences Institute Newcastle University Newcastle NE1 7RU UK Institute for Biodiversity Animal Health and Comparative Medicine University of Glasgow Glasgow G12 8QQ UK School of Natural and Environmental Sciences Agriculture Building Newcastle University Newcastle NE1 7RU UK Comparative Biology Centre The Medical School Newcastle University Newcastle NE1 7RU UK Mice are the most commonly used species for scientific research with millions bred and housed in laboratories worldwide As with all laboratory animals making refinements to their husbandry and use in scientific procedures is important for ensuring good welfare One important refinement has shown that the standard method of catching laboratory mice by the tail increases stress anxiety and depression which can be reduced by capturing them in cupped hands or with a tunnel 10 11 However tunnel handling for mice has not been widely implemented perhaps because more research is needed to provide a more comprehensive evaluation of current handling practice on rodent behaviour welfare and subsequent data collection Anhedonia is an inability to experience pleasure from rewarding stimuli It can be assessed in mice by measuring how much they consume of a rewarding substance e g a pleasant tasting sucrose solution as well as by examining lick cluster size Mice produce fast rhythmic sets of licks which can be grouped into clusters with the number of licks in a cluster increasing as the palatability of the reward increases Lower consumption of sucrose or smaller lick cluster size therefore indicates a more anhedonic state Box 1 We have previously found that different handling methods alter the hedonic value of rewards how pleasurable a reward is for laboratory mice11 see Box 1 as mice handled by the tail consumed less sucrose and had smaller lick cluster sizes than tunnel handled mice This suggests that tail handled mice perceived the reward as being lower in value than tunnel handled mice did which indicates that tail handled mice had a 103

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Report of the 2019and RSPCA UFAW Rodent Welfare Welfare Group Group meeting meeting Animal Technology Welfare Rodent RSPCA UFAW more negative affective state This difference in how valuable a reward is perceived to be is also found in human patients with negative affective states such as depression Negative affective states in humans can also mean that individuals experience more disappointment when rewards are lost and less elation or joy when rewards are gained It is therefore possible that measuring an animal s response to losing or gaining a reward could provide an objective measure of their underlying affective state The aim of our study was to understand whether handling method affected how laboratory mice expressed disappointment and elation in response to reward loss or gain with the expectation that tail handled mice would show greater disappointment to reward loss and less elation to reward gain than tunnel handled mice To explore whether handling methods changed how mice responded to losing or gaining a reward mice were handled either by their tail or using a tunnel once a day for 9 days Tail handled mice were found to interact less with their handler and were more anxious in the elevated plus maze and open field tests They also showed more anhedonia as they had lower lick cluster sizes when given sucrose than tunnel handled mice Next for ten days we rewarded mice after handling with either 4 or 32 sucrose solution After this period the concentrations of sucrose solution were swapped resulting in a reward loss condition a decrease in sucrose concentration 32 to 4 and a reward gain condition an increase in sucrose concentration 4 to 32 Control mice received the same concentration of sucrose solution throughout the study We assessed how mice responded to this change by examining lick cluster sizes We found initially that all mice showed disappointment when they lost the higher value reward as lick cluster sizes decreased when sucrose concentration decreased Whilst this disappointment was prolonged in tail handled mice tunnel handled mice recovered and lick cluster sizes increased again when tested later This suggests that tunnel handled mice may be more resilient to negative events than tailhandled mice However when the reward increased in value mice showed elation with lick cluster sizes increasing irrespective of handling method Taken together our results suggest that the handling method can affect the capacity of laboratory mice to be resilient to negative life events and that tail handling may be associated with chronic stress in mice They also suggest that laboratory mice are capable of positive emotions such as elation which highlights the importance of measuring both positive and negative emotions in laboratory mice to fully understand their welfare state 104 August 2020 To have a nest or to build a nest Mouse environmental enrichment for security and stimulation Charlotte Burn Royal Veterinary College Environmental enrichment can improve animal welfare in different ways some enrichment provides positive stimulation for animals whilst other enrichment such as nests and shelters can provide security or satisfy specific behavioural requirements There is a wide choice of commercially available cage furniture for mice so choosing enrichment can be difficult There are also practical considerations such as items being hygienic safe and not overly obstructing staff viewing of the animals To better understand mouse requirements for security we studied the behavioural effects and practicality of different shelters Shelters can provide mice with security as they allow mice to maintain contact with the walls thigmotaxis as well as providing darkness dampening of sounds and smells shelter from draughts a refuge from being chased a more natural 3D environment with something to climb on and something to gnaw Using a tunnel to catch mice is now strongly recommended for good welfare so tunnels which are placed in cages for handling may provide some of these benefits and thus could serve as a shelter However it is unclear whether a handling tunnel provides sufficient shelter We tested whether adding cardboard tunnels or orange dome shelters to mouse cages provided more benefits to mice than a colourless handling tunnel alone Using 12 cages of adult C57BL 6 mice 5 females and 7 males we provided mice with a handling tunnel alone with a cardboard tube or with an orange dome shelter in a randomised order over three weeks We found that mice used the dome shelter significantly more than the handling tunnel alone or with the cardboard tube The dome also allowed three times as many mice to simultaneously shelter and for mice to bring nesting materials inside the shelter the cardboard tunnel offered a gnawing substrate but little shelter We also conducted a study looking at the effects of different types of nesting material Nests in mouse cages can provide a more naturalistic resting area the oppor tunity for nesting behaviour and darkness Nesting materials are also important as many lab mice are housed at below optimal temperatures which can result in cold stress nesting materials therefore provide animals with warmth and provide some control over their micro environment 12 We studied the effects on behaviour nest quality which we assessed using a nest scoring system13 and practicality of compressed paper squares or soft paper strips as nest building materials versus ready made Kraft paper strip nests

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August 2020 Report Rodent Technology Welfare Group and meeting Welfare Report of of the the 2019 2019 RSPCA UFAW RSPCA UFAW Animal We used 12 cages of non breeding adult mice 3 female CBA 3 female C57BL 6 and 6 male C57BL 6 in a randomised repeated measures design over three weeks We found no significant differences in the amount of time mice spent nesting but nests produced using the soft paper strips and the ready made nests had higher nest quality scores than those produced using compressed paper squares similar to previous research 13 In terms of practicality ready made nests were easiest to standardise across cages and lasted the longest of all materials tested although paper strips were less expensive Our results therefore suggest that ready made nests were the best option of the materials tested However preference tests would need to be conducted to verify whether the mice agree Recent research on animal boredom paves the way for investigating how important cognitive occupational or sensory enrichment is for mice For example there is evidence that enrichment generally enhances mouse cognition but whether this is due to increased stimulation or increased security is unclear 14 Mice have also been found to choose to forage even when free food is available which may suggest they are looking for something to do 15 Furthermore mice in standard laboratory cages show more inactive but awake behaviour see above and more stereotypies7 this may signify boredom as bored animals have previously been shown to become drowsy but also restless and sensation seeking 16 Types of enrichment that may be stimulating for mice might include scatter feeding and puzzle feeders climbing apparatus running wheels digging materials longer tubes and multi level cages novel objects or exploration and training opportunities However more research is needed to assess the effectiveness of these different options Overnight monitoring of animals as a welfare assessment R S Bains M Stewart G Banks P Nolan and S Wells MRC Harwell Institute Harwell Science and Innovation Centre Oxford OX11 0RD UK The MRC Harwell Institute focusses on the generation and phenotyping of genetically altered GA mouse models to study the relationship between genes and disease Many of the lines created are completely novel and some animals have progressive conditions such as neurodegenerative diseases diabetes and diseases of ageing Therefore much effort is directed towards monitoring mouse welfare Laboratory mouse welfare is conventionally monitored through daily cage side assessment where the observer examines the cage and only opens it if there is an obvious cause for concern or at cage changing Whilst this is a very effective way of identifying obvious welfare concerns such as wounds and weight loss subtler indicators of welfare such as reduced activity can be missed altogether These checks also only provide a snapshot in time which can result in subtle early indicators being missed Furthermore we have shown that simply moving the cage from one rack location to another within the same room is enough to disrupt mice for up to an hour so cage side checks may not capture what is actually happening in an undisturbed cage It is also important to note that mice are nocturnal animals so many behavioural phenotypes will actually be expressed in the dark phase where they go unobserved In recognition of these issues a number of technologies have been developed to study mouse behaviour over an extended period of time However many of these technologies monitor behaviour outside the home cage which means that current understanding of normal mouse behaviour in a home cage is limited More recently there has been a shift towards automated welfare monitoring within the home cage 17 18 In our system mice have radio frequency identification RFID microchips inserted then are housed in cages which are placed on top of a baseplate containing an array of RFID antennae 19 This system allows the location of several mice to be tracked within the cage High definition infrared cameras are also used to take video footage of the mice The use of this system has allowed the detection of a number of behaviours which may be used to assess the welfare of the mice for example we have observed mice showing seizure like behaviour which had not been seen in cage side checks 20 In another case we found that one mouse was showing stereotypic or hyperactive behaviour during the dark phase 24 hours before this behaviour would have been detected with a cage side check Using this system can therefore allow us to intervene earlier and implement earlier humane endpoints this is important for the focal mouse but also for other mice in the cage who may become stressed in response to a hyperactive cage mate We have also been able to detect novel behaviours which do not currently have an activity signature recognition of these behaviours may help us to create a clearer repository of exact terms and allow information sharing between facilities to better understand the welfare implications Another benefit of our tracking system is that we are able to track several mice at once which then allows us to examine social interactions between cage mates 19 For example we can see which pairs of mice spend time together and for how long Conducting home cage monitoring allows us to make better informed decisions about a mouse s welfare 19 For example we were able to better recognise 105

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Report of the 2019and RSPCA UFAW Rodent Welfare Welfare Group Group meeting meeting Animal Technology Welfare Rodent RSPCA UFAW differences in behaviour and time budgets between different mouse strains This helps when differentiating between phenotypes and behaviours that may indicate welfare concerns We can also monitor responses to new procedures and adjust our approach if we see signs of adverse effects Overall we have found that home cage monitoring of mice has highlighted how much we do not know about mice behaviour in their home cage and has given us the opportunity to delve deeper We have been able to collect scientifically relevant data at earlier time points supporting earlier interventions are able to identify novel behaviours and create a repository of exact terms for these behaviours therefore being better placed to make informed decisions about mouse welfare The Alive Pup Project Colin Gilbert The Babraham Institute Preweaning pup losses are common in laboratory mouse colonies with some strains showing up to 50 mortality and a high rate of total litter loss 21 However little is currently known about how to ensure good mouse pup survival Received wisdom states that neonatal mor tality in mice is caused by active infanticide is associated with maternal stress is affected by the number of litters a mother has had and is worse in C57BL 6 and genetically altered mice We used these ideas as starting points to explore causes of preweaning mouse pup mor tality and better understand how mortality can be prevented To look for active infanticide we studied behavioural interactions between pups and dams 22 Whilst dams were seen eating dead offspring and interacting with both alive and dead pups no female was observed killing a pup In a later study Brajon et al in prep we found that less than 10 of cannibalism events analysed involved infanticide It therefore seems that whilst infanticide does happen it is not a major cause of mortality and that cannibalism does not necessarily indicate infanticide It has been suggested that mothers provided with more enrichment may be less stressed and therefore that pup mor tality should be lower in enriched environments 23 24 However we have not yet found effects of enrichment on mortality Another possible source of maternal stress is inspections by laboratory staff but we have examined both experimental and historical data and found that early post par tum inspections do not affect pup survival Previous research has suggested that pup survival is higher in second litters than first litters 25 but we have been unable to find evidence for this in a retrospective analysis of 344 parturitions 26 However we have found 106 August 2020 that successful mothers perform more nest building and passive maternal behaviour per form less parturition related behaviour in the labour position or giving birth pay more attention to still pups and spend less time outside the nest 27 We also investigated the effects of social environment on pup mortality and found that housing 2 females and 1 male together did not reduce overall neonatal mortality or total litter loss compared with single housing 21 However the presence of an older litter significantly increased neonatal mortality indicating that rather than providing extra warmth and protection in the nest the older pups were somehow outcompeting the newborns and increasing their chances of death So what does this high level of neonatal mortality mean for animal welfare It is unlikely that the pups suffer much a lot of pup development including brain development happens after birth so it is unlikely that pups who are stillborn or die within the first five days of life have gained sentience However mothers who lose litters are likely to experience effects on their health and wellbeing High neonatal mortalities also lead to larger numbers of mice being used for breeding if the average neonatal mortality rate is assumed to be 30 in laboratory mice based on the average EU statistics for laboratory animal use 2 3 million more mice would be needed each year to compensate The results of our project suggest there are a number of things we can do to reduce or prevent neonatal mortality These include providing nesting material avoiding pup hypothermia avoiding litter overlap in mice housed in trios and avoiding the use of aged females for breeding It is also important to know about and monitor any problems in facilities maintained for mouse breeding so reviewing inspection routines and record keeping practices is key as is awareness of social and other environmental factors which may affect neonatal mortality More research in this area will help us better understand the mechanisms and risk factors refer to the Alive Pup Project website for updates on our work https approjectweb wordpress com home the ap project Little changes big effects Caroline Chadwick University of Birmingham Of the 3Rs the one that Animal Technologists can contribute most to on an ongoing basis is refinement Refinement of experimental protocols is vital as we would all wish our experimental subjects to suffer as little as possible to provide the required scientific output However most laboratory animals spend the majority of their lives in cages as stock or breeding animals so small changes for the better with respect

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August 2020 Report Rodent Technology Welfare Group and meeting Welfare Report of of the the 2019 2019 RSPCA UFAW RSPCA UFAW Animal to housing and care can have a major positive effect on an animal s lifetime experience Within our unit we have made a number of practical changes over the years to address areas where we felt improvements could be made None of these changes are revolutionary but we have found that these simple changes can have a positive impact on animal welfare With all of these changes we have found that input from technicians is vital as is good communication between scientists and unit staff We have also found that having clear protocol plans is crucial as these provide a compliance check highlight welfare requirements and help to make staff aware of upcoming studies One area in which we have made changes is in animal housing Most laboratory species are social and do not like being on their own but researchers may be motivated to house their animals individually particularly in the case of animals who have had surgery telemetered animals or those bearing head mounts In one example from our facility we wanted to attempt to group house rats with jugular cannulas with dorsal ports We needed to find a balance between allowing sufficient surgical recovery and reducing the period of single housing but at five days post surgery grouping rats led to increased levels of seroma formation under the dorsal port However by extending the recovery period to 7 days we found seroma rates fell to 1 and rats could be housed in groups This suggests there is considerable scope to introduce group housing in many models Indeed it is not only rodents that need social housing zebrafish are social animals but are often individually housed for a period for genotyping We have successfully group housed fish in these circumstances by adding them to tanks containing different looking strains so that the focal individual is still easy to identify Another area in which we have been able to make improvements is in monitoring high risk animals or animals with special requirements such as aged animals We have found that it is effective to keep cages that need extra checks together for example high risk animals can be grouped together on the same rack for ease and efficiency which helps stop any extra checks being missed by accident Other strategies include arranging cages in order of age and using a system of colour coded paperclips to indicate the frequency of checks as an easy visual reminder to staff The last main area in which we have introduced changes is for animals which require special nursing regimes For example MediGel Clear H2O is used in our facility for post operative animals It is vital that the MediGel is introduced into cages at least 24 hours before surgery to allow the animal to become used to it as post procedure animals are often unwilling to investigate novel objects or substances This practice can also apply to HydroGel Clear H20 or DietGel Clear H20 Once again this highlights the importance of good communication between Animal Technologists and scientists as animals must therefore be identified well before surgery takes place We have also found that post operative animals are less able to utilise nesting materials to provide insulation and are often less active Raising the temperature of postsurgical rodent recovery accommodation therefore has a positive effect on recovery and reduces morbidity and mortality In summary we have found that relatively small changes can make a big difference to the lives of our experimental animals There is a clear imperative to continually reassess the daily lives of our animals and to share knowledge when we find ways of improving Positive reinforcement training with Balb C mice how quickly can they learn Samantha Izzard GSK Positive reinforcement training PRT has been used extensively in large animal species with success however the majority of animals used in research are smaller species such as rats and mice At GSK we are investigating PRT to ensure that the benefits for the animals and the science outweigh the time and effort PRT requires We trained Balb C mice to learn simple tasks that are relevant for research animals such as entering handling tunnels All animal studies were ethically reviewed and carried out in accordance with the Animals Scientific Procedures Act 1986 and the GSK Policy on the Care Welfare and Treatment of Animals Following the methods of Leidinger et al 28 we began by identifying treats which would work best as rewards for our mice We placed a choice of four certified treats Mini Yogurt Drops Bacon YummiesTM Fruit Topia TreatsTM all Bio Serv and Fruit CrunchiesTM Datesand into the home cages of six female and six male Balb C mice We found that only the yoghurt drops were eaten in every cage so intended to use these to train mice to enter tunnels To follow the natural behaviour of a mouse tunnels were placed next to the cage wall Once a mouse entered the tunnel we pressed a clicker and offered the mouse the yoghurt drop However mice would not nibble the yoghurt drop indicating that yoghurt drops did not present a high enough reward to produce the desired training effect in our mice We therefore repeated our treat trial with different uncer tified treats We found that Nestl white chocolate buttons were of high value to the mice and using these mice were successfully trained to enter the tunnels within 4 days 107

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Report of the 2019and RSPCA UFAW Rodent Welfare Welfare Group Group meeting meeting Animal Technology Welfare Rodent RSPCA UFAW Next we moved on to training our mice to enter a blood restrainer We started by placing the tube from a restrainer into the home cages to allow the mice to acclimatise to the restrainer Subsequent training sessions then took place using the restrainer from each mouse s home cage In each 5 minute training session mice would enter the restrainer and a treat was dropped through the hole in the top of the tube While a mouse was in the restrainer we gently handled their tails to familiarise them with the process of blood sampling Using this approach the mice were trained to enter the restrainer within 7 days Our results so far are promising and we have a number of next steps which we intend to address These include determining whether non certified treats can be used for experimental studies within GSK and other establishments as well as sourcing effective certified treats We intend to address whether it is realistic to train mice within their acclimatisation period Most importantly we want to delve deeper into the question of whether this approach will benefit the animals and the science In conclusion we believe that PRT could refine animal studies and increase reproducibility as it may remove differences caused by anxiety in mice This approach should also help reduce the risk of incidents within the blood restrainer tube and is likely to make blood sampling easier by having a mouse that will stay in the required position Applying the 3Rs how much evidence is enough A case study on rat cage height in the UK Penny Hawkins1 and Hibba Mazhary 2 RSPCA 2University of Oxford 1 The Home Office Code of Practice allows laboratory rats to be housed in cages 20cm high even though adult rats can rear up to 30cm if they have the headroom Evidence suggests that being able to stand upright is important for rat welfare as rats housed in higher cages frequently perform this behaviour and rats housed in 20 cm cages appear to compensate for their inability to stretch upright by performing more lateral stretches 29 However even though the Code of Practice makes clear that it defines minimum standards which can and should be improved upon some animal research establishments continue to use 20cm cages We conducted a study to better understand the perceived challenges of introducing higher rat cages and identify ways to overcome these challenges 30 We visited eight establishments across the UK of which some used 20 23 cm rat cages one used only 30cm cages and others used a number of different heights 108 August 2020 Several people were interviewed at each including Establishment Licence Holders Animal Technologists Animal Welfare Ethical Review Body AWERB members researchers and people with responsibilities for budgets and for staff health and safety Participants at establishments using minimum height cages cited a number of concerns over switching to higher cages including health and safety implications of lifting heavier cages lack of resources a belief that larger cages cause increased aggression among rats and negative effects on scientific data However establishments which were already using larger cages had been able to address these problems for example one establishment housed fewer rats per cage and removed water bottles before moving cages to reduce risks to staff Another establishment had implemented a budget for replacing rat caging over a defined time period One striking result of our survey was that many participants appeared to accept anecdotal evidence to support maintaining the status quo but wanted hard evidence to support change Some suggested that there was a lack of evidence for higher caging with clear views on the kind of evidence they would accept such as increased physiological indicators of stress in rats who are unable to stand However we found that papers demonstrating these findings have already been published We propose that there is now a stronger body of evidence for higher rat cages and that both animal welfare and science would benefit if these were provided for example by repurposing rabbit cages This shows that there is an important debate to be had around how much evidence is enough to consider changing practices We also noted that some par ticipants had knowledge gaps relating to the evidence in favour of higher rat cages the AWERB Named Information Officer and Named Training and Competency Officer can play pivotal roles in helping to address these gaps and issues of perception We encourage anyone at an establishment which houses rats in cages that do not allow them to stand up to ask why this is the case and to challenge this practice 1 For the full results of this study see our open access paper https www mdpi com 2076 2615 9 12 1104 Update from the Animals in Science Regulation Unit Charlotte Inman Home Office A major initiative for ASRU in 2018 19 has been the completion of themed inspections These are targeted inspections to address key areas where 3Rs gains can be made and risk of non compliance and associated

