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IAT Journal Animal Technology and Welfare Official Journal of the Institute of Animal Technology and European Federation of Animal Technologists ISSN 1742 0385 Vol 17 No 1 April 2018

CONTENTS Vol 17 No 1 April 2018 Editorial Jas Barley Chair of the Editorial Board ix Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method to measure internal body temperatures in invertebrates Stuart Baker Emily Knight Sarah Pellett Karen Roberts Erwin Smulders and Steven Trim 1 Synchronisation of oestrous cycle on C57BL 6J mice after the introduction of a hanging perforated container with bedding soaked by the excretions of male mice Evangelos Balafas Marianna Stasinopoulou Pavlos Alexakis Efthimios Paronis Kostantinos Paschidis and Nikolaos Kostomitsopoulos 9 PAPER SUMMARY TRANSLATIONS ANDREW BLAKE TRIBUTE AWARD WINNING ENTRY Refinement of tumour passage by optimisation of the tissue disaggregation protocol Melanie Galvin Alice Lallo Stewart Brown Paul Kelly Becky Bola Danielle Potter Cassandra Hodgkinson Kristopher Frese and Caroline Dive TECH 2 TECH Selective breeding in dogs Kerry Lavin Thomas 13 21 25 Can technologists benefit more from inter facility working personal experiences from an in vivo study during my role as a visiting worker Asha Konde 29 Identification in Xenopus laevis Cheryl Yalden 33 AS ET CONGRESS BURSARY COMPETITION ESSAYS How would you convince members of the general public that the use of animals in medical research was justifiable Jobie Broadhurst 37 How would you convince members of the general public that the use of animals in medical research was justifiable Gavin Skeels 39 How would you convince members of the general public that the use of animals in medical research was justifiable Justyna Barratt 41 POSTER PRESENTATIONS The introduction of a cable tie swing and its impact on animal welfare Laura Dean Jacqui Swan Noelia Lopez Salesansky Simon Poucher and Lisa Doar 43 Target training pigs within an isolation unit a pilot study Louise Carder 46 Improving safety assessment studies by the introduction of a refined ECG vest for dogs Peter Fisher 48 A method for repeated dosing of juvenile mice via oral gavage Peter Paccagnini 50 A barrier breakdown with potential human welfare implications while not affecting animal welfare Sarah Tamasauskas Sophie Cartwright and Stuart Stevenson 52 The importance of high quality health and phenotype information prior to importing new genetically altered GA lines Annabell Cristi and Melissa Flory 54 The life of Birdie hand rearing for comparative cognition studies Sam Melvin 60 Advances in animal welfare and scientific outcomes of the bile duct cannulated rat model utilised in ADME studies Hans van Wijk Lee Crossman Georgina Adjin Tettey Dawn Haida Emily Newell and John Kendrick 64 Are callipers obsolete A novel 3D scanning technology to measure subcutaneous tumour volume Zena Wilson Juan Delgado Michael Davies Rebecca Whiteley Jennifer Hare Amar Rahi Stephen Marshall Jarno Rall Andrew Smith Stephen Atkinson Adeala Zabair and Jane Kendrew 68 BOOK REVIEW 72 Index to Advertisers xv i

IAT REPRESENTATIVES OFFICERS President Dr Robin Lovell Badge FRS Immediate Past President Professor Sir Richard Gardner MA PhD FRSB FIAT Hon FRS Vice Presidents David Anderson MRCVS Stephen Barnett BA MSc FIAT Hon CBiol FRSB RAnTech Brian Cass CBE Miles Carroll PhD Gerald Clough BSc PhD EurBiol CBiol MRSB SFZSL Paul Flecknell MA Vet MB PhD DLAS DipLECVA MRCVS FIAT Hon Sue Houlton BVSc MA DVR DVC MRCVS 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 Jos Orellana BVSc MSc Clive Page PhD BSc Vicky Robinson CBE BSc PhD Gail Thompson RLATG Robert Weichbrod PhD RLATG Life Members 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 HonFIAT RAnTech Dorothy Woodnott FIAT Honorary Members Andy Jackson MIAT Brian Lowe MSc FIAT RAnTech Sue McHugh BSc FIAT Terry Priest MBE FIAT RAnTech Trevor Richards BEM MIAT David Spillane FIAT Pete Willan DMS FInstLM MIAT Members of Council Ken Applebee OBE Matthew Bilton Kally Booth Charlie Chambers Steven Cubitt Simon Cumming Andy Cunningham Haley Daniels Glyn Fisher Nicky Gent Cathy Godfrey Alan Graham Linda Horan Sam Jameson Elaine Kirkum Adele Kitching Theresa Langford Steve Owen Wendy Steel Allan Thornhill Lynda Westall Carole Wilson Adrian Woodhouse Council Officers Chair Ken Applebee OBE FIAT CBiol FRSB RAnTech Vice Chair Linda Horan BSc Hons MIAT RAnTech Honorary Secretary Linda Horan BSc Hons MIAT RAnTech Honorary Treasurer Charlie Chambers MIAT RAnTech Assistant Treasurer Glyn Fisher FIAT RAnTech Chair Board of Educational Policy Glyn Fisher FIAT RAnTech Chair Board of Moderators Haley Daniels MBA MSc MIAT RAnTech CIPD Chair Registration Accreditation Board Vacant TBC ATW Editor Jas Barley MSc FIAT RAnTech Bulletin Editor Carole Wilson BSc MIAT Branch Liaison Officer Lynda Westall BSc Hons FIAT DMS RAnTech EFAT Representative Charlie Chambers MIAT RAnTech Website Coordinator Allan Thornhill FIAT RAnTech Animal Welfare Officers and LABA Representatives Andy Cunningham Matthew Bilton Kally Booth Simon Cumming Nicky Gent ATW Bulletin Editorial Board Jas Barley Chair Matthew Bilton Nicky Gent Patrick Hayes Elaine Kirkum Carole Wilson Lynda Westall Board of Educational Policy Glyn Fisher Chair Steven Cubitt Secretary Adele Kitching Board of Moderators Haley Daniels Chair Moderators Anthony Iglesias Theresa Langford Jenny Parks Sarah Reed Communications Group Adrian Woodhouse Chair Elaine Kirkum Teresa Langford Allan Thornhill Lynda Westall CPD Officer Charlie Chambers Registration and Accreditation Board Chair Vacant Ken Applebee Charlie Chambers John Gregory Cathy Godfrey Gerald Clough Stuart Stevenson Carol Williams Observers Charles Gentry Certificate Holders Forum Adrian Deeny LASA Ian Mason Home Office Ngaire Dennison LAVA Congress Committee Alan Graham Chair Haley Daniels Linda Horan Adele Kitching Allan Thornhill Diversity Officer Haley Daniels MBA MSc MIAT RAnTech CIPD UK Biosciences ASG Representative Home Office Steve Owen Charlie Chambers Andy Cunningham IAT OFFICERS MAY BE CONTACTED VIA IAT Administrator admin iat org uk OR VIA THE IAT WEBSITE AT www iat org uk OR VIA THE REGISTERED OFFICE 5 South Parade Summertown Oxford OX2 7JL Advertisement Managers PRC Associates Ltd Email mail prcassoc co 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 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April 2018 Animal Technology and Welfare THE INSTITUTE OF ANIMAL TECHNOLOGY ETHICAL STATEMENT Our purpose is to advance knowledge and promote excellence in the care and welfare of animals in science and to enhance the standards and status of those professionally engaged in the care welfare and use of animals in science Editorial Jas Barley Chair of the Editorial Board As anyone who knows me will confirm it is not often that I am lost for words but I have really struggled to think of something to write about in this editorial I mulled various ideas over and then started to think as to who Animal Technology and Welfare ATW was trying to serve and of course what it was for The mission statement for this journal appears on the IAT website and states quite clearly that we aim to be the medium of Animal Technologists and all those concerned with the care and welfare of animals used for research purposes to communicate best practice In addition to our main reason for existence I consider that as a journal we should give younger technologists an opportunity to be published and this does not necessarily mean only communicating about science but includes the opportunity to share their experiences of caring for animals both those within our industry but also those of us sharing the lives of the animals we encounter at home and elsewhere Two good examples of giving younger Technologists a chance to be heard are the contributions from Kerry Lavin Thomson Asha Konde and Cheryl Yalden All these articles originate from presentations made as part of the Applied Learning and Developmental Skills Module of the Higher Education Programme Designed to develop skills in reflection research and the development of effective plans that enhance the development and delivery of learning at both an individual and organisational level Kerry s article raises some interesting points about the breeding of dogs and one cannot fail to reflect that if we were breeding animals with similar defects in our industry it would be regulated The only criteria it does not meet for such activity to require a licence is that it is not for scientific purposes Further examples of the writing abilities of younger Technologists can be seen in the entries for the AS ET Congress Bursary competition We have three very different responses to the same question but all deserving merit in their own right This issue also demonstrates the diversity of animals we Technologists work with as it includes another contribution from the team at Venomtech I should warn those of our readers who like me cannot bear the creatures this article contains photographs of spiders Obviously Steve Trim and his team feel somewhat differently about these eight legged horrors but it is very reassuring that they give as much concern about the welfare of their spiders as do the Technologists who work with more conventional animals As well as serving the UK our readership includes Technologists and related staff in over 28 countries worldwide and we always welcome articles from them The contribution from the staff at the Greek Biomedical Research Foundation of the Academy of Athens demonstrates what may seem a simple way of refining synchronisation of oestrus but as the initial study seems to indicate it could be an important way of improving animal welfare The Andrew Blake Tribute Award was a young man s way of acknowledging the service Animal Technologists provide to those with life threatening illnesses Suffering from an incurable condition known as Fredrich s Ataxia Andrew was aware of the difficulties Animal Technologists at the time were facing from the extreme animal rights campaign and wanted to show that the work we do is appreciated by those who suffer from life limiting illnesses The award was intended not only to show us that we were valued but also to exhibit to our critics that animal welfare was not being sacrificed in the name of science Melanie Galvin and her colleagues in the Clinical and Experimental Pharmacology group based at the Cancer Research UK Manchester Institute are excellent examples of this combination of fighting human disease but at the same time continually striving to improve welfare and reduce the use of animals in research It would not be ATW if we were unable to include a selection of posters from Congress and the final contributions from Congress 2017 are included here A wide range of welfare and scientific refinements and improvements are discussed ranging from swings for mice target training in pigs hand raising birds and bile duct cannulation in mice I hope you will agree that ATW is meeting its purpose of providing a medium for disseminating best practice whilst at the same time giving everyone an opportunity to be heard ix