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August 2020 Report Rodent Technology Welfare Group and meeting Welfare Report of of the the 2019 2019 RSPCA UFAW RSPCA UFAW Animal animal welfare implications can be reduced The areas for the themed inspections are provision of food and water refined handling and needle re use Failure to provide food and water for experimental animals has been a significant cause of noncompliance for several years representing around 20 of non compliance cases We asked inspectors to review the strategies used at establishments to ensure food and water are provided Some common factors associated with the lack of food and water have been identified including changes in housing poor communication between PILs and Animal Technologists confusion over who is responsible for animals especially after procedures and weekends due to reduced staffing Addressing these problems involves reducing risk for example within the animal unit risky areas such as isolators or rooms outside the main facility should be identified and addressed Other approaches include reviewing the system of checks to ensure that it is effective and ensuring staff are appropriately trained Data collection and review is ongoing and will help us to develop further potential strategies to reduce the incidence of failure to provide food and water The second area of our themed inspections is refined handling Despite resources being readily available uptake of these techniques has been patchy Our strategy in collaboration with the NC3Rs has been to engage with establishments in order to emphasise that this is a priority obtain baseline data on current use signpost resources and assist with establishment strategy development So far we have found that 59 of establishments out of 110 respondents are using only non aversive methods with 5 not attempting to use non aversive methods The primary factor for success appears to be whether Animal Technologists are engaged with the process with motivation to try these methods coming from within the technologist community Establishments which have not made changes are often affected by the opinions of influential researchers and animal staff who dispute the evidence We are continuing to collect and analyse data and are developing strategies for fur ther implementation Our final area of themed inspections focusses on needle re use Re using needles can cause unnecessary pain and tissue damage and can reduce data quality by inducing stress in animals and contributing to infection spread Whether an establishment re uses needles is also indicative of its culture of care We have used questionnaires to help establish our position and understand the impacts of our approach We found that 73 of establishments out of 86 respondents have considered the issue of needle re use Thirty four establishments reported currently re using needles with reasons cited including costs convenience staff health and safety or the reuse of needles for euthanasia We also found that around 25 establishments had no policy on needle reuse and in the absence of a policy the relative level of needle re use was higher in these establishments Presence of a policy may be indicative of more general awareness of the issue and may also indicate a generally better culture of care Further analysis and a follow up themed inspection as well as working with stakeholders is planned in order to further develop policy Interactive discussion session on sentience At the start of the day delegates were given an activity asking them to indicate which of 16 organisms were sentient Some choices were uncontroversial most participants thought that dogs are sentient while many agreed that trees are probably not sentient However there was more disagreement over inver tebrate species only some thought that honey bees are sentient and fewer still thought that crabs and spiders are sentient Delegates also disagreed about the onset of sentience whilst many thought that adult zebrafish are likely to be sentient many were unsure whether a zebrafish larva would have gained sentience at three days post fertilisation Later in the day there was a discussion session about sentience and what it means for animal welfare Some delegates pointed out that whilst discussions of sentience are important they are not always practical for Animal Technologists as a cer tain level of detachment may be needed for staff who must carry out procedures or humane killing There was also discussion about how the topic of sentience might be addressed at participants own institutions the importance of the need for strong support from management was highlighted Delegates also expressed that these may be particularly difficult discussions to have with scientists especially if they do not see the animals every day or may be more inclined to consider discussions of sentience as anthropomorphic However it was agreed that it is often par t of the responsibility of an Animal Technologist to find the right skills or language to communicate concerns to researchers in the right way and several delegates suggested ways in which accusations of anthropomorphism can be countered List of action points based on all presentations and discussions Aim to start discussions about animal sentience at your establishment for example with respect to welfare assessment the harm benefit analysis or out of scope animals Look out for mice displaying inactive but awake 109

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Report of the 2019and RSPCA UFAW Rodent Welfare Welfare Group Group meeting meeting Animal Technology Welfare Rodent RSPCA UFAW behaviour and see whether this correlates with other indicators of distress If not doing so already consider using refined handling methods such as tunnel handling rather than tail handling Know that not all enrichment is created equal where possible evaluate different forms of enrichment e g nest building materials to see which benefits the animals most Be aware that different forms of enrichment have different purposes some may provide stimulation some may provide security and try and provide animals with a range of enrichment to meet these different needs Cage side checks during the light phase may not be enough to identify welfare issues in nocturnal animals consider reviewing cage checking procedures to see whether rats and mice can be monitored during the dark phase Disturbances and moving animals from their home cages can affect behaviour and lead to welfare issues being masked consider if there are possibilities for remote monitoring of animals Review the recommendations of the Alive Pup project and see whether there is scope to further implement these Review single housing of social animals and discuss possible strategies for allowing animals to be grouphoused with your NACWO NVS and AWERB Review inspection routines for animals who may need extra checks such as high risk or aged animals Can cages which need extra checks be placed together or some other clear visual cue be used Discuss with the NVS the possibility of increasing the temperature of housing for rodents who are recovering from surgery and monitor whether this helps reduce morbidity and mortality Positive reinforcement training can work for rodents and rabbits as well as larger animals consider trying this to help refine procedures or when introducing animals to new and potentially stressful situations Review practices around making sure all animals are provided with food and water especially after cage changes or when identifying who has responsibility for animals If not already in place encourage your establishment to develop a policy on needle re use References 1 2 3 Ben Ami Bartal I Decety J and Mason P 2011 Empathy and pro social behavior in rats Science Vol 334 1427 1430 Birch J 2018 Degrees of sentience Animal Sentience Vol 3 11 Mellor D J Diesch T J Gunn A J and Bennet L 2005 The impor tance of awareness for understanding fetal pain Brain Research Reviews Vol 49 455 471 110 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 August 2020 Dawkins M S 2017 Animal welfare with and without consciousness Journal of Zoology Vol 301 1 10 Yeates J 2016 Quality of life and animal behaviour Applied Animal Behaviour Science Vol 181 19 26 American Psychiatric Association 2013 Diagnostic and statistical manual of mental disorders BMC Medicine Vol 17 133 137 Fureix C Walker M and Harper L et al 2016 Stereotypic behaviour in standard non enriched cages is an alternative to depression like responses in C57BL 6 mice Behavioural Brain Research Vol 305 186 190 Bateson M and Matheson S M 2007 Performance on a categorisation task suggests that removal of environmental enrichment induces pessimism in captive European starlings Sturnus vulgaris Animal Welfare Vol 16 33 Burman O H P Parker R Paul E S and Mendl M 2008 A spatial judgement task to determine background emotional state in laboratory rats Rattus norvegicus Animal Behaviour Vol 76 801 809 Hurst J L and West R S 2010 Taming anxiety in laboratory mice Nature Methods Vol 7 825 826 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 Scientific Reports Vol 8 2448 Johnson J S Taylor D J Green A R and Gaskill B N 2017 Effects of Nesting Material on Energy Homeostasis in BALB cAnNCrl C57BL 6NCrl and Crl CD1 ICR Mice Housed at 20 C Journal of the American Association for Laboratory Animal Science Vol 56 254 259 Hess S E Rohr S Dufour B D et al 2008 Home improvement C57BL 6J mice given more naturalistic nesting materials build better nests Journal of the American Association for Laboratory Animal Science Vol 47 25 31 W rbel H 2001 Ideal homes Housing effects on rodent brain and behaviour Trends in Neurosciences Vol 24 207 211 Inglis I R Forkman B and Lazarus J 1997 Free food or earned food A review and fuzzy model of contrafreeloading Animal Behaviour Vol 53 1171 1191 Burn C C 2017 Bestial boredom a biological perspective on animal boredom and suggestions for its scientific investigation Animal Behaviour Vol 130 141151 Spruijt B M and DeVisser L 2006 Advanced behavioural screening automated home cage ethology Drug Discovery Today Technologies Vol 3 231 237 Richardson C A 2015 The power of automated behavioural homecage technologies in characterizing disease progression in laboratory mice A review Applied Animal Behaviour Science Vol 163 19 27 Bains R S Cater H L and Sillito R R et al 2016 Analysis of individual mouse activity in group housed animals of different inbred strains using a novel automated home cage analysis system Frontiers in Behavioral Neuroscience Vol 10 106 Bains R S Wells S Sillito R R et al 2018 Assessing mouse behaviour throughout the light dark cycle using automated in cage analysis tools Journal of Neuroscience Methods Vol 300 37 47

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August 2020 21 22 23 24 25 26 27 28 29 30 Report Rodent Technology Welfare Group and meeting Welfare Report of of the the 2019 2019 RSPCA UFAW RSPCA UFAW Animal Brajon S Munhoz Morello G Teixeira M S et al 2019 Social environment as a cause of litter loss in laboratory mouse A behavioural study Applied Animal Behaviour Science Vol 218 104827 Weber E M Algers B Hultgren J and Olsson I A S 2013 Pup mortality in laboratory mice infanticide or not Acta Veterinaria Scandinavica Vol 55 83 Gaskill B N Pritchett Corning K R Gordon C J et al 2013 Energy reallocation to breeding performance through improved nest building in laboratory mice PLOS One Vol 8 e74153 Leidinger C S Th ne Reineke C and Baumgart N and Baumgart J 2019 Environmental enrichment prevents pup mortality in laboratory mice Laboratory Animals Vol 53 53 62 Brown R E Mathieson W B Stapleton J and Neumann P E 1999 Maternal behavior in female C57BL 6J and DBA 2J inbred mice Physiology Behavior Vol 67 599605 Weber E M Algers B W rbel H Hultgren J and Olsson I A S 2013 Influence of strain and parity on the risk of litter loss in laboratory mice Reproduction in Domestic Animals Vol 48 292 296 Weber E M Hultgren J Algers B and Olsson I A S 2016 Do laboratory mouse females that lose their litters behave differently around parturition PLOS One Vol 11 e0161238 Leidinger C Herrmann F Th ne Reineke C Baumgart N and Baumgart J 2017 Introducing clicker training as a cognitive enrichment for laboratory mice Journal of Visualized Experiments Vol 121 e55415 Makowska I J and Weary D M 2016 The importance of burrowing climbing and standing upright for laboratory rats Royal Society Open Science Vol 3 160136 Mazhary H and Hawkins P 2019 Applying the 3Rs A Case Study on Evidence and Perceptions Relating to Rat Cage Height in the UK Animals Vol 9 12 1104 1 Since the meeting was held the Canadian Council on Animal Care has published new guidelines on rat housing including a requirement for caging to accommodate upright rearing ccac ca Documents Standards Guidelines CCAC_Guidelines_Rats pdf 111

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August 2020 Welfare Animal Technology and Welfare Animal TechnologyAugust and Welfare 2020 Reduction of the negative effects of methionine on bone parameters in broilers embryos by intra egg injection of vitamin B12 MOHAMMAD NASER NAZEM 1 SHIMA TASHAROFI 2 NEGIN AMIRI 3 and SEPIDEH SABZEKAR4 1 2 3 4 Assistant Professor of Anatomy Histology and Embryology Department of Basic Sciences School of Veterinary Medicine Shahid Bahonar University of Kerman Kerman Iran Department of Animal Science Researches Agriculture and Natural Resources Education and Research Center of Kerman Agriculture and Natural Resources Education and Research Organization of Iran PhD student of Animal Nutrition and Breeding Department of Animal Science Shahid Bahonar University of Kerman Kerman Iran Post graduate student of Veterinary Sciences Shahid Bahonar University of Kerman Kerman Iran Correspondence shima tasharrofi gmail com Abstract Methionine is a sulphur containing amino acid In addition to its general function as a component of proteins methionine is crucial for a variety of methyltransferase reactions Although methionine is crucial for cartilage synthesis its negative effects on bone diseases such as osteoporosis and tibial dyschondroplasia must not be ignored These negative effects may be abrogated by vitamin B12 therapy This study carried out to investigate the effects of intra egg injection of some solutions including 1ml of distilled water methionine B12 methionine with additional B12 on some bone and serum parameters in broilers embryos Ross 308 Fertile chick embryos were injected on day 4 of incubation into the yolk sac On day 18 embryos were examined for left tibia parameters amount of osteocyte and osteoblast cells and their length the thickness of trabeculae and the collagen sediment and serum parameters amount of alkaline phosphatase and homocysteine Methionine alone caused a loss of osteocytes length osteoblasts heights and sedimentation of collagen whereas those groups treated in addition with vitamin B12 were comparable to the control Alkaline phosphatase concentration of serum in both B12 injected groups B12 and B12 was significantly upper than control Homocysteine concentration of serum was higher in both methionine injected groups methionine and 112 B12 methionine compared with control Data obtained in the current study suggest that supplementation with vitamin B12 may prevent defects in bone development parameters brought about by methionine Key words broiler methionine B12 embr yos in ovo injection Introduction Although methionine is an essential amino acid and crucial for cartilage synthesis it has some harmful effects on the age related diseases such as diabetes obesity osteoporosis and cancer 1 So the potential to know and reduce its negative effects is increasing 2 6 Extra dietar y supplementation of methionine has adverse effects on bone matrix in broiler chickens and 7 8 High methionine diets lead to high serum homocysteine levels which increases the risk of fracture 9 Also methionine restriction shifts bone morphology due to delays in osteoblast differentiation 6 However some of the research considers the disadvantages effects of low methionine diet on bone parameters due to increased collagen degradation In methionine restricted mice bone mass density and bone mineral content was elevated5 and in methioninerestricted rat bone mass decreased 10 In addition oral administration of methionine increased the growth of bones in rats 11 113

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Reduction of the negative effects of methionine on bone parameters in broilers embryos August 2020 Animal Technology andembryos Welfare Reduction of the negative effects of methionine on bone parameters in broilers Furthermore vitamin B12 cobalamin is a fundamental cofactor in methionine metabolism 12 13 B12 and methionine have direct and indirect relation to bone mass densitometry respectively 13 Use of B12 in diet decreases hip fracture in elders 14 15 Deficiency of B12 in rats has no effect on the methylation reaction or on bone marrow folate levels although the B12 concentration in bone marrow reduces in B12 deficiency 16 In addition no significant relation is found between the change of vertebral bone mineral density and B12 in women 17 Therefore in the current study it was hypothesised that the injection of B12 into yolk sac at day 4 of incubation period may rectify the adverse effects of methionine injection on embryos bone parameters measure the osteocyte and osteoblast cells and their length and thickness of trabeculae in the left tibia and serum metabolites measure alkaline phosphatase and homocysteine Materials and method Kerman where the experiment was done is located in the dry area with an average annual rainfall of 200 mm and maximum annual temperature of 40 C with altitude of 1500 to 2000m Design and Animals At day 4 of incubation 40 fertile eggs Mahan Farm Kerman Iran from 35 week old broiler breeder chickens Ross 308 were individually weighed and only eggs with a mean weight 57 5 1 2 g of those eggs weighed were randomly set on each of 4 treatments 1 Intra egg injection of 1 ml distilled water control 2 Intra egg injection of 1 ml distilled water containing 40 mg Methionine Scharlau Co Spain 3 Intra egg injection of 1 ml distilled water containing 100 g vitamin B12 Daroupakhsh Distribution Co Iran 4 Intra egg injection of 1 ml distilled water containing 40 mg Methionine and 100 g vitamin B12 of 10 eggs each Incubators with automatic temperature control 37 5 0 1 and rotation of the eggs every 1 hour were maintained at 60 relative humidity until day 18 of incubation when all eggs were opened to obtain the embryos for experiments In Ovo Injection Procedure The injection site broad end of the egg was disinfected with 70 alcohol Ethanol and then 1 ml of each solution was injected into the yolk sac at day 4 of incubation period using a 22 gauge needle with depth of 28 mm from the broad end of the egg The entry holes were sealed using melted paraffin wax 114 Data collection At 18th day of incubation period all 40 eggs were weighed and opened and all embryos were dried weighed and killed to measure alkaline phosphatase and homocysteine of serum and count the osteocyte and osteoblast cells and measure their lengths in the left tibia bone The experimental protocols were reviewed and approved by the Animal Care Committee of Department of Basic Sciences School of Veterinary Medicine Shahid Bahonar University of Kerman Kerman Iran For serum analysis 18 day old embryos were dried by soft tissues and decapitated to collect the blood in eppendorf tubes Bloods then were centrifuged at 3000 x g for 10 min to gain serum To measure the total homocysteine homocysteine methyltransferase transfers the methyl group of d methionine methylsulfonium to homocysteine leading to the generation of l methionine and d methionine Then d amino acid oxidase oxidizes d methionine with the simultaneous production of hydrogen peroxide to yield methylene blue Diazyme Homocystein Enzymatic Colorimetric Assay Kit Poway CA 92064 USA with an absorbance at 660 nm 18 To measure alkaline phosphatase biochemical kit Pars Azmoon Co Tehran Iran was used following by use of a spectrophotometer at 405 nm 19 After killing at 18th day of incubation period left tibias were taken from all embryos Soft tissues were removed and tibias were used to determine the amount of osteocytes in bone matrix and their length the amount of osteoblasts in bone edges and their lengths and the thickness of trabeculae In the first step a cross section was taken from the centre of the tibia using a scalpel The slices were fixed in 10 buffered formalin 100 ml of 40 formaldehyde 4 g phosphate 6 5 g dibasic sodium phosphate and 900 ml of distilled water for 24 hrs and then the 10 buffered formalin was renewed Tissues were dehydrated by transferring through a series of alcohols with increasing concentrations placed into xylol and embedded in paraffin Wax Samples were cut into 5 m sections Sections were then stained using Masson s trichrome in order to evaluate the orientation and measurement the thickness of trabeculae and collagen sediment von Kossa in order to evaluate the calcium sediment on trabeculae and haematoxylin eosin H E for standard histopathological evaluation methods and examined using light microscope It is noticeable all sections of all groups that prepared for von Kossa method were stained together It is important to omit the colour concentration effect On the Masson s trichrome stained slides the mean of diameter of 30 trabeculae in each section was measured by using a digital system Dino eye AM 7023 5Mp Taiwan Counts of osteocyte and osteoblast cells measurements of their lengths and heights and 113

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Reduction of the negative effects of methionine on bone parameters in broilers embryos Animal Technology and Welfare August 2020 Reduction of the negative effects of methionine on bone parameters in broilers embryos trabecular thicknesses magnification of 40X 20 were determined at a Statistical analysis The data were analysed using the one way analysis of variance ANOVA of SPSS 16 Chicago USA software Differences among treatments were determined with Tuki s test Statements of significant probability were based on p0 05 Table 1 Figure 2 Collagen sediments are visible in the trabeculae of all groups by green colour A control B Methionine C Vit B12 D Methionine Vit B12 Masson s trichrome 40X Table 1 Effect of intra egg injection of methionine and B12 on performance in broiler embryos Bone parameters Trabeculae in control group were formed normally They were widely expanded from the cortex to medulla In the methionine group trabeculae were discontinued very thin and developed weakly B12 and methionine B12 groups showed similar aspects as controls Figure 1 Figure 3 Calcium sedimentation in experimental groups A control B Methionine C Vit B12 D Methionine Vit B12 All groups show the presence of calcium brown to black color in trabeculae von Kosaa 40X Figure 1 Trabecular formation in the tibia The thickest trabeculae are seen in control A Vit B12 C and methionine Vit B12 D The t in methionine group B are thin and discontinued H E 40X Based on obtained results by Masson s trichrome method collagen sediment was seen in all groups 114 According to Table 2 in the current study amounts of osteocytes and osteoblasts did not change significantly by treatments P 0 05 but numerally intra egg injection of methionine reduced both of them Also methionine injection did negatively affect the length of osteocytes height of osteoblasts and trabeculae thickness at 18th day of incubation period P

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Reduction of the negative effects of methionine on bone parameters in broilers embryos Animal Technology and Welfare August 2020 Reduction of the negative effects of methionine on bone parameters in broilers embryos amount of homocysteine are four pivotal effects of extra access to methionine in animals 11 Thus like all other nutrients both deficiency and extra contents of methionine could not terminate to good bone development But because a higher dietary methionine concentration can improve growth performance in broilers it is necessary to find a solution for its adverse effects on bone 37 38 In the present study supplying B12 via in ovo injection procedure rectified bone parameters which were under disadvantage effects of extra methionine 12 13 14 In conclusion injecting methionine into yolk sac of fertile broiler eggs at 4th day of incubation adversely affects bone parameters of 18 day old embryos but B12 injection could reduce these bad effects These results will allow us to validate intra egg injection of methionine B12 as an appropriate method not only for better performance but also for more developed bones 15 16 References 1 2 3 4 5 6 7 8 9 10 Vijiyan V Khandelwal M Manglani K Gupta S and Surolia A 2013 Methionine down regulates TLR4 MyD88 NF KB signaling in osteoclast precursors to reduce bone loss during osteoporosis Br Pharm 10 19 156 158 Frankel T 1995 Sulfate incorporation into organic bone matrix of the tibiotarsus of broiler chicks is reduced by excess dietary methionine Poult Sci 74 3 510 516 Komninou D Leutzinger Y Reddy B S and Richie J P inhibits colon 2006 Methionine restriction carcinogenesis Nutr Cancer 54 202 208 Plaisance E P Greenway F L Boudreau A Hill K L Johnson W D and Krajcik R A 2011 Dietar y methionine restriction increases fat oxidation in obese adults withmetabolic syndrome J Clin Endocrinol Metab 96 836 840 Ables G P Perrone C E Orentreich D and Orentreich N 2012 Methionine restricted C57BL 6J mice are resistant to diet induced obesity and insulin resistance but have low bone density Plos One 7 e51357 Ouattara A Cooke D Gopahakrishnan R Huang T and Ables G P 2016 Methionine restriction alters bone morphology and affects osteoblast differentiation Bone Rep 5 33 42 Lohakare J Choi J Kim J Yong J Shim Y Hahn T Chae T 2005 Effects of dietary combinations of vitamin A E and methionine on growth performance meat quality and immunity in commercial broilers Asian Aust J of Anim Sci 18 4 516 523 Whiting S and Draper H 1981 Effect of a chronic acid load as sulfate or sulfur amino acids on bone metabolism in adult rats J Nutr 111 10 1721 1726 Saito M and Fuji K 2006 Degree of mineralization related collagen cross linking in the femoral neck cancellous bone in cases of hip fracture and controls Calcifi Tissue Int 79 160 168 Huang T H Lewis J L Lin H S Kuo L T Mao S W and Tai Y S 2014 A methionine restricted diet and endurance exercise decrease bone mass and extrinsic 116 17 18 19 20 21 22 23 24 25 strength but increase intrinsic strength in growing male rats Nutr 144 621 630 McGrath K R and Nakamoto T 1985 Orally administered methionine alters the growth of tooth germs in newborn rats Ann Nutr Metab 29 6 374 380 Golbahar J Hamidi A and Aminzadeh M A 2004 Association of plasma folate plasma total homocysteine but not methylene tetrahydrofolate reductase C6677 polymorphism with BMD in postmenopausal Iranian woman a cross sectional study Bone 35 760 765 Morris M S and Jacques P F 2005 Relation between Hcy and B vitamins statues indicators and BMD in older Americans Bone 37 234 42 Sato Y Honda Y Iwamoto J Kanoko T and Satoh K 2005 Effect of folate and metcobalamin on hip fractures in patients with stroke a randomized controlled trial JAMA 293 1082 1088 McLean R R Jacques P F and Selhub J 2008 Plasma B vitamins homocysteine and their relation with bone loss and hip fracture in elderly men and women J Clin Endocrinol Metab 93 2206 2212 Cheng F W Shane B and Stokstad E L R 1975 The Anti Folate Effect of Methionine on Bone Marrow of Normal and Vitamin B12 Deficient Rats Available from https doi org 10 1111 j 1365 2141 1975 tb00863 x Cagnassi A Bagni B Zini A Cannoletta M Generali M and Volpe A 2008 Relation of folates vitamin B12 and homocysteine to vertebral bone mineral density change in postmenopausal women A five year longitudinal evaluation Bone 42 314 320 Matsuyama N Yamaguchi M Toyosato M Takayama M and Mizuno K 2001 New enzymatic colorimetric assay for total homocysteine Cli Chem 47 12 2155 2157 Bar J Rosenberg A and Hurwitz S 1982 Plasma and intestinal content of 1 25 dihydroxy vitamin D3 in calcium or phosphorus restricted birds Current Advances in Skeletogenesis Pp197 200 in Proceeding of the 5th Workshop on Calcified Tissues Elsevier Science Publishing Amsterdam The Netherlands Zhao W Byrne M H Wang Y and Krane S M 2000 Osteocyte and osteoblast apoptosis and excessive bone deposition accompany failure of collagenase cleavage of collagen J Clin Invest 106 8 941 949 Tako E Ferket P R and Uni Z 2004 Effects of in ovo feeding of carbohydrates and beta hydroxy betamethylbutyrate on the development of chicken intestine Poult Sci 83 2023 2028 Uni Z Ferket P R Tako E and Kedar O 2005 In ovo feeding improves energy status of late term chicken embryos Poult Sci 84 764 770 Uni Z and Ferket R P 2005 Methods for early nutrition and their potential World s Poult Sci J 60 101111 Chamani M Tasharofi S Foroudi F Sadeghi A A and Aminafshar M 2012 Evaluation the effects of inovo injection of different nutrients on hatch percentage performance and carcass parameters of broilers Annals of Biol Res 3 7 3771 3776 Tasharofi S Mohammadi F Amiri N and Nazem M N 2018 Effects of intra yolk sac injection of dextrose and albumin on performance jejunum morphology liver and pectoral muscle glycogen and some serum metabolites of 117

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Reduction of the negative effects of methionine on bone parameters in broilers embryos August 2020 Animal Technology andembryos Welfare Reduction of the negative effects of methionine on bone parameters in broilers 26 27 28 29 30 31 32 33 34 35 36 37 38 broilers J of Anim Phys and Anim Nutr 00 1 7 https doi org 10 1111 jpn 12882 Li M Zhai L Wei W and Dong J 2016 Effect of Methionine Restriction on Bone Density and NK Cell Activity Bio Med Res Int http dx doi org 10 1155 2016 3571810 Luo E Hu J and Bao C 2012 Sustained release of adiponectin improves osteogenesis around hydroxyapatite implants by suppressing osteoclast activity in ovariectomized rabbits Acta Biomaterialia 8 2 734743 Ozkurt B Ozkurt Z N Altay M Aktekin C N Glayan O C and Tabak Y 2009 The relationship between serum adiponectin level and anthropometry bone mass osteoporotic fracture risk in postmenopausal women J Bone Miner Metab 20 2 78 84 Shinoda Y Yamaguchi M and Ogata N 2006 Regulation of bone formation by adiponectin through autocrine paracrine and endocrine pathways J of Cell Biochem 99 1 196 208 Mescher Anthony L 2013 Junqueira s basic histology text and atlas 13th edition McGraw Hill New York PP 364 384 Mansukhani A Bellosta P Sahni M and Basilico C 2000 Signaling by fibroblast growth factors FGF and fibroblast growth factor receptor 2 FGFR2 activating mutations blocks mineralization and induces apoptosis in osteoblasts J of Cell Biol 149 1297 1308 Raucci A Bellosta P Grassi R Basilico C and Mansukhani A 2008 Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways J Cell Physiol 215 2 44251 Lia J and Ward R L 2010 Four folate and one carbon metabolism and its impact on aberrant DNA methylation in cancer Adv in Genetics 71 79 121 Barzel U S and Massey L K 1998 Excess Dietary protein can adversely affect bone J of Nutr 128 6 10511053 Bonjour J 2005 Dietary protein an essential nutrient for bone health J Am Coll Nutr 24 526S 36S Heaney R P and Layman D K 2008 Amount and type of protein influences bone health Am J of Cli Nutr 87 5 1567 1570 Ahmed M E and Abbas T A 2011 Effects of Dietary Levels of Methionine on Broiler Performance and Carcass Characteristics Int J of Poult Sci 10 2 147 151 Chen Y P Chen X Zhang H and Zhou Y M 2013 Effects of dietary concentrations of methionine on growth performance and oxidative status of broiler chickens with different hatching weight Bri Poult Sci 54 4 531 537 118 117

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August 2020 Welfare Animal Technology and Welfare Animal TechnologyAugust and Welfare 2020 The care of the Children s Python Antaresia childreni ALEXANDER HOSKING and GARY MARTINIC Reptile House K1 Animal Facility Technical Support Services Western Sydney University Hawkesbury Campus Richmond NSW Australia Correspondence g martinic westernsydney edu au Abstract This article describes the care of the Children s python Antaresia childreni Figure 1 It includes information on housing feeding handling as well as the general health care of these animals The information presented is derived from Standard Operating Procedures used at the Western Sydney University which have been co written by the authors It is hoped that the information presented herewith may assist novice Animal Technologists with responsibilities for caring for these species in an animal research facility or a zoological setting Western Australia through the Northern Territory into Queensland They inhabit a variety of habitats from arid plains to rocky hills and are agile climbers on rock surfaces often hanging from stalactites in order to catch microbats mid flight but do not venture into larger trees Like other pythons these nocturnal snakes kill their prey by coiling around and suffocating them before ingesting them whole They lay eggs in clutches of around 15 25 The incubation period is approximately 55 days The sex of individuals is determined by probing the inside of the cloaca When inserting the probe into the cloaca the depth that the probe can be inserted indicates the presence or absence of hemipenes If the probe only enters 1 3 scales deep then there are no hemipenes and the snake is female If the probe can be inserted much further 9 15 scales deep then this indicates the presence of hemipenes and the snake is male The K1 reptile facility specimens are all captive bred and have an expected captive lifespan of approximately 30 years Housing and environmental conditions Figure 1 An adult Children s python coiled up within its enclosure Photo A Hoskings Background The Children s python is one of Australia s smallest pythons Fully grown adults average 0 75m in length but can grow up to 1 5m long The body is slender and the tail tapers to a thin point Children s pythons occur across the entire of Northern central Australia from 118 For one adult Children s python a 90 x 45 x 60 cm enclosure is recommended Figure 2 This is large enough for the animal to uncoil and stretch out while reducing excess open areas in the enclosure which can be a source of stress to Children s pythons Small pythons generally do better in enclosures where they feel secure They do not thrive in large enclosures A small hide box should be provided with a hole large enough for the animal to enter without scratching its scales The water dish should be heavy to prevent water being tipped over and it should be positioned at the cooler end of the enclosure away from the heat globes The water container must be large enough for the animal to soak itself in if it chooses Butcher s paper or washed sand is used as the substrate on the bottom of the enclosures In the case of Butcher s paper it is placed 2 3 layers thick spot cleaned daily and replaced weekly In the case of washed sand it covers the floor 1 inch deep is spot cleaned daily and replaced fully every 6 months The reptile rooms are 119

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The care of the Children s Python Antaresia childreni August 2020 Animal Technology andchildreni Welfare The care of the Children s Python Antaresia maintained at constant temperature of 22oC 2 C and a relative humidity of between 40 60 The air conditioning system provides positive pressure ventilation and between 10 12 changes hour hrs light dark provided by individual electronic light timer switches with manual override features to each room Figure 3 Figure 2 Children s python enclosure showing wide sliding glass panel doors shown opened sand substrate a feed and water dish a log an imitation rock platform back right on which to bask and a carboard hide front left An infra red globe to provide heating in an enclosed case Photo A Hosking Figure 3 Electronic Light Timer Switch as used in each reptile holding room to regulate room lighting Enclosure lighting Handling Being a primarily nocturnal species lighting is not as critical to Children s pythons as it is for other reptiles A UVB 25 Watt globe provides suitable artificial UVB light and is connected to a timer to create a day and night cycle with 10 hours of UVB light 7am 5pm Higher intensity UVB globes should not be used in Children s pythons enclosures Lights must be covered with a wire mesh to prevent the pythons from touching or breaking the bulbs and to minimise the spread of glass in the unlikely occurrence of the bulb bursting Children s pythons also benefit from short periods of unfiltered natural light and are taken outside 1 2 times a fortnight for supervised sunning Juvenile Children s pythons are more likely to bite due to their defensive instinct and aggressive feeding response As they mature in captivity they can become quite docile snakes especially when handled appropriately Bites are most likely to occur during feeding and as such handlers need to be aware of this and handle them accordingly to minimise risk Children s pythons can be difficult to remove while biting Once the snake has bitten the sustained injury is not likely to worsen while the animal is attached but improper or hasty attempts to remove the snake may cause a larger laceration Running cold water over their body or into the mouth can result in the python releasing its hold Alcohol from an Alco wipe located in the first aid kit is also very effective Serious injury is unlikely but bites from larger specimens can be very painful Enclosure heating Providing adequate temperature gradients within Children s pythons enclosures is essential for their health and wellbeing They require a temperature gradient so they can move around the enclosure to a temperature that suits them Children s pythons require a hot basking spot maintained at approximately 34 C The cool end of the enclosure should be maintained between 24 26 C The enclosure should not drop below 18 21 C at night Temperatures should be regulated by a thermostat and checked daily to ensure the thermostat and globes are functioning Room lighting Room lighting within the separate reptile holding rooms is maintained on an artificial photoperiod set at 12 12 120 Children s pythons can often strike out as soon as you open the enclosure especially in the days immediately preceding scheduled feeding days when the snake is hungrier It is good practice to remove the snake from its normal enclosure before you feed them This way they do not associate the enclosure opening with feeding When opening the unit to handle or remove a python the handler should first gently tap the snakes head with a gardening style glove The glove cannot be mistaken for food and the snakes in captivity have been conditioned such that this tap indicates handling rather than feeding While the python is outside the enclosure Figure 4 if it begins to coil around an arm 119

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Animal Technology and Welfare The care of the Children s Python Antaresia childreni The care of the Children s Python Antaresia childreni August 2020 or hand the handler should keep the snake moving and support the python s whole bodyweight otherwise the python may adopt feeding behaviours and bite once the arm or hand is restricted It is not recommended to handle pythons if they have been fed in the previous 24 hours It is also not recommended to handle pythons if they are blue and about to commence shedding the python s shedding is recorded and enclosures of snakes about to shed are labelled blue Snakes are identified as blue when the brille the scale covering the eye becomes a milky blue and or their scales generally become dull indicating they are about to shed After three to four days the eyes become clear again and the snake begins seeking out rough surfaces in its enclosure such as branches and rocks which should be relatively smooth not pumice and should be readily accessible this may include course or naturally rock shaped hides The shedding will progress from nose to tail and takes anywhere from seven to 14 days Children s pythons should not be handled if they are showing signs of an impending shed or are actively shedding Snakes will generally not eat during a shed Force feeding during this time is not necessary and in fact can be harmful Once complete the shed skin should be removed and the snake checked for a complete shed including the brille eye scales Incomplete sheds Figure 4 One of our Children s pythons on the grassed area outside of the K1 Animal Facility We try and provide regular periods of sunning for each of our pythons on a weekly basis Photo G Martinic Hygiene Infection control is one of the basic principles of good animal care Staff should wash their hands and change gloves between enclosures They should use a quality disinfectant cleaner like F10sc Veterinary Disinfectant F10 Products Ampholytic sur factants and sequesterants to clean cages and furniture regularly This will reduce the risk of bacterial build up and the risk of Salmonella in particular Staff should remove shed skins uneaten foods and droppings daily and change water every two days Food and water dishes should be washed thoroughly and enclosure substrates changed weekly Staff should wash their hands thoroughly before and after handling reptiles Skin shedding Healthy Children s pythons will shed regularly particularly when they are growing Pythons should not be handled during the shed especially larger ones as they can be defensive during this time For this reason 120 There are many reasons for the shedding process to be incomplete or improper referred to as dysecdysis The most common related to poor husbandry and or nutrition Dysecdysis is a symptom of another problem and not a primary problem in itself Persistent or otherwise concerning incomplete sheds should be inspected by an experienced reptile veterinarian or experienced herpetologist This will help to rule out medically treatable causes such as mites or bacterial infections of the skin Other causes of dysecdysis include trauma dermatitis malnutrition and overhandling The veterinarian will advise the appropriate treatments once the underlying cause has been determined Husbandry techniques used to assist with incomplete sheds Humidity is ver y impor tant for reptiles with requirements varying from species to species Most snakes require an environment of 50 to 70 humidity Incomplete sheds can often be managed by increasing humidity Spraying daily with luke warm water from head to tail may be beneficial The butcher s paper in the unit can also be soaked to increase the humidity of a specific enclosure Snakes which retain their shedding for an extended period of time can be lightly sprayed with Shed ezi Spray or similar product on the residual scale which can then be gently rubbed off Sometimes the snake may be placed in a large container with warm water deeper than the girth of the snake to allow submersion but shallow enough such that it can easily keep its head above water while resting on the bottom of the container Never leave a 121

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The care of the Children s Python Antaresia childreni August 2020 Animal Technology andchildreni Welfare The care of the Children s Python Antaresia soaking snake unattended After 10 15 minutes soaking in the tub the residual shed should be easily removed with gentle rubbing Retained eye caps or spectacles brille can be very dangerous for snakes They can harbour dangerous bacteria as well as make it difficult for the snake to see Removing them is not difficult but can permanently damage the cornea of the snake if not done correctly Staff must first be properly trained in this procedure before performing it or otherwise contact the supervisor to organise for a consult with an experienced herpetologist or reptile veterinarian to remedy the complication poultry Although most pythons asymptomatically carry and shed some serotypes of salmonella large gut loads of serotypes that they do not usually harbour can cause disease which is often fatal Salmonella causes gastrointestinal infections Signs of gastrointestinal infection are weight loss lack of appetite and foulsmelling diarrhoea and vomiting This illness must be treated by a veterinarian Feeding In the wild Children s pythons diets consist of small reptiles small lizards geckos frogs birds and rodents but in captivity they can be fed exclusively on rodents Newborn Children s pythons are very small but they can effectively be fed neonatal mice As they grow they are fed progressively larger feeds starting with neonatal mice then progressing to finely furred young mice and eventually adult mice The adult Children s pythons in the K1 Animal Facility are fed primarily adult mice although they can also be fed medium sized rats Adults are generally fed 5 of their bodyweight weekly or 10 fortnightly unless they are blue The feed is always frozen for at least 48 hours before use and thawed freshly in hot water Ensure the prey is fully thawed before feeding Dry the prey with paper towel and allow it to cool for 5 10 minutes before offering it to ensure the food is not too hot The prey should be presented to the snake using tongs or a gloved hand They will generally strike at it very quickly and coil around it If the snake does not strike the food item can be left in the enclosure while the other snakes are feeding If the snake does not constrict or consume the food within 30 minutes the food should be removed and disposed of in a biological waste bag Feeds and refusals are to be recorded on the room activity log Healthcare Even with proper care Children s pythons may become sick It is impor tant to seek specialist reptile veterinarian care before an illness progresses too far Observation is the key to recognise if the animal is sick Watch for changes in behaviour weight loss loss of appetite changes in colour changes in faeces and changes in muscle tone or coordination incomplete or absence of ecdysis shedding and persistent aggression A range of conditions and illnesses that can afflict Children s pythons are presented below for the benefit of the Animal Technologist Stomatitis Commonly referred to as Mouth Rot or Canker usually results from poor husbandry and sanitation practices It may appear when a snake s immune system has been weakened by a concurrent disease e g pneumonia low temperatures or excessive humidity Symptoms of stomatitis include accumulation of cheesy material along the gums and around the tongue sheath blood spots and bruising in the gums an inability to shed skin dribbling saliva in severe cases swelling of the gums and subcutaneous tissue along the jaw may be seen possibly spreading to much of the head and neck Specialist veterinary advice should be sought if stomatitis is suspected Necrotising Dermatitis Often referred to as Scale Rot and Blister Disease often results from unhygienic conditions and excessive dampness most commonly affecting reptiles that come from dry arid regions of Australia Occasionally a deficiency of vitamins A and C can be a contributing factor Symptoms of scale rot include yellow red or greenish black discolouration of the scales particularly along the underside softening or swelling of the skin surface caused by serum seeping through sloughing of affected skin exposing subcutaneous tissue fluid filled blisters in the scales again primarily on the ventral surfaces and bruising due to blood in the tissue beneath the scales in advanced cases Seek veterinary advice if dermatitis is suspected Respiratory Infections Pneumonia Respiratory disease or pneumonia is quite common when conditions are too cold or damp for reptiles While the condition is normally the result of bacterial infection lungworms fungal disease and tumours can also cause similar signs Diagnosis may require a lung wash or radiology Symptoms of pneumonia include open mouthed breathing resting of the head in elevated positions tongue tips stick together or snake is unable to flick its tongue gurgling sound while breathing accumulation of frothy mucus at the back of the throat Specialist veterinary advice should be sought if pneumonia is suspected Infections Fungal infections can be exacerbated by a warm and damp environment These infections can occur in a cut or scrape and should be treated with an antifungal ointment under the direction of a veterinarian Salmonellosis Salmonella bacteria are readily found in the environment and can cause disease when present in large volumes in food It is often associated with Helminthic internal parasite Infestations Children s pythons can become infected with internal parasites 122 121

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Animal Technology and Welfare The care of the Children s Python Antaresia childreni The care of the Children s Python Antaresia childreni August 2020 notably nematodes roundworms and cestodes tapeworms These infections can be treated with Panacur fenbendazole for the nematodes and Drontal praziquantel pyrantel pamoate and febantel dog worming tablets for the tapeworms These medications can be inserted into food and fed to the adults This should only be done under veterinary instruction Ectoparasitic external parasite Infestations Children s pythons can become infested with mites although this is unlikely in the K1 facility unless new animals are introduced However mites could be inadvertently introduced by personnel if proper hygiene controls are not followed Symptoms of ectoparasite infestation include lying in the water bowl excessive rubbing against terrarium furnishings skin shedding which is slow uneven or does not occur the presence of very small red brown insects on the skin In the case of mites when a white pillowcase is placed in terrarium overnight mites will be visible moving slowly on the bottom the following morning Mites are extremely difficult to eliminate and require a two stage response which is to treat the animal as well as the habitat under veterinary direction Acknowledgements We gratefully acknowledge the dedication and skilful assistance of our Animal Technologists from the Animal Facilities team including Erin Mosmman Amy Woodley Sophie Ball and Lauren Grote We thank Gavin McKenzie Cluster Manager Institutes at the University for reviewing the manuscript This work has been made possible under Scientific Licence number SL100342 held by Gary Martinic and issued by the National Parks Wildlife Service of New South Wales Bibliography Ehmann H 1992 Encyclopedia of Australian Animals Reptiles Australian Museum and Angus Robertson Sydney Greer A E 1989 The Biology and Evolution of Australian Lizards Surrey Beatty Sons Sydney NSW Department of Environment and Climate Change 2008 Hygiene protocol for the control of disease in captive snakes NSW Department of Environment and Climate Change Sydney OEH 2013 Code of Practice for the Private Keeping of Reptiles State of NSW and Office of Environment and Heritage Sydney ISBN 978 1 74293 323 8 Weigel J 1988 Care of Australian Reptiles in Captivity Reptile Keepers Association Gosford 122 123