April 2018 Animal Technology and Welfare Spider and chips the use of Internal Radio Frequency Identification RFID chips as a minimally invasive method to measure internal body temperatures in invertebrates STUART BAKER 1 EMILY KNIGHT 1 2 SARAH PELLETT 3 KAREN ROBERTS 4 ERWIN SMULDERS4 and STEVEN A TRIM1 1 2 3 4 Venomtech Ltd Discovery Park Sandwich Kent CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury Kent CT1 1QU Animates Veterinary Clinic Ltd The Green Thurlby Lincolnshire PE10 0EB Plexx BV PO Box 86 6660 AB Elst The Netherlands Correspondence s trim venomtech co uk Abstract Introduction Internal RFID transponders have been used in vertebrates for many years however studies into their use in invertebrates are less well represented in the literature The use of RFID transponders for internal temperature measurement represents a less invasive alternative to thermocouples and rectal thermometers Internal transponders were inser ted into three invertebrate species Salmon pink spiders Lasiodora parahybana desert locusts Schistocerca gregaria and Madagascan hissing cockroaches Gromphadorhina portentosa Animal behaviour was monitored and temperature measurements were obtained from the transponder and compared with the animals surroundings Radiographs confirmed location and relative size of the transponder implants The use of Radio Frequency Identification RFID technology in vertebrate animals is commonplace This microchipping of animals is becoming a legal requirement in an increasing number of instances The procedure usually takes the form of a subcutaneous implant or transponder being inserted under the skin of the animal Surviving animals appeared to behave normally during the study Small differences in internal temperatures compared to surface and surroundings were observed however further studies are recommended in order to validate these findings This pilot study demonstrates modern implants designed for rodents can be implanted successfully in large arthropods and this is expected to make a great impact in future invertebrate physiology research In a laboratory setting transponders are used to simplify the identification of individual animals and have been used for permanent identification of rats 1 guinea pigs rabbits and amphibians2 as well as theraphosid spiders 3 The transponders used in this study are also capable of relaying internal body temperature data Although the initial procedure is invasive this technology has replaced the use of rectal thermometers in ver tebrates and should replace thermocouples in laboratory invertebrates allowing measurements to be taken without causing any subsequent stress or injury to the subjects 4 Keywords RFID inver tebrate body temperature internal transponder Exotic animals are often fitted with an RFID transponder to comply with legislation regarding the Convention on International Trade in Endangered Species CITES RFID transponders are used in livestock animals primarily to track medical treatment and vaccinations as well as for traceability through the human food chain The use of RFID technology in invertebrates is at the 1

Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method present time far less widespread Cooper and Cooper5 produced a comprehensive list of methods of identifying live animals and considered the insertion of transponders to be suitable only for vertebrates There are instances of transponders being glued to the exterior surface of invertebrates such as honeybees Apis mellifera 6 The use of radio telemetry tags on lobsters Homarus americanus and other large crustaceans appears to be relatively commonplace but often relies on the equipment being attached to the outside of the animal 7 In longer term studies external tags can be damaged or lost if they become dislodged they may cause injury to the animal Problems are also likely to occur during Ecdysis which could prove fatal i e transponders that are tied to par ts of an invertebrate could prevent a successful moult Smaller transponders that are attached to the exoskeleton via an adhesive would simply be lost during the moulting process An internal transponder avoids these problems There are examples of internal tags being used with large crustaceans8 9 but very little with terrestrial invertebrates This study builds on previous work by Reichling and Tabaka3 by refining the implantation technique through use of the BMDS IPTT 300 transponder Plexx Elst Netherlands and proprietary delivery needle These refinements are aimed to reduce the trauma to the animals due to less tissue damage and thus reduced need for tissue glue They also allow internal temperature measurement in a repeatable and less traumatic way in arthropods as there is only a single implantation event This will allow greater research into invertebrate biology in the laboratory setting Materials and methods Ethical statement All procedures were performed in accordance with the ethos of the Animals Scientific Procedures Act 1986 even though the animals used are not protected species Animals Eight adult desert locusts Schistocerca gregaria sourced from Monkfield nutrition Royston UK were used as the pilot species in the study bodyweight 1 465g 0 18g The locusts were housed in a 3 litre l locking plastic box Really Useful Products Ltd Kingston Upon Thames UK with drilled ventilation holes Dry blue roll was used as substrate with water being provided in a small plastic dish and a half round of UPVC round line guttering Wickes Ltd Watford UK acting as a hide basking spot The box was heated from above with a 75W heat lamp approximately 30cm above the enclosure This study utilised four adult salmon pink spiders Lasiodora parahybana with mean body weight 44 46g 2 16 04g All four of these animals were long term captives at Venomtech originally sourced from commercial breeders in the UK The spiders were housed in standard 9 litre secure locking plastic boxes Really Useful Products Ltd with holes drilled for ventilation and 2 3cm depth of moistened vermiculite as substrate to maintain humidity Water was provided in small 5cm diameter dishes Vanishing World Herne Bay UK with half rounds of UPVC round line guttering provided as a hide to replicate natural burrows Individual plastic boxes were stacked on shelves with a 60W heat mat Habistat Hayes UK set to 27 3 C controlled by a standard range pulse proportional thermostat Habistat Boxes were heated from one end to provide a thermal gradient across the box with the hide at the end of the box closest to the heat mat Twelve adult Madagascan hissing cockroaches Gromphadorhina portentosa captive bred from a commercial breeder in the UK were also included The active animals had bodyweights 8 82g 0 04g They were housed in a 400mm x 200mm x 200mm acrylic enclosure The Spider s Lair UK as Really Useful Product enclosures do not provide sufficient security for this species Again dry blue roll was used as substrate with water being provided in a small plastic dish and a half round of UPVC round line guttering acting as a hide The enclosure was heated from below on one side with a 22W heat mat Ultratherm Ltd Glenrothes UK Surgical technique Pilot study One 5th instar locust weighing 1 353g was anaesthetised with a rapidly rising concentration of CO2 in a 125ml screw cap tub with a CO2 flow of 0 5 l min for 6 min 14 secs Once deeply anaesthetised the cap was removed from a new transponder needle and a BMDS IPTT 300 transponder Plexx Elst Netherlands was inserted into the left flank between the first and second abdominal segments No haemolymph leakage was observed so no wound sealing agent was used The locust successfully recovered from the anaesthesia after 9 min 47 sec in fresh air at ambient temperature The recovered locust was placed in a 3 litre enclosure as described above It was noted that the locust was making exaggerated breathing movements 24 min 5 mins post recovery it had vomited slightly and tarsus of rear legs were abnormally raised but it jumped normally when stimulated A second locust was added to the box the morning following the procedure as a control for the conditions Follow on study Following the pilot study the same procedure was carried out on a further five animals three more locusts then the two adult female salmon pink spiders

Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method All animals were anesthetised using a rising concentration of CO2 gas Each was then weighed and fitted with a subcutaneous BMDS IPTT 300 transponder Plexx Elst Netherlands In the spiders the transponder was inser ted into the opisthosoma laterally to the left of the dorsal midline using the proprietary needle supplied All other animals had minimal haemolymph leakage and had GLUture a topical tissue adhesive applied as a precautionary measure Figure 3 and Figure 4 All animals were then allowed to recover from anaesthesia in ambient conditions before being returned to their home enclosures Figure 1 In the case of the locusts this was into the abdomen Figure 2 Where necessar y a single locust damage to the animal s cuticle was repaired with GLUture World Precision Instruments Sarasota FL to prevent excessive loss of haemolymph Figure 3 Application of GLUture to L parahybana Figure 1 Insertion of transponder into L parahybana Figure 4 L parahybana post GLUture application Two of the transponders that were removed during post mortem examinations were sterilised and reused in adult Madagascan hissing cockroaches The transponders were inserted between 6th and 7th abdominal segment to the left side of the dorsal midline using a size 24 blade to make an incision about the width of the transponder The behaviour and survival of the animals was monitored following the procedures X ray imaging Figure 2 Insertion of transponder into S gregaria In order to determine the position of the transponder within their bodies whole body radiographs of the animals were taken using the Clear Vision DR 4000 digital radiography x ray system Jungwon Precision Ind Co Ltd Seoul Korea Conscious radiographs were taken anaesthesia or immobilising agents were not 3

Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method required for this procedure Dorsoventral views were taken for the spiders locusts and cockroaches Radiographs were obtained using the settings 55 kilovolts kV 100 milliamperes mA 0 1 milliamperes per second mA s for the spiders 40 kV 100 mA 0 05 mA S for the locusts and 50 kV 100 mA 0 1 mA S for the cockroaches Further images were obtained with similar clarity using the digital intraoral x ray system EVA Vet AEP Imaging New York Temperature data Ad hoc temperature data were collected for the internal temperatures using a DAS 7007S Wireless Reader with a IPT IPTT probe Plexx Elst Netherlands The probe was calibrated using an ST 8861 Infrared dual laser thermometer AML Instruments Gainsborough UK The same thermometer was used for external skin temperatures substrate and hide temperatures Data were collected at six different time points for each of the chipped spiders and the longest surviving locust The external temperature was measured by aiming the infra red thermometer at the opisthosoma of the spiders and abdomen of the locusts The substrate temperature was measured at the point closet to where the animal was sitting at the time of the measurement The hide temperature was measured at the closest point to the animal Data analysis Data were processed and analysed using MS Excel Microsoft Albuquerque USA and Minitab Minitab 17 Statistical Software 2010 All data were tested for normal distribution Paired T tests were performed for all data The statistical significance level was set at p

Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method differentiation but clearly showed the microchip 11 The radiograph images Figure 5 display the location of each transponder and the relative size of the chip compared to the overall body size of each type of animal tested substrate temperatures and hide temperatures each measured with the IR temperature gun The internal temperature was compared with each of the other three tested temperatures by paired Student s T test For each animal tested at least one IR temperature was significantly different p 0 05 from the recorded internal temperature The average temperature difference between the internal chip temperature 25 67 C and the surface temperature 25 12 C was 0 55 C Discussion Figure 5 Radiograph images showing the location and relative size of the transponder chips in a spider b cockroach c locust Temperature data Temperature data were collected and compiled for both chipped spiders L par01 and L par02 and the longest surviving locust The results displayed below Figure 6 compare the internal temperatures measured by transponder chip external skin temperatures Figure 6 Comparison of internal external substrate and hide temperatures for both chipped spiders L par01 and L par02 Ns not statistically significant p 0 05 Error bars show standard deviation based on six readings each from two individuals Figure 7 Comparison of internal external substrate and hide temperatures for the chipped locust Ns not statistically significant p 0 05 Error bars show standard deviation from five readings from one individual This work builds upon the previous use of passive integrated transponders in theraphosid spiders by Reichling and Tabaka 3 In addition to individual identification this paper describes the use of transponders for measuring internal body temperature This study demonstrates the further potential for implantation of similar transponders to measure other biological factors such as pH oxygen levels movement or heart rate The implantation technique applied to the spiders in this study was less invasive as the transponder was delivered via needle implantation rather than by cutting the opisthosoma to create a wound opening This meant that haemolymph leakage was minimal or absent Although glue was used to seal the puncture area in most cases it was considered a precaution rather than a necessity as minimal haemolymph leakage was seen The radiographs taken show the transponder location is broadly parallel with the body of the spider rather than perpendicular as shown by Reichling and Tabaka 3 It is thought that the transponders in this study are situated between the cuticle and fat tissue rather than perpendicular to the exoskeleton which has the potential for internal organ damage All test animals were anesthetised using CO2 prior to insertion of transponders It is thought that this would reduce the potential stress for the animal particularly regarding restraint stress It also allowed more precise insertion of transponder as the animals were immobile throughout the procedure The use of an anaesthetic also served to reduce the risk of a bite to the person implanting the transponder There was no discernible change in feeding habits or other behaviour following transponder insertion implying that anaesthesia did not have any long term effects on the animals following the recovery period Prior to the availability of temperature sensitive transponders more invasive techniques were required to obtain internal body temperatures from animals Examples include the use of rectal thermometers in vertebrates4 and the use of copper wire thermocouples in invertebrates 12 While the initial insertion of the transponder may cause stress to the animal subsequent readings can be taken through the walls of an enclosure and unlike the insertion of a rectal 5

Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method thermometer should not cause any additional stress Pers obs 13 The fitting of thermocouples as used by Bartholomew and Bernd 12 is a more invasive procedure Two incisions insertions need to be made to attach the thermocouple These then remain in place with fixed wires protruding from the animal In contrast a transponder can remain in place indefinitely without causing any apparent issues for the animal No significant difference between the internal and external temperatures were recorded in the locust Contrary to what was expected it was found that there was a significant difference between the internal and external temperatures of the spiders There was also a significant difference found between the internal temperatures and the substrate temperatures in both cases Several possible explanations for this are discussed below As the spiders are kept in boxes on racks the boxes had to be removed from the racks and the lids opened before any of the external temperatures were measured On occasion during the process the spiders appeared to be startled and moved from one end of their enclosure to the other It is possible that the spider had been basking near to the heat mat and after running away from it appeared warmer than its surroundings Assuming the rate of heat loss would be more rapid from the surface of the animal than from its interior this could also explain the difference between the internal and external temperatures Relatively small variations such as those observed could possibly be the result of heat generated from muscle movements when the spider moved rapidly upon being disturbed Bar tholomew and Bernd 12 observed using thermocouples significantly larger temperature differences generated by rapid muscle movement in invertebrates than shown in this study This trial clearly demonstrates improved techniques for studying such physiology in invertebrates as still very little is known Significant differences were found between the temperature of the hides and the substrates and the above biological explanations obviously cannot account for this It suggests the possibility that the infra red thermometer experiences some variation in the temperature measurement of varying surfaces due to different resistivity This could account for the differences in temperature readings obtained from the transponder compared to readings obtained from the infra red thermometer It is also possible that one or more of the transponders was incorrectly calibrated The manufacturer recommends that the transponders are calibrated 6 using a temperature controlled water bath In an attempt to minimise any discrepancies caused by measurements taken using two different devices the transponders were calibrated using the infra red thermometer rather than a water bath for this study It is possible that this deviation from the recommended practice was enough to cause inaccurate readings from one or more of the transponders Karen Roberts and Erwin Smulders pers com This study widens the potential for using other types of transponders in inver tebrate model animals Transponders currently available or in development that could measure pH O2 heart rate and respiration rate14 are of similar size Standard transponders in conjunction with fixed sensors can be used to monitor the animal s movement 15 This would provide information on basking behaviour and thermal regulation Use of these transponders could improve the current understanding of invertebrate health and biology by providing baseline biological data which to the authors knowledge is not currently presented in the literature One particularly relevant example would be a transponder capable of measuring pH Although the use of CO2 as an anaesthetic did not appear to cause significant harm or stress to the invertebrates used in this study questions have been raised concerning acidosis associated with CO2 which have been difficult to answer in the past A pH sensing transponder could answer any such questions in the future CO2 is routinely used at Venomtech for anaesthesia of arthropods with a high success rate The use of various types of transponders may allow the use of invertebrate animals in the laboratory where only vertebrates are currently used This would be of benefit to scientists as there are fewer legal and licensing implications when working with invertebrates as the law currently stands Further investigation of large ar thropod physiology will not only aid our understanding but could reduce the numbers of rodents used in severe studies of evolutionarily conserved physiology such as pain In conclusion although some small but significant differences between internal transponder temperatures and external measurements were found fur ther studies with more individuals are required to build on this pilot study The work presented here in combination with the work presented by Reichling and Tabaka3 demonstrates the safe and effective use of internal transponders in theraphosid spiders However the authors agree that locusts are too small for the currently available transponders due to the high mortality rate The lower limit of successful implantation in this study is 8 8g bodyweight for invertebrates The authors feel further studies are required to confirm the lower safe limit for