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August 2020 2020 Animal Technology and Welfare Feline assisted therapy a promising part of animal assisted therapy AAT ELISKA MI KOVA AND KRISTYNA MACHOV Department of Ethology and Companion Animal Science Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kam ck 129 16500 Praha 6 Suchdol Czech Republic Correspondence E Mi kov mickova eliska seznam cz Abstract The amount of literature describing the effect of human animal interactions is quite large The available sources describe its impact in many different areas environments and effects The majority of studies in this area are focussed on the use of the dog Moreover there are many works dedicated to therapeutic interaction with horses and only a small portion of studies is reporting usage of small or exotic mammals or farm animals It is very surprising that although the cat is the second most owned animal there is little literature describing the use of a cat as a beneficial assistant in human animal intervention The question is why Key words cat animal assisted therapy felineassisted therapy human animal interaction human cat interaction Introduction Human animal interactions are gaining popularity all over the world especially in the domain of animals effect on human health Marino 2012 describes the positive influence of these interactions in every human life stage 1 The review suggests that for example in childhood interactions with an animal can improve emotional stability in children with emotional disorders or increase the frequency of social contact in autistic children During middle age human animal interaction could be beneficial for example for adults with psychiatric disorders to enhance self efficiency and coping skills In older adults who sometimes suffer from loneliness the presence of an animal can also support social contact and there is also evidence that companion animals increase physical activity and physical functioning in healthy older adults 2 Nevertheless older adults with pathological conditions such as dementia also benefit from interactions with animals The positive effect was found in treating depressive symptoms apathy agitation and improving the quality of social interaction 3 Most research is focussed on canine assisted and equine assisted inter ventions however the number of published papers with the use of husbandry animals small mammals birds fish and other animals is growing 1 One of the animals that can be used as a therapeutic animal is the cat Surprisingly only a little research has been done on this topic although the cat is the second most owned animal 4 Reviewing the literature and using keywords like cat and animal assisted therapy cat and animal assisted activity or cat and human animal interaction we reviewed seven articles Few authors include cats as well as other animals in their work Somerville et al 2008 report no effect of dog or cattherapist on blood pressure or pulse 5 In this study authors used cats obtained from a shelter thus none of them had been kept in a home setting or even trained as a therapy animal Barak et al 2001 describe positive effect of a dog and a cat session on social functioning of geriatric schizophrenic patients but they did not differ results between these two animals so it is not possible to tell whether this effect was brought on by a dog or a cat or both 6 A study by Hart et al 2018 focussing specifically on human cat interaction suggests that cats can be social mediators for children with less severe forms of autistic spectrum disorder ASD 7 Moreover some family cats are more affectionate towards ASD children than towards adults or other children in the family Cats improve ASD children s quality of life mood and encourage them to talk or express emotions 7 Cats can decrease the negative mood of their owners although the effect on positive mood was not observed 8 Fur thermore adults aged 50 with previous cat ownership are at a lower risk of developing cardiovascular disease and myocardial infarction however no protective effect has been associated with present cat ownership 9 Stasi et al 2004 reported that geriatric patients undergoing a 6 week therapy session with a cat showed lower depressive symptoms and a decrease in blood pressure values 10 Another study that must be considered on this topic is a clinical note published by Wells et al 1997 11 Authors 125 123

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Feline assisted therapy a promising part part of of animal assisted animal assisted therapy therapy AAT AAT Animal Technology and Welfare describe changes in communication of psychotherapy clients after feral cats started entering mental health facility Persons who were seriously vulnerable such as persons with schizophrenia multiple personality disorders or severe character pathology bonded with the animal and started speaking to the therapist about themselves through the cats An important change was observed in victims of childhood sexual physical and emotional abuse These clients paid par ticular attention to the ways the cats manage to survive in the winter when they face other hardships and natural enemies They quickly identified with the cats who were also seen as victims and survivors because of their own perceived experiences of hardship The authors then describe the situation of an obsessive compulsive client who was distressed by her obsessive conviction that she could easily hurt others with whom she interacted After interacting with the cat she realised the cat s superior survival capabilities and found out the cat sensed no danger from her This resulted in restructuring the client s faulty thinking Conclusion According to available literature it seems that felineassisted therapy has great potential and the authors hope to conduct future studies on this subject Unfortunately there is currently little literature relating to this area of interest and it is very difficult to followup on other works Conflict of interests The authors have no conflicts of interests to declare References 1 2 3 4 5 6 7 Marino L 2012 Construct validity of animal assisted therapy and activities How important is the animal in AAT Anthrozo s Vol 25 sup1 s139 s151 Mi kov E Machov K Dad ov K Svobodov I 2019 Does Dog Ownership Affect Physical Activity Sleep and Self Repor ted Health in Older Adults International journal of environmental research and public health Vol 16 No 18 3355 Bernabei V De Ronchi D La Ferla T et al 2013 Animal assisted interventions for elderly patients affected by dementia or psychiatric disorders a review Journal of psychiatric research Vol 47 No 6 762 773 Staats S Wallace H Anderson T 2008 Reasons for companion animal guardianship pet ownership from two populations Society Animals Vol 16 No 3 279 291 Somerville J W Kruglikova Y A Robertson R L Hanson L M MacLin O H 2008 Physiological responses by college students to a dog and a cat Implications for pet therapy Barak Y Savorai O Mavashev S Beni A 2001 Animal assisted therapy for elderly schizophrenic patients a one year controlled trial The American Journal of Geriatric Psychiatry Vol 9 No 4 439 442 Hart L A Thigpen A P Willits N H Lyons L A HertzPicciotto I Hart B L 2018 Affectionate interactions 126 124 8 9 10 11 August 2020 of cats with children having autism spectrum disorder Frontiers in veterinary science Vol 5 39 Turner D C Rieger G Gygax L 2003 Spouses and cats and their effects on human mood Anthrozo s Vol 16 No 3 213 228 Qureshi A I Memon M Z Vazquez G Suri M F K 2009 Cat ownership and the Risk of Fatal Cardiovascular Diseases Results from the Second National Health and Nutrition Examination Study Mortality Follow up Study Journal of vascular and interventional neurology Vol 2 No 1 132 135 Stasi M F Amati D Costa C et al 2004 Pettherapy a trial for institutionalized frail elderly patients Archives of gerontology and geriatrics Vol 38 Supplement 9 407 412 Wells E S Rosen L W Walshaw S 1997 Use of feral cats in psychotherapy Anthrozo s Vol 10 No 2 3 125 130

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August 2020 2020 Animal Technology and Welfare The care of Central and Pygmy Bearded Dragons ALEXANDER HOSKING and GARY MARTINIC Reptile House K1 Animal Facility Technical Support Services Western Sydney University Hawkesbury Campus Richmond NSW Australia Correspondence g martinic westernsydney edu au Abstract This article describes the care of central bearded dragons Pogona vitticeps Figure 1 as well as pygmy bearded dragons Pogona henrylawsoni It includes information on housing feeding handling and sexing technique as well as the general health care of these animals The information presented is derived from Standard Operating Procedures which have been cowritten by the authors It is hoped that the information presented herewith may assist novice Animal Technicians with responsibilities for caring for these species in an animal research facility or a zoological setting Both males and females can show this behaviour To appear even more menacing the bearded dragon may also gap or open its mouth very wide A common way bearded dragons show dominance is through head bobbing Bearded dragons can also show submission through slowly waving around one arm The bearded dragons in the K1 Reptile Facility are captive bred very calm and used to being handled Housing and environmental conditions Central bearded dragons can grow to a length of between 40 60cm while adult pygmy bearded dragons reach 20 25cm in length and a suitably sized enclosure is essential to ensure their wellbeing For one adult central or pygmy bearded dragon an enclosure of at least 90x 45x 60cm is recommended Figures 2 and 3 Adults are often territorial and are single housed to reduce stress and aggression The enclosure should be dry and include a basking rock or log as well as branches for the animals to climb on Figures 2 4 The water dish should be heavy to prevent Figure 1 A male central bearded dragon shown in its enclosure Photo A Hosking Background Bearded dragons are active semi arboreal lizards which are socially interactive and have a variety of communication behaviours including head bobbing beard displays eye bulging and leg waving Bearded dragons get their name from the dark coloured neck skin that they use as part of their displays The central bearded dragon occurs in a band across the semi arid interior of eastern Australia including western New South Wales and the Riverina region whereas the pygmy beared dragons are found on the black soil plains of the central western parts of Queensland During breeding season to display dominance or if startled or threatened a dragon may puff out its beard Figure 2 This photograph shows a suitably sized enclosure for a central bearded dragon containing a wide sliding viewing window and the front of the enclosure and wire mesh air inlet above The white light source is a 25W compact globe and the infrared globe is a 150W heat globe The sand substrate in use can be clearly seen as well as enrichment devices such as wooden log branch and basking rock at left and the food and water dishes at right Courtesy A Hosking 127 125

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The care of central Centraland andWelfare Pygmybearded Beardeddragons Dragons Animal Technology and pygmy being tipped over and should be positioned at the cooler end of the enclosure away from any heat globes Butcher s paper or washed sand is used as the substrate on the bottom of the enclosures In the case of Butcher s paper it is placed 2 3 layers thick Figure 3 spot cleaned daily and replaced weekly In the case of washed sand it covers the floor 1 inch deep Figure 2 is spot cleaned daily and replaced fully every 6 months While sand is preferred some pygmy dragons were found to be ingesting the substrate and so in those cases butcher s paper is used The reptile rooms are maintained at constant temperature of 22 C 2 C and a relative humidity of between 40 60 The air conditioning system provides positive pressure ventilation and between 10 12 changes hour August 2020 compact globe should be used as a source of artificial UV lighting in the dragon s enclosure A higher intensity globe may be required depending on the average basking distance of the lizard The lighting should be set to a day and night cycle with UV lights running for approximately 10 12 hours each day set on an electronic light timer switch The dragons should also have access to unfiltered natural light at least once a week This is achieved by 30 60 minutes in enclosed pens outdoors with water and shade cover available Figure 5 Figure 3 This photograph depicts the enclosure set up for a pygmy bearded dragon using butcher s paper as a substrate instead of sand Courtesy A Hosking Figure 5 Central bearded dragon and shingleback lizard sunning in an enclosed pen top opened for photo Photo A Hosking Figure 4 Close up view inside the enclosure showing a central bearded dragon perched high on a rock platform used as a form of environmental enrichment Courtesy A Hosking Enclosure lighting Ultraviolet light UV plays an important role in a bearded dragon s growth and development and so correct lighting is crucial Both UVA and UVB light must be available to ensure normal bodily functions and for the production of vitamin D A 25 watt UVB tube or 128 126 Enclosure heating Providing adequate temperature gradients within a dragon s enclosure is essential for their health and wellbeing They need a temperature gradient so they can move around the enclosure to a temperature that suites them Bearded dragons require a hot basking spot maintained between 35 40 C Figure 4 They should have access to an elevated piece of timber or a rock ornament to allow them to bask within 30cm of the heat source The cool end of the enclosure should be maintained between 24 26 C and should not drop

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August 2020 below 18 21 C at night Temperatures are regulated by a thermostat in enclosures and this should be checked daily to ensure the thermostat and the globes are functioning as they should Figure 6 A young pygmy bearded dragon shown observing the front of its enclosure as it enjoys basking in the heat produced by the infra red globe inside its enclosure Courtesy G Martinic Room lighting Room lighting within the separate reptile holding rooms is maintained on an artificial photoperiod set at 12 12 hrs light dark provided by individual electronic light timer switches with manual override features to each room Figure 7 Figure 7 Electronic Light Timer Switch as used in each reptile holding room to provide room lighting The and Bearded Animal Technology andDragons Welfare Thecare careofofCentral central andPygmy pygmy bearded dragons Feeding Bearded dragons are omnivorous eating both insects and vegetables Adult central bearded dragons will also eat newborn mice on occasion At the K1 Animal Facility our dragons are fed a vegetable mix dusted with calcium two days per week usually on Mondays and Fridays and a meal of live crickets mealworms and cockroaches once a week usually on Wednesdays This is part of a feeding schedule that we have developed in our animal facility A suitable mix of vegetables for bearded dragons includes raw shredded carrot collard greens dandelion greens mustard greens kale peas and beans Spinach and broccoli should be avoided as too much can be harmful to the lizards Fruits such as apples pears strawberries and grapes may also be fed Vegetables should be finely chopped when feeding to bearded dragons especially in the case of juveniles or pygmy bearded dragons The vegetable fruit mix dusted with calcium is offered on a food dish and insects are offered live The crickets and cockroaches will roam and hide within the enclosure providing behavioural enrichment for the bearded dragons while the mealworms are placed in the food dish to avoid them burrowing into the sand All food items including insects should be smaller than the space between the lizard s eyes to reduce the risk of lizards choking on food items All insects used for feeding must also be fed a nutritious diet to ensure the nutrients are passed on to the dragon Uneaten food should be removed daily Handling Bearded dragons are hardy semi arboreal lizards and are tolerant of handling especially individuals raised in captivity that have been handled often Being a semi arboreal species means they prefer to be held more vertically than horizontally As such they should be held with their head higher than their body tail with their tail supported Figure 8 Bearded dragons are often comfortable clinging onto a handler s arm or shoulder Figure 8 Correct handling of adult central bearded dragon left and pgymy bearded dragon right Courtesy A Hosking 129 127

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The care of central Centraland andWelfare Pygmybearded Beardeddragons Dragons Animal Technology and pygmy Sexing technique They have a captive lifespan of approximately 10 15 years and can be sexed by examination of their perianal area to visualise the hemipenal bulges that are present in male animals Figure 9 or the lack thereof in females Figure 10 Sexing hatchling bearded dragons can be difficult as they are so small and the hemipenes are harder to expose In a bearded dragon that is 4 or more weeks old the process is a little easier To safely and effectively expose the hemi penal bulges one needs to place the dragon in the palm of one s hand or on a sturdy surface where the body and legs of the dragon will be supported and with the tail facing towards you and then gently bending the tail straight up and looking just above the ventral opening Figures 9 10 In addition to the hemipenes an August 2020 examination of the femoral pores may be valuable The femoral pores are located around the thigh area and they contain pheromone carrying wax On a male dragon the femoral pores will be larger than they are on female dragons Figure 11 Figure 11 Comparison of femoral pores in male left and female right bearded dragons Hygiene Infection control is one of the basic principles of good animal care Staff must wash their hands and change gloves between enclosures They should use a quality disinfectant cleaner like F10sc to clean cages and furniture regularly This will reduce the risk of bacterial build up and the potential risk of Salmonella infection Staff must remove shed skins uneaten foods and droppings daily and change water every two days Food and water dishes must be washed thoroughly and enclosure substrates changed weekly Staff should wash hands thoroughly before and after handling reptiles Figure 9 Hemipenal bulges evident in a male P vitticeps Courtesy beardeddragon org Skin shedding Healthy bearded dragons will shed regularly particularly while they are growing It is recommended not to handle animals while they are in shed Animals should be examined carefully after each shed paying particular attention to the animal s feet toes and tail During the shed old skin can sometimes get stuck and cause constriction around these delicate areas Either misting the dragon lightly with shed spray or bathing the dragon in warm shallow water will help soften old skin and lift it from the body Do not leave a bearded dragon unsupervised in any depth of water Healthcare Figure 10 Absence of hemipenal bulges appearing as one bulge above the vent in a female P vitticeps Courtesy beardeddragon org 130 128 Bearded dragons in captivity can live for approximately 10 15 years Even with proper care they may become sick and require specialist reptile veterinary care Observation is the best way to recognize if an animal is sick Watch for changes in behaviour weight loss loss of appetite changes in colour change in faeces and change in muscle tone and coordination A range of conditions and illnesses that can afflict Bearded dragons have been presented for the benefit of the Animal Technicians

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August 2020 Genetic disorders Genetic disorders are hereditary health problems that some bearded dragons are born with They often occur in the offspring of dragons that are closely related Some genetic diseases include deformed limbs tails and possibly extra appendages Dietary deficiencies When a bearded dragon has vitamin deficiencies it can cause seizures stunted growth deformities poor bone growth brittle bones and death Vitamin D3 and calcium problems can be avoided by making sure that the bearded dragon receives both proper lighting and the required dietary supplements A deficiency in betacarotene will cause the coloration of bearded dragons to fade over time This is especially prevalent in lizards with bright colour variations The easiest way to rectify the problem is to include carrots and yellow vegetables in the dragon s diet Overfeeding Overfeeding is of particular risk in baby and juvenile dragons It can cause paralysis in the hindquarters and is usually fatal It results from the pressure of the food bolus on the spinal nerves As such it is imperative that animal technicians do not overfeed young dragons or offer them insects that are too large Egg binding Egg binding occurs when a female cannot pass the mature eggs developed in her reproductive system It can be caused by calcium deficiency being under or overweight or if the bearded dragon is unable to find a suitable egg nest If bearded dragons are gravid ensure that they have a deep soil substrate where they can dig a nest If there is a suitable spot to dig a nest and the animal stops eating for days and keeps looking for a different place to lay its eggs there is a problem which could be egg binding Staff should not attempt to treat egg binding and should consult a specialist veterinarian Injuries It is not uncommon for bearded dragons that are housed together to inflict injury upon less aggressive lizards They may lose toes or tail tips from combat behaviour Any injuries should be treated with a disinfectant ointment If aggression persists separate caging is the only way to stop the fighting Infections Respiratory Infections Signs of a respiratory infection include gaping noisy breathing and mucus discharge from the nose and mouth It is usually caused by low temperatures high humidity or both Bearded dragons must be kept warm and at relatively low humidity If any of these symptoms occur consult a specialist veterinarian without delay The and Bearded Animal Technology andDragons Welfare Thecare careofofCentral central andPygmy pygmy bearded dragons Gastrointestinal Infections Signs of gastrointestinal infections include weight loss lack of appetite and foul smelling diarrhoea and vomiting This type of illness must be treated by a specialist veterinarian Fungal Infections Fungal infections may be caused cryptosporial and microsporidial fungi and are usually exacerbated by warm damp environments These infections can often occur in a cut or scrape and should be treated with an anti fungal ointment under the direction of a specialist veterinarian Coccidial Infections Coccidia are a microscopic parasite that bearded dragons normally have in their intestinal tract in small amounts This is a normal occurrence both in the wild and in captivity A minimal coccidian count is usually not troublesome in a healthy dragon Their immune system is able to keep the amount of coccidia in their system in check Some dragons with coccidiosis may not show any clinical signs These individuals often act as carriers of the parasite and can be a source of the disease spreading However if the animals are stressed or sick their immune system becomes compromised they can become over whelmed by the coccidian parasite Symptoms include diarrhoea with or without blood weight loss and loss of appetite Faecal microscopy is normally employed to determine the parasite load and consultation with a specialist veterinarian may be required to establish a suitable treatment plan Protozoal Infections Flagellated protozoans also called flagellates are common endoparasites of bearded dragons Low numbers are considered normal in healthy individuals but treatment should commence if moderate to severe numbers are present Higher loads have been shown to cause gastrointestinal disease The flagellates are acquired by exposure to infected cysts in contaminated food and water as well as during copulation Helminthic internal parasite Infestations These infections can occur from Roundworms Hookworms and Pinworms A severe infection of internal parasites can starve a dragon of nutrients and can cause anaemia if left unchecked Most infested Bearded dragons only show general clinical signs if infested with internal parasites Symptoms can vary but may include partial or complete anorexia weight loss and or poor body condition dehydration weakness lying flat and lethargy or dullness and difficulty digesting which all lead to a loss of appetite Other more specific symptoms may include diarrhoea blood tinged stools and vomiting The additional stress on the immune system can leave the bearded dragon susceptible to secondary infections Faecal microscopy is employed to determine the parasite load and consultation with a specialist veterinarian may be required to establish a suitable treatment plan 131 129

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The care of central Centraland andWelfare Pygmybearded Beardeddragons Dragons Animal Technology and pygmy Ectoparasitic external parasite Infestations Mites are extremely difficult to eliminate and often require that an infested animal and its habitat be treated under specialist veterinary advice Appendix For information of sexing bearded dragons please refer to the link below https www beardeddragon org articles sexing Acknowledgements We gratefully acknowledge the dedication and skilful assistance of our Animal Technicians from the Animal Facilities team including Erin Mosmman Amy Woodley Sophie Ball and Lauren Grote We also thank Gavin McKenzie Cluster Manager Institutes at the University for critically reviewing the manuscript This work has been made possible under Scientific Licence number SL100342 held by Gary Martinic and issued by the National Parks Wildlife Service of New South Wales Bibliography https www bushheritage org au species beardeddragons gclid EAlolQobChM Ehmann H 1992 Encyclopedia of Australian Animals Reptiles Australian Museum and Angus Roberston Sydney Greer A E 1989 The Biology and Evolution of Australian Lizards Surrey Beatty Sons Sydney NSW Department of Environment and Climate Change 2008 Hygiene protocol for the control of disease in captive snakes NSW Department of Environment and Climate Change Sydney OEH 2013 Code of Practice for the Private Keeping of Reptiles State of NSW and Office of Environment and Heritage Sydney ISBN 978 1 74293 323 8 Vosjoli P Mailloux R Donoghue S Klingenberg R and Cole J 2001 The Bearded Dragon Manual Advanced Vivarium Systems USA Weigel J 1988 Care of Australian Reptiles in Captivity Reptile Keepers Association Gosford Wilson S K and Knowles D G 1988 Australia s Reptiles A Photographic Reference to the Terrestrial Reptiles of Australia William Collins Sydney 132 130 August 2020

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August 2020 2020 Animal Technology and Welfare CONTENU DE LA REVUE Rapport de la r union de 2019 du Groupe de protection des rongeurs de la RSPCA UFAW CHLOE STEVENS EMILY FINNEGAN JASMINE CLARKSON 3 CHARLOTTE BURNS SONIA BAINS COLIN GILBERT CAROLINE CHADWICK SAMANTHA IZZARD CHARLOTTE INMAN PENNY HAWKINS HUW GOLLEDGE R sum Le Groupe de protection des rongeurs de la RSPCA UFAW tient chaque automne depuis 26 ans une r union d une journ e afin que ses membres puissent discuter de la recherche actuelle sur le bien tre changer des points de vue sur les questions de bien tre et partager des exp riences de la mise en uvre des 3R de remplacement r duction et raffinement en ce qui concerne l utilisation de rongeurs L un des principaux objectifs du groupe est d encourager la r flexion concernant l exp rience de vie enti re des rongeurs de laboratoire en veillant ce que chaque impact n gatif potentiel sur leur bien tre soit examin et minimis La 26e r union s est tenue en novembre 2019 l Universit de Birmingham et a r uni plus de 60 d l gu s issus de tout le Royaume Uni Le th me de la r union de cette ann e tait La sensibilit le bien tre positif et le bien tre psychologique La r union s est ouverte par un discours sur la sensibilit des animaux et sur ce qu elle signifie pour leur bien tre Ce th me a ensuite t repris dans la session de discussion la fin de la journ e les d l gu s discutant de leurs propres points de vue sur la sensibilit des animaux Certaines discussions abordaient divers raffinements y compris l enrichissement les m thodes de manipulation affin es et la formation au clicker ainsi que la mani re dont ces raffinements affectent le comportement et l tat affectif motionnel des animaux D autres discussions ont abord de nouvelles fa ons de surveiller les animaux pour assurer une meilleure valuation du bien tre traiter la mortalit n onatale chez les souris de laboratoire et comment de petits changements peuvent avoir un effet positif cumulatif sur la vie des animaux de laboratoire La journ e s est termin e par une mise jour de l unit de r gulation des animaux dans la science ASRU du Home Office concernant leurs inspections th matiques li es au manque de nourriture et d eau la manipulation raffin e des souris et la r utilisation des aiguilles Ce rapport r sume la r union et se termine par une liste de points d action que les lecteurs peuvent envisager de soulever dans leurs propres tablissements Mots cl s rongeurs lapins bien tre exp rience de vie sensibilit bien tre psychologique R duction des effets n gatifs de la m thionine sur les param tres osseux des embryons de poulets de chair par injection intra uf de vitamine B12 MOHAMMAD NASER NAZEM SHIMA TASHAROFI NEGIN AMIRI SEPIDEH SABZEKAR Correspondance Shima tasharrofi gmail com R sum La m thionine est un acide amin contenant du soufre En plus de sa fonction g n rale en tant que composante des prot ines la m thionine est essentielle pour diverses r actions la m thyltransf rase Bien que la m thionine soit essentielle la synth se du cartilage ses effets n gatifs sur les maladies osseuses telles que l ost oporose et la dyschondroplasie tibiale ne doivent pas tre ignor s Ces effets n gatifs peuvent tre vit s par un traitement la vitamine B12 Cette tude a donc analys les effets de l injection intra uf de certaines solutions y compris 1 ml d eau distill e de m thionine de vitamine B12 de m thionine avec vitamine B12 suppl mentaire sur certains param tres osseux et s riques des embryons de poulets Ross 308 Le sac vitellin d embryons de poussins fertiles a t inject au 4e jour d incubation Au 18e jour les param tres des tibias gauches des embryons ont t examin s quantit de cellules d ost ocyte et d ost oblaste et mesure de leurs longueurs paisseur des trab culaires et des s diments de collag ne ainsi que les param tres s riques quantit de phosphatase alcaline et d homocyst ine La m thionine seule a caus une perte de longueur des ost ocytes de hauteur des ost oblastes et de s dimentation du collag ne tandis que les groupes trait s en plus avec de la vitamine B12 taient comparables au groupe t moin La concentration s rique en phosphatase alcaline des deux groupes inject s la vitamine B12 B12 et B12 m thionine tait significativement sup rieure celle du groupe t moin La concentration s rique en homocyst ine tait plus lev e dans les deux groupes inject s la m thionine m thionine et vitamine B12 m thionine que dans le groupe t moin Les donn es obtenues dans la pr sente tude sugg rent que la suppl mentation en vitamine B12 peut pr venir les d fauts dans les param tres de d veloppement osseux li s la m thionine Mots cl s embryons de poulets de chair injection in ovo m thionine vitamine B12 133 131

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Paper Translations AnimalSummary Technology and Welfare August 2020 Soins prodigu s aux pythons de Children Antaresia childreni ALEXANDER HOSKING et GARY MARTINIC Correspondance G Martinic westernsydney edu au R sum Cet article d crit la prise en charge des pythons de Children Antaresia childreni Figure 1 Il comprend des informations sur le logement l alimentation et la manipulation ainsi que les soins de sant g n raux de ces animaux Les informations pr sent es ont t cor dig es par les auteurs et proviennent des proc dures op rationnelles standard utilis es la Western Sydney University Il est esp rer que les informations pr sent es ci apr s peuvent aider les techniciens animaliers novices en charge de ces esp ces dans un centre de recherche sur les animaux ou dans un cadre zoologique Mots cl s Python de Children soins de sant logement conditions environnementales Th rapie assist e par f lin un aspect prometteur de la th rapie assist e par animal TAA ELISKA MI KOV et KRISTYNA MACHOV R sum La quantit de litt rature d crivant l effet des interactions entre l homme et l animal est assez g n reuse Les sources disponibles d crivent son impact dans de nombreux domaines environnements et effets La majorit des tudes men es dans ce domaine sont ax es sur l utilisation du chien Il existe galement de nombreux ouvrages consacr s l interaction th rapeutique avec les chevaux et seule une petite partie des tudes rapporte l utilisation de petits mammif res d animaux exotiques ou de ferme Il est tr s surprenant que bien que le chat soit le deuxi me animal le plus poss d il n existe que peu de litt rature d crivant l utilisation d un chat comme assistant b n fique dans l intervention humain animal La question est de savoir pourquoi Mots cl s chat th rapie assist e par animal th rapie assist e par f lin interaction homme animal interaction homme chat Soins prodigu s aux agames barbus et aux agames barbus nains ALEXANDER HOSKING et GARY MARTINIC R sum Cet article d crit les soins prodigu s aux agames barbus Pogona vitticep Figure 1 ainsi qu aux agames barbus nains Pogona henrylawsoni Il comprend des informations sur le logement l alimentation la manipulation la technique de sexage ainsi que les soins de sant g n raux de ces animaux Les informations pr sent es proviennent des proc dures op rationnelles standard qui ont t cor dig es par les auteurs Il est esp rer que les informations pr sent es ci apr s peuvent aider les techniciens novices en charge de la prise en charge de ces esp ces dans un centre de recherche sur les animaux ou dans un cadre zoologique 134 132

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August 2020 2020 Animal Technology and Welfare INHALTVERZEICHNIS Bericht zur Tagung der RSPCA UFAW Tierschutzgruppe f r Nagetiere 2019 CHLOE STEVENS EMILY FINNEGAN JASMINE CLARKSON CHARLOTTE BURNS SONIA BAINS COLIN GILBERT CAROLINE CHADWICK SAMANTHA IZZARD CHARLOTTE INMAN PENNY HAWKINS HUW GOLLEDGE Abstract Die RSPCA UFAW Tierschutzgruppe f r Nagetiere veranstaltet seit 26 Jahren jeden Herbst ein eint giges Treffen bei dem die Mitglieder ber Themen der aktuellen Tierschutzforschung diskutieren und Erfahrungen und Meinungen ber nagerrelevante Tierschutzfragen sowie ber die Umsetzung der 3R Prinzipien Vermeidung Verringerung und Verbesserung des Einsatzes von Nagern austauschen k nnen Ein Hauptziel der Gruppe ist es zum Nachdenken ber die Gesamtheit der Lebenserfahrungen von Nagern als Laborversuchstiere anzuregen und sicherzustellen dass jede potenzielle Beeintr chtigung ihres Wohlergehens gepr ft und auf ein Minimum gesenkt wird Die 26 Tagung fand im November 2019 an der Universit t von Birmingham mit mehr als 60 Teilnehmern aus ganz Gro britannien statt Das Thema der diesj hrigen Veranstaltung war Empfindungsf higkeit aktiver Tierschutz und seelisches Wohlbefinden Er ffnet wurde sie mit einem Vortrag ber die Empfindungsf higkeit von Tieren und die damit verbundenen Konsequenzen f r den Tierschutz Dieses Thema wurde in der Diskussionsrunde am Ende des Tages erneut aufgegriffen bei der Teilnehmer ihre Ansichten zur Empfindungsf higkeit von Tieren darlegten Weitere Vortr ge befassten sich mit verschiedenen Verbesserungsmethoden wie Anreicherung optimierte Handhabung und Clicker Training sowie damit wie diese Verbesserungen Verhalten und Gem tszustand emotionale Befindlichkeit von Tieren beeinflussen Dar ber hinaus wurden neue M glichkeiten der Beobachtung von Tieren zur besseren Beurteilung des Wohlergehens sowie die Mortalit t neugeborener Laborm use diskutiert und er rtert inwiefern bereits kleine nderungen einen positiven kumulativen Effekt auf das Leben von Versuchstieren haben k nnen Der Tag endete mit einem Update der Home Office Animals in Science Regulation Unit ASRU Regulierungsstelle des britischen Innenministeriums f r in der Wissenschaft verwendete Versuchstiere ber ihre Kontrollen in Bezug auf Nahrungs und Wassermangel die verbesserte Handhabung von M usen und die Wiederverwendung von Nadeln Dieser Bericht fasst die Tagung zusammen und endet mit einer Liste von Aktionspunkten die Leser f r ihre eigenen Einrichtungen in Betracht ziehen k nnen Schlagw rter Nagetiere Kaninchen Tierschutz Lebenserfahrungen Empfindungsf higkeit seelisches Wohlbefinden Reduzierung der negativen Wirkungen von Methionin auf Knochenparameter in Broilerembryonen durch Intra EiInjektion von Vitamin B12 MOHAMMAD NASER NAZEM SHIMA TASHAROFI NEGIN AMIRI SEPIDEH SABZEKAR Korrespondenz Shima tasharrofi gmail com Abstract Methionin ist eine schwefelhaltige Aminos ure Zus tzlich zu seiner allgemeinen Funktion als Bestandteil von Proteinen ist Methionin f r eine Vielzahl von Methyltransferase Reaktionen von entscheidender Bedeutung Obwohl Methionin f r die Knorpelsynthese u erst wichtig ist d rfen seine negativen Auswirkungen auf Knochenerkrankungen wie Osteoporose und tibiale Dyschondroplasie nicht ignoriert werden Diesen negativen Folgen kann m glicherweise durch eine Vitamin B12 Therapie entgegengewirkt werden Die vorliegende Studie wurde daher durchgef hrt um die Auswirkungen der Intra Ei Injektion einiger L sungen darunter 1 ml destilliertes Wasser Methionin B12 Methionin mit zus tzlichem B12 auf einige Knochen und Serumparameter bei Broilerembryonen Ross 308 zu untersuchen Es wurden befruchtete H hnereizellen am Tag 4 der Inkubation in den Dottersack injiziert Am 18 Tag wurden die Embryonen auf Parameter der linken Tibia Menge an Osteozyten und Osteoblastenzellen und Messung ihrer L nge Dicke der Trabekel und Kollagenablagerung und Serumparameter Menge an alkalischer Phosphatase und Homocystein untersucht Methionin allein verursachte einen Verlust an Osteozytenl nge Osteoblastenh he und Kollagenablagerung w hrend die Gruppen die zus tzlich mit Vitamin B12 behandelt wurden mit der Kontrollgruppe vergleichbar waren Die Konzentration der alkalischen Phosphatase im Serum war in beiden mit B12 injizierten Gruppen B12 und B12 Methionin signifikant h her als in der Kontrollgruppe Die Homocysteinkonzentration des Serums war in beiden mit Methionin injizierten Gruppen Methionin und B12 Methionin h her als bei der Kontrollgruppe Die in der vorliegenden Studie gewonnenen Daten legen nahe dass eine Supplementation mit Vitamin B12 durch Methionin verursachte Defekte in den Knochenentwicklungsparametern verhindern k nnte Schlagw rter Broilerembryonen In ovo Injektion Methionin B12 135 133

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Paper Translations AnimalSummary Technology and Welfare August 2020 Pflege gefleckter Pythons Antaresia childreni ALEXANDER HOSKING und GARY MARTINIC Korrespondenz G Martinic westernsydney edu au Abstract Dieser Artikel befasst sich mit der Pflege von gefleckten Pythons Antaresia childreni Abb 1 Er enth lt Informationen ber Unterbringung F tterung Handhabung sowie ber Fragen allgemeiner Gesundheitsf rsorge f r diese Tiere Die hier vorgelegten Informationen stammen aus an der Western Sydney University verwendeten Standardarbeitsanweisungen die von den Autoren mitverfasst wurden Sie sollen jungen Tiertechnikern Unterst tzung bieten die in einer Tierforschungseinrichtung oder einem zoologischen Umfeld f r die Pflege dieser Arten verantwortlich sind Schlagw rter Gefleckter Python Gesundheitsf rsorge Unterbringung Umweltbedingungen Katzengest tzte Therapie ein vielversprechender Teil tiergest tzter Therapie AAT ELISKA MI KOV und KRISTYNA MACHOV Abstract Es existiert eine F lle an Literatur die die Effekte von Mensch Tier Beziehungen beschreibt Die verf gbaren Quellen skizzieren die diesbez glichen vielf ltigen Auswirkungen in vielen verschiedenen Bereichen und Umgebungen Die Mehrzahl der Studien in diesem Bereich konzentriert sich auf den Einsatz des Hundes Dar ber hinaus widmen sich zahlreiche Arbeiten der therapeutischen Beziehung mit Pferden und ein geringer Teil der Studien berichtet ber die Verwendung von kleinen oder exotischen S uge bzw Nutztieren Es berrascht in diesem Zusammenhang dass sich kaum Literatur mit der Verwendung der Katze als n tzlicher Helfer im Rahmen von Mensch Tier Beziehungen befasst obwohl die Katze das zweith ufigste Haustier ist Die Frage ist warum Schlagw rter Katze tiergest tzte Therapie katzengest tzte Therapie Mensch Tier Beziehung Beziehung zwischen Mensch und Katze Pflege von streifenk pfigen Bartagamen und Zwergbartagamen ALEXANDER HOSKING und GARY MARTINIC Korrespondenz G Martinic westernsydney edu au Abstract Dieser Artikel befasst sich mit der Pflege von streifenk pfigen Bartagamen Pogona vitticeps Abb 1 und von Zwergbartagamen Pogona henrylawsoni Er enth lt Informationen ber Unterbringung F tterung Handhabung und Geschlechtsbestimmung sowie ber Fragen allgemeiner Gesundheitsf rsorge f r diese Tiere Die hier vorgelegten Informationen stammen aus Standardarbeitsanweisungen die von den Autoren mitverfasst wurden Sie sollen jungen Tiertechnikern Unterst tzung bieten die in einer Tierforschungseinrichtung oder einem zoologischen Umfeld f r die Pflege dieser Arten verantwortlich sind 136 134

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August 2020 2020 Animal Technology and Welfare INDICE DELLA REVISTA Resoconto dell incontro del RSPCA UFAW Rodent Welfare Group del 2019 CHLOE STEVENS EMILY FINNEGAN JASMINE CLARKSON CHARLOTTE BURNS SONIA BAINS COLIN GILBERT CAROLINE CHADWICK SAMANTHA IZZARD CHARLOTTE INMAN PENNY HAWKINS HUW GOLLEDGE Abstract Ogni autunno da ormai 26 anni il RSPCA UFAW Rodent Welfare Group Gruppo sul benessere dei roditori organizza un incontro di un giorno per consentire ai suoi membri di discutere degli studi attuali di ricerca sul benessere di scambiarsi opinioni sulle questioni legate al benessere e di condividere esperienze di applicazione del principio delle 3 R sostituzione replacement riduzione reduction e perfezionamento refinement in relazione all uso di roditori Uno degli obiettivi primari del Gruppo quello di invitare a prendere in considerazione l intero percorso di vita dei roditori da laboratorio accertandosi che venga valutato e minimizzato ogni possibile impatto negativo sul loro benessere Il 26 incontro ha avuto luogo a novembre 2019 presso l Universit di Birmingham e ha visto la partecipazione di oltre 60 delegati britannici Il tema di quest anno era senzienza welfare positivo e benessere psicologico L incontro si aperto con una discussione sulla senzienza degli animali e su cosa significhi per il benessere animale un argomento poi ripreso nella sessione di fine giornata che ha permesso ai delegati di esprimere le loro opinioni su tale aspetto Alcune discussioni erano improntate su vari perfezionamenti tra cui l arricchimento metodi di gestione pi affinati e il clicker training e su come essi incidano sul comportamento e sullo stato affettivo emotivo degli animali Altri discorsi hanno posto l enfasi su nuovi metodi di monitoraggio degli animali volti a garantire una migliore valutazione del benessere sulla gestione della mortalit neonatale nei topi da laboratorio e su come piccoli cambiamenti possano avere un effetto positivo cumulativo sull esistenza degli animali da laboratorio La giornata si conclusa con un aggiornamento da parte dell Animals in Science Regulation Unit ASRU del Ministero dell Interno britannico sulle loro ispezioni a tema relative alla mancanza di cibo e acqua a una gestione pi perfezionata dei topi e al riutilizzo degli aghi Il presente resoconto riassume l incontro e termina con un elenco di punti di intervento che i lettori possono portare all attenzione delle loro strutture Parole chiave Roditori Conigli Benessere Percorso di vita Senzienza Benessere psicologico Riduzione degli effetti negativi della metionina sui parametri ossei di embrioni di pollo attraverso l iniezione di vitamina B12 nelle uova MOHAMMAD NASER NAZEM SHIMA TASHAROFI NEGIN AMIRI SEPIDEH SABZEKAR Corrispondenza Shima tasharrofi gmail com Abstract La metionina un amminoacido contenente zolfo Oltre alla sua funzione generale di costituente delle proteine essenziale per una variet di reazioni di metiltransferasi Bench la metionina sia cruciale per la sintesi del tessuto cartilagineo si raccomanda di non ignorare i suoi effetti negativi sulle malattie ossee come l osteoporosi e la discondroplasia tibiale Questi effetti negativi possono essere annullati con la terapia di somministrazione della vitamina B12 A tal proposito questo studio si proposto di indagare gli effetti dell iniezione in ovo di certe soluzioni tra cui 1 ml di acqua distillata metionina vitamina B12 metionina con vitamina B12 addizionale su alcuni parametri ossei e sierici degli embrioni di polli da carne Ross 308 Durante il quarto giorno di incubazione sono stati iniettati embrioni di pulcino fertile nel sacco vitellino Al 18 giorno gli embrioni sono stati esaminati per controllare i parametri della tibia sinistra quantit di osteociti e osteoblasti e misurazione della loro lunghezza lo spessore delle trabecole e il sedimento di collagene e i parametri sierici concentrazione di fosfatasi alcalina e omocisteina La sola metionina ha causato una diminuzione della lunghezza degli osteociti dell altezza degli osteoblasti e la sedimentazione del collagene mentre i gruppi trattati con l aggiunta di vitamina B12 erano comparabili al braccio di controllo La concentrazione di fosfatasi alcalina nel siero di entrambi i gruppi iniettati con vitamina B12 B12 e B12 metionina era notevolmente pi elevata rispetto al braccio di controllo La concentrazione di omocisteina nel siero era pi alta in entrambi i gruppi iniettati con metionina metionina e B12 metionina rispetto al braccio di controllo I dati ottenuti dallo studio attuale indicano che l integrazione di vitamina B12 pu prevenire difetti nei parametri di sviluppo osseo causati dalla metionina Parole chiave embrioni di pollo iniezione in ovo metionina vitamina B12 137 135