Spider and chips the use of internal Radio Frequency Identification RFID chips as a minimally invasive method transponders of this size which is expected to be between 8 8g and 1 4g To the best of the authors knowledge these are the smallest arthropods to be successfully implanted with RFID chips Acknowledgements 15 progress and challenges Current Opinion in Chemical Biology 2002 6 5 633 641 K nig B Lindholm A K Lopes P C Dobay A Steinert S and Buschmann F J U 2015 A system for automatic recording of social behavior in a free living wild house mouse population Animal Biotelemetry 2015 3 1 39 Thanks to Craig Mackinlay for assistance with radiography References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ball D J Argentieri G Krause R et al 1991 Evaluation of a microchip implant system used for animal identification in rats Laboratory Animal Science 1991 41 2 185 186 Mrozek M Fischer R Trendelenburg M and Zillmann U 1995 Microchip implant system used for animal identification in laboratory rabbits guinea pigs woodchucks and in amphibians Laboratory Animals 1995 29 3 339 344 Reichling S B and Tabaka C 2001 A technique for individually identifying tarantulas using passive integrated transponders Journal of Arachnology 2001 29 1 117118 Chen P H and White C E 2006 Comparison of rectal microchip transponder and infrared thermometr y techniques for obtaining body temperature in the laboratory rabbit Oryctolagus cuniculus J Am Assoc Lab Anim Sci 2006 45 1 57 63 Cooper J E and Cooper M E 2013 Wildlife Forensic Investigation Principles and Practice CRC Press 2013 Henry M Beguin M and Requier F et al 2012 A common pesticide decreases foraging success and survival in honey bees Science 2012 336 6079 348350 Schmiing M and Afonso P 2009 Acoustic tag retention of the Mediterranean slipper lobster Scyllarides latus Latreille 1802 in the Azores Life and Marine Sciences 2009 26 37 43 Frusher S D Hall D Burch P and Gardner C 2009 Combining passive integrated transponder tags with conventional T bar tags to improve tag reporting rates in a rock lobster trap fishery New Zealand Journal of Marine and Freshwater Research 2009 43 1 347 353 Sato T Yoseda K Abe O et al 2013 Growth of the coconut crab Birgus latro estimated from mark recapture using passive integrated transponder PIT tags Aquatic Biology 2013 19 2 143 152 Bennie N A Loaring C D Bennie M M and Trim S A 2012 An effective method for terrestrial arthropod euthanasia J Exp Biol 2012 215 Pt 24 4237 4241 Pizzi R 2012 Spiders In Lewbar t G A ed Invertebrate Medicine Chichester UK Wiley Blackwell 2012 187 221 Bartholomew G A and Bernd H 1978 Endothermy in African Dung Beetles During Flight Ball Making and Ball Rolling J Exp Biol 1978 73 65 83 Cilia J Piper D C Upton N and Hagan J J 1999 A comparison of rectal and subcutaneous body temperature measurement in the common marmoset J Pharmacol Toxicol Methods 1999 40 1 21 26 Frost M C and Meyerhoff M E 2002 Implantable chemical sensors for real time clinical monitoring 7

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April 2018 Animal Technology and Welfare Synchronisation of oestrous cycle on C57BL 6J mice after the introduction of a hanging perforated container with bedding soaked by the excretions of male mice EVANGELOS BALAFAS MARIANNA STASINOPOULOU PAVLOS ALEXAKOS EFTHIMIOS PARONIS KOSTANTINOS PASCHIDIS and NIKOLAOS KOSTOMITSOPOULOS Laboratory Animal Facility Biomedical Research Foundation of the Academy of Athens Athens Greece Correspondence nkostom bioacademy gr Abstract Synchronisation of oestrous cycle is a commonly used method to manage pregnancies either for breeding or for research purposes Based on Whitten effect the aim of our study was to introduce a new refined method for oestrous synchronisation and to evaluate its efficacy Our results clearly show that the use of a hanging perforated container with bedding soaked by the excretions of male mice can be used as an effective method for the induction of oestrous in mice housed in IVCs Our intervention apart from being noninvasive constitutes a refinement for the environment of the animals because it gives the opportunity to the animals to interact with the container and promotes their socialisation Introduction Synchronisation of oestrous is important to improve animal breeding management and to meet the requirements of studies that need large numbers of pregnant mice with accurate identification of the date of the mating and pregnancy The method used to achieve synchronisation varies from institution to institution Bedding from cages with male mice is often mixed with the females bedding in order to exploit the Whitten effect by which air diffused pheromones from the males excretions induce a synchronisation of oestrous cycles in exposed females 1 2 The housing of the animals in Individually Ventilated Cages IVCs with high rates of air renewal 70 air changes per hour may affect the efficacy of this approach and group housing of females may suppress the oestrous cycle 3 Another method of choice for the synchronisation of the oestrous cycle is the use of vasectomised mice 1 This procedure is invasive while anaesthesia and postoperative complications and side effects may occur In addition this approach requires time specialised equipment and trained personnel all of which may delay study starts and increase study costs Induction of the oestrous cycle has also been accomplished through the use of pharmacological agents specifically exogenous hormones Hormones may be luteolytic prostaglandin F PGF2 and its analogues or progestative progesterone and its analogues These substances are administered either as subcutaneous implants or by intraperitoneal injection 4 5 As with vasectomisation this approach may also be considered as invasive and raises animal safety concerns The aim of the present study was to successfully induce synchronisation of oestrous cycle with a noninvasive refined procedure that takes into account the wellbeing of the animals The study was performed in the Laboratory Animal Unit of the Biomedical Research Foundation of the Academy of Athens in accordance to the Greek and European legal framework for the protection of animals used for scientific purposes Presidential Decree 56 2013 and European Directive 2010 63 Materials and methods Eighty four 10 week old C57BL 6J female virgin mice originated from 10 12 different litters from the breeding colony of our facility were housed in polysulfone individually ventilated cages IVCs 1284L 9

Synchronisation of oestrous cycle on C57BL 6J mice Blue Line Sealsafe Tecniplast Buguggiate Italy All animals were housed under Specific Pathogen Free SPF conditions in the same animal room supplied with HEPA filtered air at 15 ACH at a temperature of 22 2 C and relative humidity 55 10 a 12 12 hour light dark cycle lights on at 0700 light intensity of 300 lux at one metre above the floor in the middle of the room and positive air pressure of 0 6 Pa within the room During the dark cycle rooms were completely devoid of light All mice had ad libitum access to filtered tap water in drinking bottles and a pelleted chow 4RF22 Mucedola Milan Italy Autoclaved corncob was used as bedding material Rehofix MK 2000 J Rettenmaier Sons Rosenberg Germany Female mice were randomly allocated in two equal groups Before the start of the study vaginal smears of the female mice were taken on two consecutive days to determine whether all animals showed a normal oestrous cycle as well as the exact stage of the cycle of each animal For the collection of vaginal smear we used a cotton swab wetted with normal saline 6 The vaginal swab was rolled across a dry glass slide Slides were stained with methylene blue and obser ved immediately under a microscope 200x The stage of oestrus cycle was determined based on the presence or absence of leukocytes keratinised epithelial and nucleated epithelial cells 7 In the treated group n 42 a perforated container with soiled bedding material corncob moistened by biological excretions during a period of 10 days collected from the 3 different cages containing 5 13 to 15 week old C57BL 6J male mice was hung from the metal lid of the female cage Figure 1 all cages were placed in the middle rows of the racks approximately one metre above the floor Light intensity within the cage varied between 51 and 60 lux Light intensity was measured using a calibrated lux meter Lux Meter LX1010BS China Room conditions were continuously monitored through the central Building Management System BMS of the animal facility After 48 and 72 hrs from the placement of the perforated container the swab smear technique was repeated for all animals in order to ascertain if oestrous cycle synchronisation had occurred as well as the percentage of mice in which this was achieved In all cases vaginal smears were taken between 09 00 and 10 00 am At the end of the study all animals were kept for breeding purposes The analysis of vaginal smears was run under blind conditions Results were recorded and data from each day were analysed using the Pearson s chi square test In the statistical evaluation p 0 05 was defined as significant All analyses were performed using the SPSS version 22 00 SPSS Inc Chicago IL Results At the first day D0 of the experiment comparison between the two groups revealed no statistically significant difference for any oestrous cycle stage p 0 05 Figure 2 Figure 2 Percentage of mice oestrous phase during the study in both control and treated groups on D0 just before the introduction of the container Figure 1 A perforated container with used bedding material was hung from the metal lid of the female cage In the control group n 42 a perforated container with clean bedding material was similarly introduced Female mice were housed in groups of 4 5 animals per cage To avoid any bias due to different light intensity 10 Figure 3 Percentage of mice oestrous phase during the study in both control and treated groups on D2 48 hours after the introduction of the container