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Paper Translations AnimalSummary Technology and Welfare August 2020 La cura del pitone di Children Antaresia childreni ALEXANDER HOSKING e GARY MARTINIC Corrispondenza G Martinic westernsydney edu au Abstract Questo articolo descrive la cura del pitone di Children Antaresia childreni Fig 1 Include informazioni sulla stabulazione l alimentazione e la gestione di questi animali nonch sulla cura generale della loro salute Le informazioni presentate si basano sulle procedure operative standard in uso presso la Western Sydney University che sono state co redatte dagli autori Ci si augura che le informazioni ivi fornite possano rivelarsi utili agli stabularisti alle prime armi incaricati di occuparsi di questa specie in un centro di ricerca per animali o in un contesto zoologico Parole chiave Pitone di Children cura della salute stabulazione condizioni ambientali Terapia assistita con felini un aspetto promettente della terapia assistita da animali AAT ELISKA MI KOV e KRISTYNA MACHOV Abstract Esiste una vasta letteratura incentrata sull effetto delle interazioni tra umani e animali Le fonti disponibili descrivono il suo impatto su svariate aree ambienti ed effetti La maggioranza degli studi relativi a tale area pone l enfasi sull uso dei cani Inoltre svariate pubblicazioni sono dedicate all interazione terapeutica con i cavalli mentre solo una piccola percentuale di studi fa riferimento a mammiferi piccoli o esotici o ad animali da fattoria Bench il gatto sia l animale posseduto dalla maggior parte di persone sorprendente vedere che il suo coinvolgimento come forma di trattamento negli interventi tra uomini e animali esplorato in una letteratura limitata spontaneo chiedersi perch Parole chiave gatto terapia assistita da animali terapia assistita con felini interazione uomo animale interazione uomo gatto La cura di draghi barbuti dalla testa striata e pigmei ALEXANDER HOSKING e GARY MARTINIC Corrispondenza G Martinic westernsydney edu au Abstract Questo articolo descrive la cura di draghi barbuti dalla testa striata Pogona vitticeps Fig 1 e draghi barbuti pigmei Pogona henrylawsoni Include informazioni sulla stabulazione l alimentazione e la tecnica di sessaggio di questi animali nonch sulla cura generale della loro salute Le informazioni presentate si basano sulle procedure operative standard co redatte dagli autori Ci si augura che le informazioni ivi fornite possano rivelarsi utili agli stabularisti alle prime armi incaricati di occuparsi di questa specie in un centro di ricerca per animali o in un contesto zoologico 138 136

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August 2020 2020 Animal Technology and Welfare INDICE DE LA REVISTA Informe sobre la reuni n del Grupo para el bienestar de roedores RSPCA UFAW 2019 CHLOE STEVENS EMILY FINNEGAN JASMINE CLARKSON CHARLOTTE BURNS SONIA BAINS COLIN GILBERT CAROLINE CHADWICK SAMANTHA IZZARD CHARLOTTE INMAN PENNY HAWKINS HUW GOLLEDGE Resumen El Grupo para el bienestar de roedores RSPCA UFAW ha celebrado una reuni n de un d a cada oto o durante los ltimos 26 a os para que sus miembros puedan debatir sobre la investigaci n actual sobre bienestar intercambiar opiniones sobre temas relacionados con el bienestar y compartir su experiencia respecto a la implementaci n de las 3 R reemplazo reducci n y refinamiento en relaci n al uso de roedores Uno de los objetivos primordiales del Grupo es fomentar que las personas piensen sobre la experiencia vital de los roedores de laboratorio garantizando a su vez que cualquier impacto negativo posible en su bienestar sea revisado y reducido La vig simo sexta conferencia se celebr en noviembre de 2019 en la Universidad de Birmingham a la que asistieron m s de 60 delegados de todo el Reino Unido La tem tica de la conferencia de este a o era conciencia bienestar positivo y bienestar psicol gico La conferencia empez con un coloquio sobre la conciencia animal y su impacto sobre el bienestar de los animales y este tema volvi a tocarse durante la tertulia del final del d a en la que varios delegados expusieron sus opiniones sobre la conciencia animal Algunas charlas trataron varios temas sobre refinamiento como los m todos de gesti n refinados el enriquecimiento y el adiestramiento con clicker y el modo en que estos refinamientos afectaban al comportamiento y al estado afectuoso emotivo del animal Otras tertulias debatieron nuevas formas de controlar a los animales para realizar una mejor evaluaci n de su bienestar tratando la mortalidad de neonatos en ratones de laboratorio y c mo unos cambios peque os pueden tener un efecto positivo acumulativo en la vida de los animales de laboratorio El d a finaliz con una actualizaci n de la Home Office Animals in Science Regulation Unit ASRU sobre sus inspecciones espec ficas en relaci n a la falta de alimentos y agua la gesti n refinada de roedores y la reutilizaci n de jeringuillas Este informe resume la reuni n y finaliza con una lista de acciones para que los lectores consideren presentarlas en sus propias instalaciones Palabras clave Roedores conejos bienestar experiencia de por vida conciencia bienestar psicol gico Reducci n de los efectos negativos de la metionina en par metros seos de embriones de pollos mediante inyecci n intrahuevo de vitamina B12 MOHAMMAD NASER NAZEM SHIMA TASHAROFI NEGIN AMIRI SEPIDEH SABZEKAR Correspondencia Shima tasharrofi gmail com Resumen La metionina es un amino cido que contiene sulfuro Adem s de su funci n general como componente de prote nas la metionina es crucial para una serie de reacciones de metiltransferasa Aunque la metionina es crucial para la s ntesis de cart lago sus efectos negativos en enfermedades de los huesos como la osteoporosis y la discondroplasia tibial no deben ignorarse Estos efectos negativos pueden ser suprimidos mediante una terapia con vitamina B12 Por tanto este estudio se llev a cabo para investigar los efectos de la inyecci n in ovo de algunas soluciones como 1 ml de agua destilada metionina B12 metionina con B12 adicional en algunos par metros seos y de suero en embriones de pollos Ross 308 Se inyectaron embriones f rtiles de cr as el d a 4 de incubaci n en el saco vitelino El d a 18 los embriones fueron examinados para comprobar si hab a restos de par metros de tibia cantidad de c lulas oste citas y osteobl sticas y medida de sus dimensiones el grosor del sedimento col geno y trabeculares y los par metros de suero cantidad de fosfatasa alcalina y homociste na La metionina por s sola provoc una p rdida de la longitud de los osteocitos la altura de los osteoblastos y la sedimentaci n de col geno mientras que los grupos tratados adem s con vitamina B12 fueron comparables al grupo de control La concentraci n fosfatasa alcalina de suero en ambos grupos inyectados con B12 B12 y B12 metionina fue significativamente superior a la del grupo de control La concentraci n de homociste na de suero fue superior en ambos grupos inyectados con metionina metionina y B12 metionina en comparaci n al grupo de control Los datos obtenidos en este estudio sugieren que el suplemento de vitamina B12 puede evitar defectos en los par metros del desarrollo seo producidos por la metionina Palabras clave embriones de pollos inyecci n in ovo metionina B12 139 137

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Paper Translations AnimalSummary Technology and Welfare August 2020 El cuidado de la pit n de Children Antaresia childreni ALEXANDER HOSKING y GARY MARTINIC Correspondencia G Martinic westernsydney edu au Resumen Este art culo describe el cuidado de la pit n de Children Antaresia childreni Imagen 1 Incluye informaci n sobre jaula alimentaci n manipulaci n as como los cuidados generales de salud de estos animales La informaci n presentada procede de los Procedimientos de Operaci n Est ndar de la Universidad de Western Sydney que han sido escritos por ambos autores Se espera que la informaci n aqu expuesta pueda ayudar a tecn logos de animales noveles que puede que tengan que ocuparse del cuidado de esta especie en instalaciones de investigaci n con animales o en un zool gico Palabras clave Pit n de Children cuidado de salud enjaulado condiciones ambientales Terapia asistida por felinos una parte prometedora de la terapia asistida por animales AAT por sus siglas en ingl s ELISKA MI KOV y KRISTYNA MACHOV Resumen La cantidad de publicaciones que describen el efecto de las interacciones entre humanos y animales es abundante Las fuentes disponibles describen su impacto en diferentes reas ambientes y efectos La mayor a de los estudios de este rea se centran en el uso de perros Asimismo existen muchos proyectos dedicados a la interacci n terap utica con caballos y solo un peque o porcentaje de estudios registra un uso de mam feros peque os o ex ticos o animales de granja Es bastante sorprendente que a pesar de que el gato es el segundo animal de compa a del hombre no haya muchas publicaciones que describan el uso del gato como un asistente beneficioso en la interacci n entre humano y animal La pregunta es por qu Palabras clave gato terapia asistida por animales terapia asistida por gatos interacci n entre humano y animal interacci n entre humano y gato El cuidado de Pogonia vitticeps y Pogona henrylawsoni ALEXANDER HOSKING y GARY MARTINIC Correspondencia G Martinic westernsydney edu au Resumen Este art culo describe el cuidado de Pogona vitticeps Imagen 1 as como el cuidado de Pogona henrylawsoni Incluye informaci n sobre jaula alimentaci n manipulaci n t cnica sexual as como los cuidados generales de salud de estos animales La informaci n presentada procede de los Procedimientos de Operaci n Est ndar que han sido escritos por ambos autores Se espera que la informaci n aqu expuesta pueda ayudar a tecn logos de animales noveles que puede que tengan que ocuparse del cuidado de esta especie en instalaciones de investigaci n con animales o en un zool gico 140 138

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August 2020 2020 Animal Technology and Welfare LOOKING BACK Editor s introduction During the 70 year history of the Institute s Journal our overseas contributors have been an important source of articles They provide an interesting insight into both the differences and similarities between Animal Technology in different countries and continents and a fascinating reminder of the way things have changed or in some cases have remained the same These papers have not been edited and appear in their original style The first article from an author in France is taken from the Journal of the Institute of Animal Technicians Volume 23 4 December 1972 and was presented at the IAT Congress held that year in Newcastle upon Tyne It predates the passing of the UK legislation The Health Safety at Work Act 1974 which was a forerunner to other similar Acts concerning safety in the workplace outside of the UK The second paper discusses an incidence of Pseudomonas in a laboratory rabbit colony Taken from The Journal Volume 20 2 June 1969 Many laboratory animals at this time were bred in house and disease outbreaks in laboratory animals was often a common problem for technicians and research workers alike Some organisms made replication of results difficult and the loss of animals through disease especially virulent outbreaks was extremely upsetting for Animal Technicians and Researchers In addition losses were also of considerable economic impact both due to the loss of animals and in the case of animals already on experiment reagents and research time was also lost Thankfully thanks to the efforts of animal technicians globally commercial breeding companies and research workers disease in laboratory animals is much less common although when it does occur the effects remain the same Physical hazards in the laboratory animal house R T CHARLES World Health Organisation International Agency for Research on Cancer Lyon France Although it is an employer s responsibility to keep working conditions as safe as possible for his employees the Superintendent or Chief Animal Technician in charge of animal units should be responsible for the safety of the personnel in his charge In all laboratory house manipulations common sense experience and genuine desire to work safely are the basic requirements In all matters of safety perhaps the most important thing is to visualise the hazard and to take adequate precautionary measures in advance When an accident does occur it is important to know what to do Accidents do not just just happen They all have a cause To eliminate the possible danger of an accident it is necessary to find out exactly what these causes are and to realise that there are two basic factors involved in most accidents 1 personal factors 2 two mechanical factors It has become common practise in most large industrial organisations to employ a Safety Officer who is a trained specialist in this field but if there is no establishment within your own organisation for such a person who would be responsible under normal circumstances for the formation of a safety committee directly answerable to the management the need for such a committee is still of paramount importance The Super visor of a large animal breeding or experimental unit should be on such a safety committee or be able to report to the person directly responsible to the management for the safety of the unit Too often technical staff are work orientated either in the production of animals or the housing of large groups of experimental animals without giving a 141 139

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Looking Back Animal Technology and Welfare thought to the safety of themselves their staff and in particular to that of the animals under their care who are literally imprisoned in their cages Fire Of course it may never happen but fires have been known to break out in or next animal quarters This type of fire hazard has a threefold aspect of safety 1 the necessity to prevent the spread of fire to other animal rooms and to the surrounding areas 2 the protection of personnel within the building 3 the protection of the animals who are not able to escape unless assisted by personnel by removing cages from the danger zone The humane responsibility should be the top priority when designing and planning new animal houses so that the evacuation of animal cages could be undertaken efficiently in the case of a fire in or near to the animal accommodation Smoking within the animal rooms is not advisable and certainly should not be permitted in or near to a bedding store If smoking is allowed within the animal rooms it should be considered a privilege and should not on any account be abused by carelessness with matches and cigarette ends Flooding is a not uncommon hazard due to burst overhead pipes or leaky joints which may result in the distribution of not only cold water but possibly hot water and perhaps steam over animal cages Care should be taken in the design of animal quarters to avoid potential hazards of flooding as the result of badly placed overhead pipes There could also be danger from the escape of toxic gases such as formal gas ethylene oxide butane ordinary town gas or ether into the animal rooms or into a service facility within the area At one laboratory recently two serious fires were initiated by anaesthetic ether which was being used during the course of an animal experiment The explosion was triggered off by a spark from a thermostat control unit of a nearby refrigerator For tunately the use of halothane which is noninflammable is now gradually replacing ether as an anaesthetic 1 Mechanical equipment People unfamiliar with mechanical equipment such as autoclaves or cage washers should not interfere with them They should know how to operate them and what to do in the case of emergencies It should be clear which valve should be opened or closed and which switch cuts off the source of electricity The machine should be insured and receive regular inspection by qualified engineer 142 140 August 2020 Particular attention should be paid to connecting pipes electric wires and cables and if they show any sign of wear they should be replaced or repaired promptly To avoid the possibility of burns or electric shock from frayed wires regular maintenance checks of all the electrical equipment used by members of staff should be made Such things as the wire flex to desk lamps magnifying dissection lamps electric hair clippers vacuum cleaners etc should be checked regularly Water bottles For tunately glass water bottle feeders are now gradually being replaced by plastic bottles usually with metal caps and drinking spouts Many glass bottles however are still in use and great care is needed when inserting glass tubing into cork or rubber bungs Cuts on the knuckles are still one of the most common injuries All cracked and chipped bottles should be thrown away rather than remain as a potential danger to the technician handling them or to the person responsible for washing them Broken glass should be swept up and not picked up with the hand Fallen objects There is a tendency to use racking which is too high for the accommodation of animal cages This makes handling difficult for the technician and also presents him with the potential danger of falling objects such as misplaced bottles badly fitted cage covers or even the cage itself The use of chairs boxes and stools should be discouraged and if some aid is needed to reach the top shelf of a high rack then a well balanced and wellconstructed step ladder should be used Preferably animal cage manufacturers should be encouraged to make tracking that can be serviced by a person of average height without having to resort to aids of any sort It is already noticeable that some manufacturers are paying more attention to this aspect of rack design and are advising clients accordingly against the hazards of either high mobile racks or high wall racks Reaching too far or too high can cause muscular strain and can be avoided by the use of properly designed equipment In the case of a fall from a ladder or stool the resulting injury could be serious as most animal room floors are constructed of a hard durable material Not only just the hard floor present danger but in the course of a fall cuts and abrasions can be received from the rack itself or from the sharp corners of a cage Ugly lacerations of the face can be sustained by a technician falling into a rack of white top cages especially if the wire has been galvanised and left poorly finished

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August 2020 Should a metal scoop used for filling food hoppers fall from the hand of someone at the height of 7 to 8 feet 2 to 2 5 metres onto the head of a person in the direct Line of descent it could cause a nasty bruise or laceration Plastic cages are not such a potential hazard as metal cages but even so the metal or wire lid of a plastic cage is still very dangerous and it is often possible to see loose unused cage tops requiring only the slightest movement before they fall stored on the top shelf of a rack Precautions should therefore be taken and loose cages lids or bottles should not be stored on the top shelf of a rack they look unsightly as well as being a potential risk to personnel It would be more sensible to use the bottom shelf of a rack for loose material where it could be more stable or even better to find a storage area outside the animal room or at least to stack carefully in the corner of the room all spare cage not in use and all those which may have become damaged suitably labelled to show that they have been put on one side to await repair General considerations When lifting heavyweights cartons of glass bottles cages sacks of diet etc the back should be straight and the knees bent A sack truck if provided should be used to avoid unnecessary feats of strength If protective clothing such as goggles gauntlet s mask caps gowns etc are provided for use within the animal quarters they should be used All bites and scratches received from animals must be reported and any other injury must be recorded in an accident book which should be placed near the first aid box The contents of the accident book should be brought to the notice of the management periodically All working areas in the animal quarters should be kept clean Floor should be kept free if spilt diet pellets etc which could cause a fall and possibly a sprained ankle When floors have been left wet after washing it is it is always advisable to wear proper non slip shoes or boots The technician should always be responsible for keeping his own work area clean and free of encumbrances There should be sufficient space between racks working tables and benches trolleys etc and passageways and corridors should not be blocked by racks or other items of equipment There should be no running in the corridors and trucks should not be moved too fast Doors should be designed so that they do not open into the corridors If space is limited then sliding doors should be provided Doors in corridors Back Animal TechnologyLooking and Welfare should have glass panelling if they form part of a partitioning of corridors In the course of an animal technicians work he is obliged to handle various species of animals Caution is necessary if he is not familiar with a particular species and does not know how to handle it properly It can be seen the Animal Quarter Supervisor has a great responsibility and he must make sure that every member of his staff realises the importance of observing safety precaution measures He must also make sure that he knows where fire extinguishers fire hoses buckets hydrants goggles and respirators are kept and not only should he know how to use a particular type of fire extinguisher but he should make sure that his staff is trained in the proper use of this equipment Technicians who are working alone inside buildings must have telephone communication with some responsible person outside who could come to their aid in an emergency A technician entering a barrier controlled SPF facility can slip while taking a shower and receive an injury Apart from safety grips being provided within the shower unit someone should be responsible to see that he has actually entered the protected zone without mishap Also when passing items into an entry lock where ultraviolet light is used special goggles should be worn or the ultraviolet light switched off for a time Similarly when passing items through a dunk tank gauntlets should be worn to protect the arms from any possible effect of disinfectants especially if a person is particularly sensitive to these substances When tipping sawdust into a container particularly if the sawdust is fine and dusty care should be taken to protect the eyes or to ensure that a better form of bedding is used which is dust free and which will be advantageous both to the technician and to the animal in his care Toxic chemicals Gas cylinders should be treated with caution and stiff valves manipulated carefully Cylinders should not be banged and no oil or grease should be used to free a stiff valve If toxic material is being used within the animal quarters special precautions must be taken to prevent splashes on the clothing or skin They should be diluted immediately with the proper solvent and if possible the laboratory clothing discarded and replaced by a clean garment Aerosol cans containing insecticides deodorisers etc when placed on a window sill in direct sunlight can be dangerous if they explode Hazards 143 141

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Looking Back Animal Technology and Welfare from toxic chemicals are adequately dealt with in the publication Hazards in the chemical laboratory 2 The Chemical Society or the Institute of Science Technologies publication The care handling and disposal of dangerous chemicals 3 and these should be consulted Other aspects of safety must be applied to prevent accidental transmission of disease to technicians and research workers The proper care necessary for handling infected animals and contaminated material is adequately dealt with by Darlow 4 When cuts and abrasions are received in animal rooms which house animals suffering from infectious diseases possibly of unknown viral origin the dangers to health cannot be overestimated It is strongly recommended that all staff working with animals should be actively immunised against tetanus In conclusion it can be seen some general rules for the protection of personnel working in laboratories animal quarters are essential and to prevent accidents and to know what safety measures are necessary is the moral responsibility and duty of every technician Some general rules must be born in mind and appropriate notices displayed for all employees to see and familiarise themselves with To summarise you should know i Where all emergency exits are situated ii The location of all fire fighting equipment iii The position of the fire alarm if any or the telephone number to call in event of a fire iv The basic rules of first aid to the injured v In all emergency situations the telephone number of your nearest doctor or qualified medical practitioner vi The safety regulations enforce which should be read and remembered vii No smoking notices mean no smoking References 1 2 3 4 Sebesteny A 1971 Fire risk free anaesthesia of rodents with halothane Lab Animal 5 225 231 The Chemical Society 1971 Hazards in the chemical laboratory Gaston P J 1970 The care handling and disposal of dangerous chemicals Institute of Science Technology Darlow H M 1967 Safety in the animal house Lab Animal 1 35 42 144 142 August 2020

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August 2020 2020 Animal Technology and Welfare The incidence of a pathogenic strain of pseudomonas in a rabbit colony G R ALPEN and K MAERZ Research Division Riker Laboratories Australia Pty Ltd Sydney New South Wales Australia Type of rabbit The stock rabbit selected for the colony included a Lop rabbit b Multi colour c Great white d New Zealand White e Lab White and f Californian Such strains were selected animated to establish the laboratory stock Housing The rabbits were housed in stainless steel cages in multiples of 24x15 x 13 in a room having 65 humidity 70 2 F and receiving 8 10 changes of air per hour The water supply was an automatic nipple system and the diet was a pellet supplemented with greens The cages were sterilised every four weeks and the trays every three days Clinical symptoms The rabbit rabbits had forced spasmodic respiration and a mucopurulent nasal discharge Sneezing lethargy and an ocular discharge were common In the terminal stages the animals failed to eat the diet and death soon followed Post mortem The abdominal organ showed no pathological abnormalities On examination of the thoracic cavity however a large volume of pleural fluid was seen Massive absences were present involving whole lobes of the lungs and adhering to the pleura Red hepatinisation was present as serofibrinous pleurisy with fibrin covering much of the affected lobe Areas of necrosis were present in which traces of the preexisting structure remain and which are surrounded by a narrow inflammatory zone containing neutrophils and much granular debris The heart appeared normal although the pericardium showed miliary infection The airways from the nostrils through the trachea to the bronchi were inflamed and filled with a mucopurulent discharge Haemorrhage areas will also present Bacterial isolation Swabs from the bronchi trachea throat and nose as well as lung slices were incubated in cooked meat medium at 37 C overnight after which the broths were Gram stained and subcultured onto sheep blood agar and incubated aerobically at 37 C overnight Gram stain In all cases pure growth of a gram negative bacillus approximately 1 5 x 0 5 From the sheep blood agar subculture s abundant moist spreading large irregular colonies with a metallic green colour resulted The characteristic pseudomonas odour was present Sugar fermentation reactions were all negative except glucose which produced acid only however green single pellicles were present in all tubes The bacilli were motile grew on McConkey agar and in KCN broth utilised citrate but were indole negative Starch was not hydrolysed From the identification procedures performed the organism was typed as Pseudomonas aeruginosa Sensitivities Antibiotic sensitivities were tested on nutrient agar and neomycin as well as polymyxin B were found to be successful The tube sensitives for neomycin titred to 10mg ml Treatment Known carriers were inoculated with neomycin sulphate 200 mg ml intramuscularly i m They received a five day course of 1ml i m Throat swabs were again taken five days after the course had finished and were cultured by the usual procedures If the cultures were positive then another course of neomycin sulphate was given A vaccine was offered so prepared with a slight modification 1 Method The bacteria were grown in you nutrient broth for 24 36 hours past the log phase and into the stationary phase and then killed by heating to 56 C for one hour This longer incubation period permitted the bacteria to secrete exotoxin 145 143

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The incidence of a pathogenic pathogenic strain of of pseudomonas pseudomonas in in aa rabbit rabbitcolony colony Animal Technology and Welfare strain The culture was then centrifuged at 4 500 rpm for 30 minutes and the bacteria as well as the exotoxins aggregated as gelatinous mass A viability test was carried out on sheep blood agar No growth resulted so that bacterial suspension was diluted in normal saline to a concentration of 1000 million organisms per millilitre counted on a haemocytometer and a pH to 7 6 Inoculation All stock particularly newly weaned rabbits were given 3 by 1 5 ml intramuscularly over a period of three weeks this was boosted at 6 00 weeks by a further 2ml intramuscular Antibody titre The agglutination reaction gave a positive title of 1 64 and after boosting a titre of 1 256 2 Haemagglutination also gave the same titres 3 In this case the antigen was tagged to a group O human red cells 37 C for one hour Adaptation of the Lancefield grouping also gave a positive precipitin reaction 4 inoculated rabbit serum being tested against the vaccine Conclusion With the aid of immunisation it was possible to protect rabbits from Pseudomonas aeruginosa Neomycin sulphate 200mg ml was found to be only bacteriostatic to the condition of bacilliary pneumonia in older animals Unprotected cases were fatal Summary To build a laboratory rabbit colony of good quality experimental animals and in substantial numbers rabbits were obtained from 6 different suppliers throughout Australia Following quarantine and on commencement of the breeding programme cases of pneumonia were discovered within the colony which were all fatal The bacteria rapidly spread and within five days six of the stock had died Deaths were also reported due to the bacterial pneumonia for the ensuing four months Post mortems revealed massive lung abscess within the thoracic cavity Treatment was administered to the remaining stock by a neomycin sulphate 200 mg ml intramuscularly b vaccination and immunisation The animals were checked by nose and throats swabbings which were incubated in cooked meat medium at 37 C overnight Such swabbings were done every five days over a period of 15 days 146 144 References 1 2 3 4 August 2020 Cruickshank R Medical Microbiology 1965 Cruickshank R Modern trends in immunology 1963 Middlebrook G and Dubos R F Specific serum agglutination erythrocytes sensitised with extracts of tubercule bacilli J Exp Med 88 521 Lancefield C Serological differentiation of human and other groups of haemolytic streptococci J Exp Med 57 571

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August 2020 2020 Animal Technology and Welfare TECH 2 TECH Haven t the time to write a paper but want to get something published Then read on This section offers readers the opportunity to submit informal contributions about any aspects of animal technology Comments observations descriptions of new or refined techniques 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 be presented as technical notes and do not need to be structured and can be as short or as long as is necessary Accompanying illustrations and or photos should be high resolution NB Descriptions of new products or equipment submitted by manufacturers are welcome but should be a factual account of the product However the Editorial Board gives no warranty as to the accuracy or fitness for purpose of the product Development of a sifting cage change method for rats to improve welfare SEONAGH HENDERSON Cardiovascular Research Facility University of Glasgow 126 University Place Level 5 BHS Glasgow G12 8TA UK Correspondence seonagh henderson glasgow ac uk Introduction Long term goal I would like to explain to you about some improvements in relation to the 3Rs with a development of a sifting cage change method for rats to improve welfare To continue to provide the animals with a high level of welfare and to ease stress when changing cages by introducing them back into their own familiar smell while retaining olfactory cues for the rats I came up with the idea after seeing how skittish and stressed the rats in my unit were and wanted to develop a way to improve how they interacted with my colleagues and myself Normally during a cage change a small handful of dirty bedding is placed into the clean cage but I felt this was not enough to comfort the rats After trialling a few methods I finally came up with a sieve as the best tool for the job The technique can be easily adopted with minimal cost saving on consumables and is good for the environment but the biggest impact has been on the rats themselves they are so calm and naturally inquisitive instead of hiding for cover when I enter the room Figure 1 Image shows a cage of 4 adult male rats prior to being cleaned out after a period of 1 week 147 145

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Tech 2 Tech Animal Technology and Welfare August 2020 Disadvantages A case study Process is dusty and use of a pure flow helmet is advised but not essential However a face mask must be worn Best carried out in a change station Can take a little longer to change cages shortterm Some cages need a full change every week e g large males as they produce more waste products 50 sifted and 50 fresh is used instead Reported benefit from one of our researchers Putting the method into practice 5 6 bags of corn cob less used each week Saving approx 1656 per year 12 15 black bags used less each week Cages not put through cage wash as often benefits to staff welfare and environmental damage In addition to the benefits to the rats I have also noticed that there is a considerable difference in the cost of consumables saving an approximate of 1656 per annum on corn cob alone Each bag costs around 11 50 alternate full change cages from weekly to fortnightly saves 69 a week which is 138 a month hence the annual saving There is also a reduction in waste going to landfill electricity used in cage wash machines detergents manpower freeing up staff to carry out other tasks as well as many other benefits including those to the research team 150 148 The number of successful matings that result in offspring in WKY and SHRSP female rodents prior to PRE and following POST Seonagh Henderson s arrival to the CVRU on first attempt based on personal records In both strains of successful matings significantly increased following Ms Henderson s start date p 0 001 PRE vs POST Data expressed as mean SEM Data analysed by one way ANOVA

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August 2020 2020 Animal Technology and Welfare POSTER PRESENTATIONS Originally presented at IAT Congress 2019 Assessing pain in models of Rheumatoid Arthritis SAMUEL SINGLETON 1 MERIAM NEFLA 1 NGAIRE DENNISON 1 SIMON ARTHUR2 and TIM HALES 1 2 School of Life Sciences Division of Cell Signalling and Immunology University of Dundee Dundee DD1 5EH UK MRSU and Institute of Academic Anaesthesia Division of Systems Medicine Ninewells Hospital University of Dundee DD1 9SY UK Correspondence s z singleton dundee ac uk Introduction A hallmark symptom of rheumatoid arthritis in humans is painful swollen joints Pain can manifest before any inflammation is noticeable1 2 as well as persist long after inflammation has resolved 3 Measuring footpad width assumes that increased swelling is proportional to enhanced pain A mild arthritis phenotype in which there is minimal swelling may therefore inaccurately reflect the extent of pain and discomfort In rodent models of ar thritis ankle or footpad width is a commonly used surrogate marker of pain see Figure 1 Aim We aimed to determine how well pain correlated to footpad widths using the collagen antibody arthritis model A B C Figure 1 Footpad width as a surrogate measure of pain in arthritis models Commonly used methods to assess pain are footpad width A ankle width B or footpad ankle length C 151 149

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Poster Animal Presentations Technology and Welfare Conclusions Footpad widths poorly reflect pain experienced in arthritis models Observing the posture and activity of an animal might better indicate whether or not they are in pain Qualitative and semi quantitative measures of affective pain can be useful in determining when an animal should receive analgesia Further qualitative refinements should be added to identify better humane endpoints Acknowledgements References 1 2 3 4 McWilliams D F Rahman S James R J E et al 2019 Disease activity flares and pain flares in an early rheumatoid arthritis inception cohort characteristics antecedents and sequelae BMC Rheumatol 3 49 2019 https doi org 10 1186 s41927 019 0100 9 ten Klooster P M de Graaf N and Vonkeman H E 2019 Association between pain phenotype and disease activity in rheumatoid ar thritis patients a noninterventional longitudinal cohort study Arthritis Res Ther 21 257 2019 https doi org 10 1186 s13075019 2042 4 Sandor K Nandakumar K S Holmdahl R et al 2012 Collagen antibody induced arthritis CAIA leads to evoked and ongoing persistent pain like behavior but transient joint inflammation Annals of the Rheumatic Diseases 2012 71 A32 Coppard C Bonnefoy F Hannani D et al 2019 Photopheresis efficacy in the treatment of rheumatoid arthritis a pre clinical proof of concept J Transl Med 17 312 2019 154 152 August 2020

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August 2020 2020 Animal Technology and Welfare Refinements to health monitoring HANNAH JONES and REBECCA KING Core BRU Cancer Research Institute Robinson Way Cambridge CB2 0RE UK Correspondence rebecca king cruk cam ac uk Poster prepared for AST2020 Green health monitor Weight weekly if cage is on a hand check already this will be done then No other clinical signs Phenotype related to strain Amber health monitor Weight 10 loss Body condition 2 5 Sores skin lesions present but dry and no signs of scratching Papilloma present on face or genital region Anal prolapse Up to 3mm protruding Still pink moist surface not bleeding Respiration rate and effort increased or decreased Tumours visual or palpable or both Abnormal gait wobbly or splayed or issues with movement Intermittent hunching Pain Signs of pain but responds to analgesia Seizure observed duration 3mm protruding or Dark scabbed bleeding Respiration laboured very increased or decreased Tumour 10mm diameter may vary depending on PPL Multiple tumours palpable Gait complete paralysis one or more limb Permanent hunched posture Very jittery inhibiting normal movement Seizure third seizure observed HE If a mouse shows any of the listed amber signs then monitoring is increased to either once or twice weekly green HC Mice with 3 amber signs should be on red HC daily check Mice which develop more than 3 amber signs or any one red sign should be considered for humane endpoint HE 155 153

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August 2020 2020 Animal Technology and Welfare Biosecurity risks and the pre implantation embryo lessons from the mouse JEAN COZZI MENDY VERRIER and JIMMY MANCIP Charles River Laboratories Domaine des Oncins BP109 69592 L Arbresle cedex France Correspondence jean cozzi crl com Introduction Today the technique of embryo transfer is the most widely used method to rederive livestock that are microbiologically contaminated or to recover valuable animal lines archived by assisted reproduction techniques ARTs 1 In the mouse standard process consisting in per forming serial washings of preimplantation embryos in antibiotic supplemented media before embryo transfer ET has proved efficient in eliminating viral bacterial or parasitic infections 2 6 Contamination may occur during embryos production and storage processes or could be mediated by gametes and embryos themselves 7 Continuous training of the team in techniques and principles of disease controls adequate facilities and equipment are thus mandatory to avoid accidental contamination However embr yos may become contaminated in vivo at the oocyte stage prior to ovulation or following ovulation by exposure to microbial pathogens during transit through the oviduct and uterus and in vitro at insemination stage by spermatozoa when assisted fertilisation techniques are used Through analysis of scientific data relating to mouse species we discuss here the contamination risks linked to the oocyte embryo and ways to prevent them The embryo and contamination risks The pre implantation embryo is a proper entity in itself since it is present in the oviduct and uterus for only a Figure 1 Mouse ovulated eggs stained with MABs specific to mouse ZP1 mouse ZP2 and ZP3 glycoproteins constituting the ZP Confocalmicroscopy 32 very short time before collection without any vascular link to the recipient female at these stages In addition the embryo is surrounded by the zona pellucida ZP Figure 1 which acts as a natural barrier against pathogens The ZP is composed of a complex fibrous network of glycoprotein interspersed with numerous spores that are largest at the outer surface and decrease in size centripetally Figure 2 Figure 2 Scanning electron micrograph of the surface of the ZP of a hamster oocyte Micropores formed by the complex structure of ZP1 ZP2 and ZP3 glycoproteins association are visible http www cellimagelibrary org images 12624 Because of their size viruses are the most likely pathogens to be transmitted during ET since they may traverse the ZP or adhere firmly to the ZP by remaining lodged in its micropores thus rendering washing procedures less efficient in decreasing viral load Very small viruses 27 30nm such as autonomous parvovirus can traverse the ZP 8 10 Mouse Minute Virus MMV 20 nm in size can contaminate mouse embryos as confirmed by in vivo studies 11 12 even when repeated embr yos washing was per formed 11 In contrast recipients of embryos arising from IVF with oocytes and MMV exposed spermatozoa remained seronegative and were virus free as well as their offspring 11 13 In this last study prevalence of virus detected by real time PCR was lower in male than in 157 155

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Poster Animal Presentations Technology and Welfare female reproductive organs and in 2C embryos derived from infected males than derived from infected females For Mouse Hepatitis Virus MHV 80 160 nm in size no infection was observed after washing of embryos 10 times 11 In endemically infected mice Mouse Parvo Virus MPV1 was detected in ovarian tissue sperm cells embr yos and oocytes by Polymerase chain reaction PCR corroborating the transmission of viral particles 14 Presence of Lymphocytic choriomeningitis LCMV 60300nm in mouse oocytes and embryos has also been documented providing evidence of transmission via the oocyte 15 Sendaivirus 100 200nm size was found in the murine ZP 16 17 Finally standard washing or trypsin treatment were not sufficient to remove Sendai virus adhering to the ZP of mouse blastocysts The in vitro produced embryo in vitro produced embryos may be exposed to specific contamination risks Infected sperm Presence of prevalent pathogens Helicobacter typhlonius MHV MPV and retroviruses in gonads and gametes has been demonstrated 14 18 20 Efficient methods to remove pathogens from mouse sperm are not yet available However the risk of sperm mediated contamination in IVF using cumulus enclosed oocytes from non infected females seems negligible 13 18 19 Cumulus cells seem to exert a protective effect against pathogens Embryos arising from IVF of cumulus enclosed oocytes with MMV exposed sperm did not lead to seropositive recipients and pups in contrast to IVF of cumulus denuded oocytes 22 When cumulus cells are removed from the ZP micropores to approximately 140 1000nm 23 arise thus allowing entrapment of a higher quantity of the virus from the fertilization media Cumulus cells may also absorb some of the viruses thus contributing to decrease of the viral load 14 In addition standard washing procedures were effective in avoiding MHV transmission when sperm and oocytes were incubated with high levels of virus 23 Additional studies are needed to determine whether other prevalent viruses can be transmitted by sperm and IVF Invasive ARTs such as laser assisted IVF and intracytoplasmic sperm injection ICSI use cumulusfree oocytes for facilitating the micromanipulation and lead to ZP rupture Thus the oocyte protection against potential contamination is lower However the number of sperm cells in contact of the oocyte is also far less than in conventional IVF On the other hand for some viruses such as MHV contamination could be avoided by standard repetitive washing procedure when zona pellucida was par tially disrupted by laser 158 156 August 2020 microdissection 24 More research is needed to assess the risks with other pathogens since very few data in mouse are available in this field ES cell injection following transfection with desired gene constructs coupled with ET is a standard method for producing transgenic mice Mouse ES cells mESC may harbour infectious pathogens The most prevalent mESC contaminating agents are mycoplasma and viruses such as lactic dehydrogenase elevating virus LDEV polyomavirus LCMV and par vo viruses Mycoplasma may affect various mESC and parameters germ line transmission and postnatal development of resulting chimeras 25 Few data are available regarding pup contamination through injection of contaminated EScells Pup contamination was obser ved after injection of mESC infected by MMV but not by MHV MHV being cytolytic form ESCs 26 Infection risk can be significantly reduced by generating ES cells from mice free of relevant pathogens and by testing the ES cells before injection ZP structural alteration may arise from in vitro culture of mammalian preimplantation including the mouse 27 28 In pigs and bovines some pathogens seem to adhere more readily to IVP embryos 23 29 Such interactions might differ from one pathogen to another and from one species to another No data has yet been published in the mouse supporting this effect Embryos gametes storage and transportation Liquid nitrogen during its storage and distribution may become an effective media for the cryopreservation of fungal spores yeasts bacteria and viruses and may lead to contamination of embryo and gametes stocks 30 Sealed samples of embryos stored in contaminated liquid nitrogen tanks tested negative for the presence of bacteria or viruses 31 Similarly the vapour phase of liquid nitrogen is a safe means for the short term storage and transportation of embryos in dry shipper dewars 31 For review on the risks of contaminating germplasm during cryopreservation and cryobanking see Yauger et al 2011 32 It is important therefore to prevent direct contact of germplasm and reproductive tissues with LN during cryopreservation and their storage This includes the usage of hermetically sealed high quality straws or cryotubes A periodic disinfection of cryo Dewars should be considered as an additional precaution to diminish the potential for inadvertent cross contamination Separation of LN Dewars to quarantine embryos derived from infected donors of valuable genotypes or from unknown health status is also advisable Conclusion Rederivation procedure using embryo transfer is an efficient technique for pathogens removal in livestock

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Poster Presentations Animal Technology and Welfare August 2020 animals and the mouse Maximum efficiency is reached when special care is taken using zona intact preimplantation embryos and sufficient washing of oocytes and embryos at least 10 wash steps dilution factor 1 100 per wash step Nevertheless viral infection may occur since viral particle size may allow them to traverse or adhere firmly to the ZP thus rendering washing procedures less efficient The risk seems restricted to very small prevalent viruses such as MPV and MMV Viral contamination risk seems higher when using biological material oocytes embr yos originating from contaminated mouse females than from males By consequence when using oocytes embr yos from contaminated females a quarantine should allow dramatic reduction of viral load in reproductive organs and safe rederivation procedures through ART Contamination through sperm in IVF procedure appears to be negligible so this approach should be favoured when possible The use of 2 cell stage embryos is also recommended for ET because viral DNA could not be detected by PCRin2 cell embryos derived from MMV in vivo infected mice after sufficient washings 12 More and more studies are using PCR based techniques for detection of pathogens Detection of viral DNA by a ver y sensitive method does not necessarily imply the presence of sufficiently infectious virions at embryo level 25 13 14 15 16 17 18 19 20 21 22 23 24 26 27 28 Nonetheless because seroconversion to virus ex parvovirus depends on several factors including mouse strain stringent hygienic measures should be implemented including recipients being kept in IVCs or isolators with screening before release of the rederived pups References 1 2 3 4 5 6 7 8 9 10 11 D A Stringfellow S M Seidel I E T 1998 Society Manual of the International EmbryoTransfer Society a proceduralguide and general information for the use of embr yotransfer technology emphasizing sanitar y precautions The Society Savory Ill ed 3rd 1998 I C Reetz M Wullenweber Schmidt V Kraft H J Hedrich 1988 Lab Anim Sci 38 696 701 1988 H Suzuki K Yorozu T Watanabe M Nakura J Adachi 1996 Exp Anim 45 33 38 1996 M L Van Keuren T L Saunders 2004 TransgenicRes 13 363 371 2004 P Carthew M J Wood C Kirby 1983 J Reprod Fertil 69 253 257 1983 P Carthew M J Wood C Kirby 1985 J Reprod Fertil 73 207 213 1985 B Le Tallec C Ponsart B Marquant Le Guienne B Guerin 2001 Reprod Nutr Dev 41 439 50 2001 R B L Gwatkin J Reprod Fertil 13 577 578 1967 D P Bane J E James C M Gradil T W Molitor 1990 Theriogenology 33 553 561 1990 A D Heggie L Gaddis 1979 Pediatr Res 13 937 941 1979 E Mahabir et al 2007 Biol Reprod 76 189 197 2007 29 30 31 32 L M Janus A Smoczek H J Hedrich A Bleich 2009 Biol Reprod 81 1010 1015 2009 E Mahabir D Bulian R Schmoller J Needham J Schmidt 2007 Biol Reprod 78 2007 doi 10 1095 biolreprod 107 060467 2007 Comp Med 57 51 56 2007 C A Mims 1966 J Pathol Bacteriol 91 395 402 1966 M Tuffrey B Zisman R D Barnes 1972 Br J Exp Pathol 53 638 1972 C Lavilla Apelo H Kida H Kanagawa 1991 Jpn J Vet Res 39 133 141 1991 F Scavizzi M Raspa 2004 Laboratory Animals 38 189 199 2004 F Scavizzi M Raspa 2006 Laboratory Animals 40 70 79 2006 A A Kiessling R Crowell C Fox 1989 Proc Natl Acad Sci 86 5109 5113 1989 E Mahabir D Bulian J Needham J Schmidt Biol Reprod 81 531 538 2009 A Soom 2006 CAB Rev Perspect Agric Vet Sci Nutr Nat Resour 3 2008 doi 10 1079 PAVSNNR20083060 D D Peters 2006 Biol Reprod 74 2006 doi 10 1095 biolreprod 105 045112 K Markoullis et al 2009 TransgenicRes 18 71 87 2009 E Mahabir et al 2009 TransgenicRes 18 45 57 2009 G Vanroose et al 2000 Biol Reprod 62 463 469 2000 C P Silva V Silva K Kommineni D Keefe 1997 Biol Bull 193 235 236 1997 D A Stringfellow M D Givens J G Waldrop 2004 Reprod Fertil Dev 16 93 102 2004 B W W Grout G J Morris 2009 Theriogenology 71 1079 1082 2009 A Bielanski H Bergeron P C K Lau J Devenish 2003 Cryobiology 46 146 152 2003 A Bielanski G Vajta 2009 Hum Reprod 24 245767 B Yauger N A Boggs J Dean 2011 Reprod 141 313 319 2011 159 157