Synchronisation of oestrous cycle on C57BL 6J mice Forty eight hours after the placement of the container D2 42 9 of the animals in the control group were at the dioestrous stage 19 0 at metoestrous 23 8 at proestrous and 14 3 at oestrous There was a statistically significant difference between the control and treatment group where 52 4 of the animals were exhibiting oestrous and 28 6 proestrous Figure 3 On the third day of the experiment D3 a significant difference was also observed between the two groups Most animals in the control group were exhibiting dioestrous and proestrous 28 6 and 33 3 respectively On the contrary in the treatment group oestrous consists the dominant stage with a percentage even higher than the previous day 61 9 Figure 4 All results are presented in Table 1 results the use of a hanging perforated container with bedding soaked by the excretions of male mice can be used as an effective method for the induction of oestrous in mice housed in IVCs Our intervention apar t from being non invasive constitutes an environmental refinement of the animals because it gives an opportunity for the animals to interact with the container and promotes their socialisation as they can use the device and play together with the other mice in the cage According to our knowledge this is the first time this method has been implemented Additionally it is widely applicable and does not require sophisticated equipment or trained personnel References 1 2 3 Figure 4 Percentage of mice oestrous phase during the study in both control and treated groups on D3 72 hours after the introduction of the container 4 5 6 7 Table 1 Percentage of mice estrous phase during the study in both control and treated groups D0 just before the introduction of the container D2 48 hours and D3 72 hours after the introduction of the container In parenthesis the exact number of animals tested Whitten W K 1958 Modification of the oestrous cycle of the mouse by external stimuli associated with the male changes in the oestrous cycle determined by vaginal smears Journal of Endocrinology Vol 17 No 3 307 313 Whitten W K Bronson F H and Greenstein J A 1968 Estrus inducing pheromone of male mice transport by movement of air Science Vol 161 No 3841 584 585 Kostomitsopoulos N Alexakos P Eleni K Doulou A Paschidis K and Baumans V 2012 The effects of different types of individually ventilated caging systems on growing male mice Lab Animal NY Vol 41 No 7 192 197 Pallares P and Gonzalez Bulnes A 2009 A new method for induction and synchronisation of oestrus and fertile ovulations in mice by using exogenous hormones Laboratory Animals Vol 43 No 3 295 299 Hasegawa A Mochida K Inoue H Noda Y Endo T Watanabe G and Ogura A 2016 High yield superovulation in adult mice by anti inhibin serum treatment combined with estrous cycle synchronisation Biology of Reproduction Vol 94 No 1 1 8 Byers S L Wiles M V Dunn S L and Taft R A 2012 Mouse estrous cycle identification tool and images PLoS ONE Vol 7 No 4 e35538 Havenaar R Meijer D B Morton D B RitskesHoitinga J and Zwart P 2001 Biology and husbandry of laboratory animals p 19 25 In Van Zutphen L F M Baumans V Beynen A C editors Principles of Laborator y Animal Science 2nd edition Elsevier Amsterdam Discussion and conclusions In every day practice of a laboratory animal facility the widely used method for induction and synchronisation of oestrous is the use of different combinations of exogenous hormones Although the success rate of this method is high it is considered as an invasive and stressful procedure for the animals Utilising the Whitten effect the aim of our study was to develop a non invasive and refined method for the synchronisation of oestrous cycle Based on our 11

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April 2018 Animal Technology and Welfare PAPER SUMMARY TRANSLATIONS INHALTVERZEICHNIS Spinnen und Chips Nutzung interner RFID Chips als minimal invasive Methode zur Messung der K rperinnentemperatur bei Wirbellosen STUART BAKER 1 EMILY KNIGHT 1 2 SARAH PELLETT 3 KAREN ROBERTS 4 ERWIN SMULDERS4 und STEVEN A TRIM1 1 2 3 4 Venomtech Ltd Discovery Park Sandwich Kent CT13 9ND Gro britannien School of Human and Life Sciences Canterbury Christ Church University Canterbury Kent CT1 1QU Gro britannien Animates Veterinary Clinic Ltd The Green Thurlby Lincolnshire PE10 0EB Gro britannien Plexx BV P O Box 86 6660 AB Elst Niederlande Korrespondenz s trim venomtech co uk Abstract Interne RFID Transponder werden seit vielen Jahren bei Wirbeltieren eingesetzt Studien zu ihrer Verwendung bei Wirbellosen sind in der Literatur jedoch weniger stark vertreten Der Einsatz von RFID Transpondern zur Messung der K rperinnentemperatur stellt eine weniger invasive Alternative zu Thermoelementen und Rektalthermometern dar Interne Transponder wurden in drei wirbellose Arten eingesetzt Brasilianische Riesenvogelspinnen Lasiodora parahybana W stenheuschrecken Schistocerca gregaria und Madagaskar Fauchschaben Gromphadorhina portentosa Das Verhalten der Tiere wurde berwacht Temperaturmessungen wurden mit dem Transponder durchgef hrt und mit der Umgebung der Tiere verglichen R ntgenaufnahmen best tigten die Position und die relative Gr e der Transponderimplantate berlebende Tiere schienen sich w hrend der Studie normal zu verhalten Es wurden geringf gige Unterschiede zwischen Innentemperaturen und Oberfl che sowie Umgebung beobachtet jedoch empfiehlt sich die Durchf hrung weiterer Studien um diese Ergebnisse zu validieren Diese Pilotstudie zeigt dass moderne f r Nagetiere konzipierte Implantate erfolgreich in gro e Arthropoden implantiert werden k nnen was einen erheblichen Einfluss auf die zuk nftige Erforschung der Physiologie von Wirbellosen haben d rfte Schlagw rter RFID Wirbellose K rpertemperatur interner Transponder 13

Paper Summary Translations Synchronisation des struszyklus bei C57BL 6JM usen nach Einf hrung eines h ngenden perforierten Beh lters mit von Ausscheidungen m nnlicher M use getr nkter Einstreu EVANGELOS BALAFAS MARIANNA STASINOPOULOU PAVLOS ALEXAKOS EFTHIMIOS PARONIS KOSTANTINOS PASCHIDIS und NIKOLAOS KOSTOMITSOPOULOS Labortierhaltungseinrichtung Biomedizinische Forschungsstiftung der Akademie von Athen Athen Griechenland Korrespondenz nkostom bioacademy gr Abstract Die Synchronisation des struszyklus ist eine weit verbreitete Methode zur Steuerung der Tr chtigkeit f r Zucht und Forschungszwecke Basierend auf dem Whitten Effekt war es Ziel unserer Studie eine neue verfeinerte Methode zur strus Synchronisation einzuf hren und deren Erfolg zu bewerten Unsere Ergebnisse zeigen klar dass die Verwendung eines h ngenden perforierten Beh lters mit von Ausscheidungen m nnlicher M use getr nkter Einstreu als effektive Methode zur Einleitung des strus bei in IVCs untergebrachten M usen dienen kann Abgesehen davon dass sie nicht invasiv ist stellt unsere Ma nahme eine Verbesserung der Umgebung der Tiere dar da sie ihnen die M glichkeit bietet mit dem Beh lter zu interagieren was wiederum ihre Sozialisation f rdert Schlagw rter Maus strus Synchronisation C57Bl 6J Individually Ventilated Cages einzeln bel ftete K fige IVCs nicht invasiv Verbesserung 14

April 2018 Animal Technology and Welfare CONTENU DE LA REVUE Araign es et puces Utilisation de puces RFID internes comme m thode mini invasive pour mesurer la temp rature corporelle interne des invert br s STUART BAKER 1 EMILY KNIGHT 1 2 SARAH PELLETT 3 KAREN ROBERTS 4 ERWIN SMULDERS4 et STEVEN A TRIM1 1 2 3 4 Venomtech Ltd Discovery Park Sandwich Kent CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury Kent CT1 1QU Animates Veterinary Clinic Ltd The Green Thurlby Lincolnshire PE10 0EB Plexx BV P O Box 86 6660 AB Elst Pays Bas Correspondance s trim venomtech co uk R sum Bien que les transpondeurs RFID internes soient utilis s chez les vert br s depuis de nombreuses ann es les tudes portant sur leur utilisation chez les invert br s sont moins bien repr sent es dans la litt rature L utilisation de transpondeurs RFID pour mesurer la temp rature interne repr sente une alternative moins invasive que les thermocouples et les thermom tres rectaux Des transpondeurs internes ont t ins r s chez trois esp ces d invert br s la mygale saumon e Lasiodora parahybana le criquet p lerin Schistocerca gregaria et les blattes de Madagascar Gromphadorhina portentosa Le comportement animal tait surveill et les mesures de temp rature ont t obtenues gr ce au transpondeur et compar es l environnement de l animal Des radiographies ont confirm l emplacement et la taille relative des transpondeurs implant s Les animaux survivants semblaient se comporter normalement pendant le cours de l tude De petites diff rences de temp ratures internes par rapport la surface et l environnement ont t observ es mais d autres tudes sont recommand es afin de valider ces r sultats Cette tude pilote d montre que les implants modernes con us pour les rongeurs peuvent tre utilis s avec succ s chez les grands arthropodes ce qui devrait avoir l avenir un impact important sur la recherche men e en physiologie des invert br s Mots cl s RFID invert br s temp rature du corps transpondeur interne 15

Paper Summary Translations Synchronisation du cycle stral des souris C57BL 6J apr s introduction d un r cipient perfor accroch imbib par de la liti re contenant des excr tions de souris m les EVANGELOS BALAFAS MARIANNA STASINOPOULOU PAVLOS ALEXAKOS EFTHIMIOS PARONIS KOSTANTINOS PASCHIDIS et NIKOLAOS KOSTOMITSOPOULOS Installation pour les animaux de laboratoire Fondation pour la recherche biom dicale de l Acad mie d Ath nes Ath nes Gr ce Correspondance nkostom bioacademy gr R sum La synchronisation du cycle stral est une m thode couramment utilis e pour g rer les grossesses soit pour l levage soit des fins de recherche Bas e sur l effet Whitten le but de notre tude tait d introduire une nouvelle m thode plus perfectionn e permettant la synchronisation strale et d valuer son efficacit Nos r sultats montrent clairement que l utilisation d un r cipient perfor accroch imbib par de la liti re contenant des excr tions de souris m les peut s av rer une m thode efficace pour l induction du cycle stral chez les souris vivant en IVC Notre intervention en plus d tre non invasive constitue une am lioration de l environnement des animaux parce qu elle permet l animal d interagir avec le r cipient et favorise la socialisation Mots cl s Souris Synchronisation du cycle stral C57BL 6J Cages ventil es individuellement non invasif raffinement 16

April 2018 Animal Technology and Welfare INDICE DE LA REVISTA Ara as y chips el uso de chips RFID internos como m todo m nimamente invasivo para medir la temperatura corporal interna en invertebrados STUART BAKER 1 EMILY KNIGHT 1 2 SARAH PELLETT 3 KAREN ROBERTS 4 ERWIN SMULDERS4 y STEVEN A TRIM1 1 2 3 4 Venomtech Ltd Discovery Park Sandwich Kent CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury Kent CT1 1QU Animates Veterinary Clinic Ltd The Green Thurlby Lincolnshire PE10 0EB Plexx BV P O Box 86 6660 AB Elst Pa ses Bajos Correspondencia s trim venomtech co uk Resumen Los transpondedores RFID internos llevan utiliz ndose en vertebrados muchos a os sin embargo los estudios que eval an su uso en invertebrados est n menos representados en la compilaci n documental El uso de transpondedores RFID para la medici n de la temperatura interna representa una alternativa menos invasiva a los termopares y los term metros rectales Se insertaron transpondedores internos en tres especies de invertebrados ara as rosa salm n Lasiodora parahybana langosta del desierto Schistocerca gregaria y cucarachas siseantes de Madagascar Gromphadorhina portentosa Se sigui el comportamiento animal y se obtuvieron mediciones de temperatura del transpondedor que se compararon con el entorno de los animales Las radiograf as confirmaron la localizaci n y el tama o relativo de los implantes de transpondedores Los animales que sobrevivieron parecieron comportarse de forma normal durante el transcurso del estudio Se observaron peque as diferencias en las temperaturas internas en comparaci n con el entorno y la superficie sin embargo se recomienda llevar a cabo m s estudios para validar estas conclusiones Este estudio piloto demuestra que pueden implantarse satisfactoriamente implantes modernos dise ados para roedores en artr podos grandes y se espera que esto tenga un gran impacto en la investigaci n fisiol gica futura de invertebrados Palabras clave RFID invertebrados temperatura corporal transpondedor interno 17