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Animal Technology and Welfare Welfare August 2020 Environmental enrichment for a small colony of rats NICK BLACKBURN GEMMA CRONSHAW and MIKE MITCHELL Cancer Research UK Cambridge Institute University of Cambridge Li Ka Shing Centre Robinson Way Cambridge CB2 0RE UK Correspondence Gemma Cronshaw cruk cam ac uk Mike Mitchell cruk cam ac uk Introduction The Biological Resources Unit at the Cancer Research UK Cambridge Institute predominantly houses mice with a small number of rats used for brain cancer studies due to limited space and resource for equipment we came up with some easy and costeffective ways to improve enrichment for the rat colony Since implementing our protocol for forage feeding and out of cage enrichment we have found the rats easier to handle and more confident This has proven invaluable with regards to limiting stress whilst they are visiting our imaging facility A small amount of forage mix is buried in the bedding material of the rat s home cages during cage cleaning and daily checks as they spend time foraging until they have found and eaten all of the food We also hand feed the rats to encourage human interaction and this helps us monitor health effectively as they have to reach up to the cage door to take it We provide our rats with irradiated LBS forage mix LBS Biotechnology which contains ingredients known to be appetising to rodents such as sunflower seeds banana maize flakes and locust beans Figure 2 Rats are provided with super vised out of cage enrichment in a larger cage that is small enough to fit inside a downdraft cabinet Introducing this new environment regularly provides a positive association with leaving the home cage Figure 1 Approximately twenty rats are housed in IVCs in the same holding room as mice Figure 3 Rats in out of cage environment Figure 2 Forage Mix LBS Biotechnology 160 158 All enrichment must be easily cleaned or disposable to avoid cross contamination We use disposable fun tunnels also Perspex Jolley balls and screens that can be easily disinfected with Vaporised Hydrogen Peroxide VHP or autoclaved Rats also enjoy playing with empty glove boxes a green and cost effective way to provide extra enrichment for your animals

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August 2020 Animal Technology and Welfare Oestrus checking increasing productivity and embracing the 3R s SAMANTHA HOSKINS and JACK BROWN A2 Breeding Unit The Francis Crick Institute 1 Brill Place London NW1 1BF UK Correspondence alan palmer crick ac uk Overview Much like the menstrual cycle in humans mice also have a cycle where the eggs ripen ovulation takes place the females come into heat and are receptive to mate with the male this occurs over the space of 4 5 days and is called the oestrous cycle The oestrous cycle in mice has four stages pro oestrus oestrus met oestrus and di oestrus The appearance of the epithelium of the external genitalia is used to identify the stage of the oestrous cycle in a female mouse Females are selected in the afternoon when they are in pro oestrus and mated overnight The females are then plug checked in the morning and if a plug is found this confirms mating has taken place Previous results and aim of the study An original trial per formed at our legacy site in 2011 showed the rate of successful matings after implementing this method of oestrus checking increased from 13 to 54 when compared with randomly selecting females for mating This method was then used in successfully re deriving all of our mouse strains in preparation for moving to The Francis Crick Institute in 2017 The Crick Initially at The Francis Crick timed matings were set up using dirty bedding to induce the Whitten Effect and had a low success rate It was decided to perform a new trial of oestrus checking with the aim of increasing the number of confirmed matings and improving consistency in fulfilling timed mating orders for the Genetic Modification Service GeMS and other users If successful it would allow us to reduce the number of mice used and the need to repeat unsuccessful matings in accordance with the 3Rs whilst also allowing us to decrease the waiting times for projects of work through the GeMS team This trial in early 2018 showed an average successful timed mating rate of 81 in CD 1 females and it was decided to adopt this method for all A2 timed mating orders going forward We have seen a large increase in success rates with other mouse strains by using this method as well What is oestrus checking Oestrus checking as we call it is a visual check of the female s genitalia to assess her readiness to mate It is done by holding each female by the base of the tail in your left hand and using the thumb of your right hand gently touch the stomach of the mouse to open the vagina to assess which stage of the cycle she is in Females that are at the correct stage are then mated with the males for one night only Oestrous cycle There are 4 stages of the oestrous cycle in mice and altogether the cycle lasts 4 5 days For mating purposes we are looking for the pro oestrus stage Whitten Effect the stimulation by male pheromones of synchronous oestrus in a female population in this case the addition of dirty bedding containing male mouse pheromones to the female mouse cage 159

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Animal Technology and Welfare Poster Presentations August 2020 Plug checking When mating occurs the male ejaculate forms a solid mass inside the female s vagina We call this a plug We check for this plug to confirm that mating has taken place We use a thin metal stick to feel for the plug but often you can see it without using the stick Figure 1 Visual appearance at each stage of the oestrous cycle Signs to look for in pro oestrus Figure 3 Plugged female very swollen puffy tissue Results bright pink colour Random selection of females striations or lines moist tissue Figure 4 Chart showing the percentage of successful matings without using the Whitten effect or oestrus checking Use of Whitten Effect Figure 2 Female in pro oestrus Pairing takes place in the afternoon this is because with a normal 12 12 light cycle the female and male are put together at the end of the light period The mating is expected to take place early in the dark period plug checking is performed first thing in the morning to confirm that mating has taken place 160 Figure 5 Chart showing the percentage of successful matings using the Whitten Effect

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August 2020 Use of Oestrus checking Animal Technology and Welfare Poster Presentations Conclusion Selecting females for mating by visually checking for oestrus vastly increases successful plugging rates in CD 1 females Our results show an increase from 13 to 81 by introducing this method into our daily routines This allows us to greatly reduce the number of matings needed to produce the desired number of plugs in accordance with the 3R s and to consistently hit plugging targets to fulfil orders Figure 6 Chart showing the percentage of successful matings using oestrus checking What are the benefits of oestrus checking Oestrus checking allows us to only select females that are ready to mate thereby reducing the number of mice being used for mating This means that we can be very efficient ensuring we have plugged females on the requested days filling orders for a variety of users and strains It also allows us to tailor the numbers being mated to fulfil our orders without over breeding GeMS require large numbers of both donor and recipient females daily at both E0 5 and E2 5 These need to be supplied as ordered so that GeMS can plan and execute their work Oestrus checking allows us to supply these consistently and meet the ever increasing demand How oestrus checking impacts GeMS work Consistently obtaining the plug numbers required allows the GeMS team to plan their work more efficiently Birth rates are improved if embryo thaws and implantation takes place quickly Embryo numbers are predictable allowing GeMS to have the correct number of injectable blastocysts ready Predictable yields allow GeMS to schedule when a cell line is likely to be injected minimise culture times and give accurate timelines to users Choosing only females that are ready to mate also reduces the stress levels of having females not suitable for mating being introduced into male cages This is a good refinement to the process as it minimises the risk of injuries caused by aggression from the males Anecdotally it results in a higher confirmed pregnancy rate from plugged females Providing a stable and dependable service to the GeMS team and other users allowing them to plan work based on predictable yields has allowed them to give accurate timelines to users for their projects reduce waiting times and clear the backlog that had built up before oestrus checking was introduced Oestrus checking all of our plugging orders is very time consuming and initially caused scheduling issues with having trained people available during weekends However it is relatively easy to train staff to use this method and once they have picked up the skill and are proficient it allows us to provide a consistent high plugging success rate even at weekends and holidays Despite the time taken and increase in workload that oestrus checking brings it has proven to be invaluable in allowing us to consistently fulfil our plugging orders and provide this service to the GeMS team and our other users Acknowledgements Thanks to Ian Rosewell and Sophie Wood for their input on how oestrus checking impacts GeMS work Thanks also to Helen Bailey for help with poster design and content and Peter Miller for technical help with PowerPoint to produce the diagrams References Figure 1 taken from https journals plos org plosone article id 10 1371 journal pone 0035538 It is essential to allow GeMS to deliver a good service and reduce waiting times E0 5 and E2 5 refer to days post fertilisation 161

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Welfare Animal Technology and Welfare August 2020 Using habituation to reduce stress for rats being transported short distances SARAH TAYLOR University of Cambridge Department of PDN Anatomy Building Downing Street Cambridge CB2 3DY UK Correspondence st685 cam ac uk Introduction Method Transport in rats has been shown to be a big stressor this results in some major changes in their physiology 1 According to Arts et al 2012 stressed animals can affect the reliability and validity of research result 2 To reduce the stress experienced by the rats during transport I felt that the rats needed to be familiar with the transport box I was informed by a researcher that her Lister Hooded rats were hard to work with after travelling in a box even for the short distance from the facility to the laboratory on the floor below More recent research has found that the immune system can also be affected in addition to increased corticosterone level which affects heart rate blood pressure and immune function as well as behaviour 2 3 Animals need to return to their normal baseline physiological parameters of homeostasis before they are used for experimental procedures 2 The physical stress of movement can cause muscle damage during travel Moreover their behaviour is affected in that their exploration and grooming behaviour is reduced 3 Figure 3 Rats in transport box Research shows that even husbandry procedures cause acute amounts of stress to laboratory animals It has also been shown that moving rats to another room for a procedure is more stressful than if they are in their holding room having the same procedure 4 Swallow et al 20055 states that potential sources of stress for animals that are undergoing transport can also include handling separation from cage mates movement vibrations during transport causing physical stress with adjusting balance Figures 1 2 Rat habituating with transport box in the ball pool 162

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August 2020 Animal Technology and Welfare Poster Presentations To familiarise the rats to the transport box I built an empty one and placed it into the ball pool allowing the rats to get some exercise and play I placed it in the ball pool with the lid open and hid some treats amongst the bedding The rats were keen to investigate and each time the rats went close to the box or touched it I gave them a small treat After several days of repeating the procedure the rats were climbing into the box and jumping in and out of it Once the rats showed no signs of fear of the box in the ball pool I then moved it into their home cage and repeated the procedure After doing this for several more days the rats became familiar with the box and were no longer fearful of it and used it as part of their enrichment within their cage Figure 5 Transport box placed in home cage Results References When the rats were needed and the researcher took the rats to her laboratory to carry out procedures she found that the rats were a lot calmer and easier to handle after being trained with the transport box 1 2 Conclusion In order to find out if the stress of being transported has been reduced by the acclimatisation to the transport box I would need to do further investigations and collect data This would involve a scoring system of the behaviour of the rat and their interaction with the box at each stage of the training process and the experimental procedure once they have been transported On initial investigation it seems that if the rats are familiar with the transport box this can reduce the stress they experience when being transported Further investigation is needed to find out if this works in other strains of rats and for longer periods than that of just being transported from one floor to another in the same building 3 4 5 Arts J W M Ohl F and Kramer K 2008 Transportation as major life event in rats Effects on welfare and limits of adaptation Measuring Behavior 2008 p 356 Arts J W Kramer K Arndt S S and Ohl F 2012 The Impact of transportation on physiological and behavioural parameters in Wistar rats Implications for acclimitisation periods ILAR journal 53 1 ppE82 E98 Accessed 7 1 20 Arts J W M 2016 Transportation in Laboratory Rats Effects of a Black Box Doctoral dissertation Utrecht University Castelhano Carlos M J and Baumans V 2009 The impact of light noise cage cleaning and inhouse transport on welfare and stress of laboratory rats Laboratory animals 43 4 pp 311 327 Swallow J Anderson D Buckwell A C Harris T Hawkins P Kirkwood J Lomas M Meacham S Peters A Prescott M and Owen S 2005 Guidance on the transport of laboratory animals Laboratory Animals 39 1 pp 1 39 Figure 4 Rats searching for hidden treats 163

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Animal Technology and Welfare Welfare August 2020 Shining a light on rearing pigmentless Zebrafish JACQUELINE GLOVER THOM BERRIMAN DIMITRA MANTZOROU WILLIAM HAVELANGE SAM BERRY and BRUNO CORREIA DA SILVA King s College London Zebrafish Facility Biological Services Unit Hodgkin Building Guys Campus London SE1 1UL UK Correspondence jacqueline glover kcl ac uk Introduction Zebrafish Danio rerio strains with mutated pigment cells allow clear in vivo imaging to be carried out In 2016 Crystal mutants which lack most pigment cells in both their body and eyes were first produced at King s College London 1 However evidence shows strains with mutated pigment cells exhibit reduced survival during the early rearing stages in comparison to wildtype strains 2 3 The Crystal strain in particular has poor survival in the nursery compared to wildtypes and anecdotal evidence suggests lower spawning success compared to other strains Light has been shown to influence the survival of wildtype zebrafish larvae 4 however research of its effect on other zebrafish strains is limited 3 Light is also well known to influence spawning behaviour in zebrafish as their reproductive cycle is photoperiod dependant with most spawning at dawn 5 This experiment aimed to look at the impact of light intensity as to refine methods of rearing pigmentless Zebrafish and to reduce the number of fish needed to maintain important genetic lines such as the Crystal line Materials and Methods Three strains of Zebrafish varying in their degree of pigmentation were selected Wildtype AB Albinob4 and Crystal Mutant nacrew2 w2 albb4 b4 roya9 a9 Figure 1 Average light intensity LUX of tanks across two racks High medium and low light intensity used for the purpose of this study are shown in red Twelve rows on the same recirculating system were filled with empty tanks A lux meter was used to calculate the average light intensity LUX of each row from inside the tanks Three rows were selected to represent high 259 LUX medium 128 LUX and low 38 LUX light intensity levels Figure 1 Three tanks of each Zebrafish strain were then assigned to each of the three rows At 5dpf fry were transferred into 3 5 litre tanks n 25 per tank containing 1 litre of water All fish were fed according to methods previously described by Mantzorou et al 2017 6 Upon reaching sexual maturity between 58 84dpf the survival rate of each tank was calculated All tanks were then reduced to 17 fish per tank Remaining fish were then paired to spawn a total of three times to measure spawning success Eggs from each pair were collected in separate petri dishes and the fertile eggs produced by each pair were counted Figure 2 Wildtype AB left Albino centre and Crystal Mutant right Zebrafish strains 164

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Welfare Animal Technology and Welfare August 2020 The Jacket with pulling power a novel approach to early stage evaluation of magnetic nanoparticles ALISON RITCHIE 1 JAMES DIXON 2 PHIL CLARKE 1 and ANNA GRABOWSKA1 1 2 Division of Cancer and Stem Cells School of Medicine University of Nottingham Division of Regenerative Medicine and Cellular Therapies School of Medicine University of Nottingham Correspondence Alison Ritchie nottingham ac uk Introduction As a cancer research group one of our aims is to develop more efficient methods of compound delivery One area we are investigating is that of the delivery of theranostic nanoparticles which combine both therapeutic and diagnostic agents that directly target a tumour thus reducing potential side effects from systemic therapies However it is very difficult to reproduce reported results as methodology details are scant and often rely on cartoons rather than photographs Figure 2 One delivery method is to magnetise the nanoparticles during formulation and after injection they are exposed to a magnetic field located close to the tumour Figure 1 Figure 2 Representations of in vivo testing of theranostic particles Materials and methods We wanted to develop an early stage testing system which would allow us to fine tune the formulation of our nanoparticles prior to full translationally relevant testing and which would demonstrate the efficiency of the targeting without the need for invasive damaging or uncomfortable procedures Figure 1 Principles of theranostic particle delivery In order to study the targeting efficiency as well as the theranostic properties of these magnetised nanoparticles they need to be tested in vivo Researchers have used a variety of methods to expose the particles to a magnetic source including i MRI expensive therefore impractical for early stage testing requires lengthy anaesthesia and specialist equipment ii Sticking a magnet to the animal s skin with tape or superglue can damage the skin and is difficult to remove iii Surgically implanting subcutaneously requires full aseptic surgery and remains in place until termination 166 To this end we decided to develop a novel inexpensive and non harmful way to present the magnet to the tumour and took our inspiration from the already widelyavailable infusion jacket Working with the manufacturer we designed a Spandex jacket Figures 3 and 5 with a pocket to house the magnet Figure 4 and proceeded to test our nanoparticles By developing this we hoped to deliver the particles intravenously and then direct them towards an established subcutaneous tumour using the magnet After injecting the magnetic nanoparticles intravenously via the tail vein the mouse was lightly anaesthetised with an injectable anaesthetic the jacket was fitted and remained in place for one hour Figure 6

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August 2020 Animal Technology and Welfare Poster Presentations After this time the jacket was removed and the animal was imaged for fluorescence in the IVIS Spectrum Figure 7 under the same anaesthetic event Figure 3 Skin side of jacket Figure 7 IVIS Spectrum imaging system Results I hr post injection fluorescent images Figure 4 Magnet Figure 8 With magnet Figure 5 Outside of jacket Figure 9 Without magnet It can be seen from the images that the Cy5 fluorophore conjugated magnetic nanoparticles have accumulated in the region of the tumour in the presence of the magnet Figure 8 but not in its absence Figure 9 However ex vivo analysis showed that the particles had not actually penetrated the tumour which we believe is due to the encapsulated nature of the subcutaneous tumour Conclusions Figure 6 Mouse wearing jacket Preliminary data is very promising in that it demonstrates the ability of the magnet to attract the magnetic nanoparticles when housed in the jacket Moreover this pilot study has also identified a number of areas for improvement particularly in facilitating tumour uptake of the nanoparticles To this end we intend to further test the system against other tumour models including an orthotopic intraperitoneal model 167

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AS ET Congress Bursary Competition Patron Professor Lord Naren Patron Naren Patel Patel KT KT ChairofofTrustees Trustees Professor Professor Sir Sir Richard Richard Gardner Gardner FIAT Chair FIAT Hon Hon FRS FRS Secretaryto tothe theTrust Trust Ken Ken Applebee Applebee OBE Secretary OBE FIAT FIAT FRSB FRSB Trustees Stephen StephenBarnett Barnett MSc MSc Hon Hon FRSB FRSB Jasmine Trustees Jasmine Barley Barley MSc MSc FIAT FIAT Karen J Gardner Wendy Steel BSc Hons Karen J Gardner Wendy Steel BSc Hons FIAT FIAT Registered Charity Number 113319 Registered Charity Number 113319 Registered Office 5 South Parade Summertown Oxford OX2 7JL Registered Office 5 South Parade Summertown Oxford OX2 7JL The Animals in Science Education Trust is pleased to announce the AS ET Congress seventh Bursary Competition to attend Congress 2021 Bursary Competition The intention of the bursary is to encourage early career animal technical staff to extend their knowledge and experience by The Animals in Science Education Trust is pleased to announce the attending the largest Animal Technology meeting in the UK seventh Bursary Competition to attend Congress 2021 Applications are invited from animal technical staff based in the The intention of the bursary is to encourage early career animal UK or the Republic of Ireland who are studying for or were technical staff to extend their knowledge and experience by awarded the IAT Level 2 Diploma in Laboratory Animal Science attending largest Animal Technology meeting in the UK andthe Technology in 2018 2019 or 2020 The successful applicant will be selected on staff the basis of a in 1000 Applications are invited from animal technical based the word essay on of theIreland following UK or the Republic who are studying for or were awarded the IAT Level 2 Diploma in Laboratory Animal Science What challenges and rewards have you experienced as and Technology in 2018 2019 or 2020 an Animal Technician The successful applicant will be selected on the basis of a 1000 The essays must be received by AS ET by 20th October 2020 They word essay on the following should be sent via email together with the application form to contact as et org uk What challenges and rewards have you experienced as an Animal Technician Full conditions and application forms can be found on the bursary page of the AS ET website www as et org uk Also see the privacy The essays must be AS ETpage by 20th statement on received the AboutbyAS ET of ourOctober website 2020 They should be sent via email together with the application form to contact as et org uk Full conditions and application forms can be found on the bursary page of the AS ET website www as et org uk Also see the privacy statement on the About AS ET page of our website