Paper Summary Translations Sincronizaci n del ciclo reproductor en ratones C57BL 6J despu s de la introducci n de un contenedor perforado colgante con nidos empapados de excreciones de ratones macho EVANGELOS BALAFAS MARIANNA STASINOPOULOU PAVLOS ALEXAKOS EFTHIMIOS PARONIS KOSTANTINOS PASCHIDIS y NIKOLAOS KOSTOMITSOPOULOS Instalaci n para Animales de Laboratorio Fundaci n de Investigaci n Biom dica de la Academia de Atenas Atenas Grecia Correspondencia nkostom bioacademy gr Resumen La sincronizaci n del ciclo reproductor es un m todo utilizado habitualmente para gestionar embarazos ya sea para la cr a o para fines de investigaci n Con base en el efecto Whitten el objetivo de nuestro estudio era introducir un nuevo m todo refinado de sincronizaci n reproductora y evaluar su eficacia Nuestros resultados demuestran claramente que el uso de contenedores perforados colgantes con nidos empapados de excreciones de ratones macho puede utilizarse como un m todo eficaz para la inducci n reproductora en ratones encerrados en jaulas ventiladas individualmente Nuestra intervenci n aparte de no ser invasiva constituye un refinamiento para el entorno de los animales porque ofrece la oportunidad para que los animales puedan interactuar con el contenedor y fomentar la socializaci n Palabras clave rat n sincronizaci n reproductora C57Bl 6J jaulas ventiladas individualmente no invasiva refinamiento 18

April 2018 Animal Technology and Welfare INDICE DELLA REVISTA Ragni e microchip l utilizzo di microchip RFID come metodo minimamente invasivo per misurare la temperatura corporea interna degli invertebrati STUART BAKER 1 EMILY KNIGHT 1 2 SARAH PELLETT 3 KAREN ROBERTS 4 ERWIN SMULDERS4 e STEVEN A TRIM1 1 2 3 4 Venomtech Ltd Discovery Park Sandwich Kent CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury Kent CT1 1QU Animates Veterinary Clinic Ltd The Green Thurlby Lincolnshire PE10 0EB Plexx BV P O Box 86 6660 AB Elst Paesi Bassi Corrispondenza s trim venomtech co uk Riassunto I trasponder RFID sono usati da molti anni nei vertebrati mentre gli studi che ne prendono in considerazione l utilizzo negli invertebrati sono meno argomentati nella letteratura L utilizzo di transponder RFID per la misurazione della temperatura interna rappresenta un alternativa meno invasiva alle termocoppie e ai termometri rettali I transponder interni sono stati inseriti in tre specie di invertebrati la Lasiodora parahybana le locuste del deserto Schistocerca gregaria e le blatte fischianti del Madagascar Gromphadorhina portentosa Tramite il transponder si monitorato il comportamento degli animali e si sono ottenute le misurazioni della temperatura poi confrontate con l ambiente circostante Le radiografie hanno confermato la posizione e le relative dimensioni degli impianti di trasponder Per la durata dello studio il comportamento degli animali sopravvissuti apparso normale Si sono comunque osservate piccole differenze nelle temperature interne rispetto alla superficie e ai dintorni Si consigliano ulteriori studi per convalidare questi risultati Questo studio pilota dimostra che i moderni impianti progettati per i roditori possono essere impiantati con successo nei grandi artropodi e si prevede che ci avr un notevole impatto nelle future ricerche sulla fisiologia degli invertebrati Parole chiave RFID invertebrati temperatura corporea transponder interno 19

Paper Summary Translations Sincronizzazione del ciclo estrale nei topi C57BL 6J in seguito all introduzione di un contenitore perforato sospeso contenente una lettiera imbevuta di escrezioni di topi maschi EVANGELOS BALAFAS MARIANNA STASINOPOULOU PAVLOS ALEXAKOS EFTHIMIOS PARONIS KOSTANTINOS PASCHIDIS e NIKOLAOS KOSTOMITSOPOULOS Struttura per gli Animali da Laboratorio Fondazione di Ricerca Biomedica dell Accademia di Atene Atene Grecia Corrispondenza nkostom bioacademy gr Riassunto La sincronizzazione del ciclo estrale un metodo di comune utilizzo per la gestione delle gravidanze a scopo di allevamento o di ricerca Basandosi sull effetto Whitten il nostro studio aveva lo scopo di introdurre un nuovo e raffinato metodo per la sincronizzazione estrale e di valutarne l efficacia I nostri risultati mostrano chiaramente che l uso di un contenitore perforato sospeso contenente una lettiera imbevuta di escrezioni di topi maschi adottabile come metodo efficace per l induzione dell estro nei topi stabulati in gabbie individuali ventilate IVC Il nostro intervento oltre a essere non invasivo costituisce un affinamento dell ambiente degli animali ai quali d l opportunit di interagire con il contenitore e ne promuove la socializzazione Parole chiave topo sincronizzazione estrale C57Bl 6J gabbie individuali ventilate non invasivo affinamento 20

April 2018 Animal Technology and Welfare ANDREW BLAKE TRIBUTE AWARD WINNING ENTRY Refinement of tumour passage by optimisation of the tissue disaggregation protocol MELANIE GALVIN ALICE LALLO STEWART BROWN PAUL KELLY BECKY BOLA DANIELLE POTTER CASSANDRA HODGKINSON KRISTOPHER FRESE and CAROLINE DIVE Clinical and Experimental Pharmacology Group Cancer Research UK Manchester Institute University of Manchester UK Correspondence melanie galvin manchester ac uk Winner of the 2018 Andrew Blake Tribute Award Abstract Surgical implantation of tumour pieces has been used regularly in the passage of tumour models This involves an invasive surgical procedure carried out under anaesthesia ensuring mice have adequate pain relief and peri operative care The procedure also requires multiple tumour bearing donor mice to generate sufficient tissue for surgical implantation particularly for large scale efficacy studies We sought to refine our tumour passage methods and subsequently reduce the number of mice required for studies by implanting viable dissociated tumour cells instead of tumour fragments Tumours are disaggregated and depleted for red blood cells and any dead residual mouse cells This allowed the enrichment for human tumour cells that were mixed with Matrigel and implanted subcutaneously in the hind flank of mice Cell viability was successfully maintained with this method and mice developed tumours Due to the high number of cells generated using this protocol we are able to implant more mice per tumour thus reducing the number of donor mice required for large scale efficacy studies Moreover subcutaneous injection is a less invasive procedure with a milder severity than surgical implant under general anaesthesia Not only does this process improve animal welfare from a 3Rs perspective it also improves other aspects of the experiment by reducing intertumour heterogeneity thus improving the science Sponsored by The Association of the British Pharmaceutical Industry Keywords Mouse Tumour passage Refinement Reduction 3Rs Animal Welfare Introduction Small cell lung cancer SCLC represents 15 20 of lung cancers with 30 of patients presenting with limited stage LS or localised disease and 70 presenting with extensive stage ES metastatic disease Median survival time with treatment for these patients is 15 20 months for LS and 8 13 months for ES High response rates including complete responses are obtained with platinum based chemotherapy radiotherapy regimens however 5 year survival remains at 5 Drug resistant relapse is common and there is little benefit from second line therapy Since 2007 more than 100 clinical trials have started in SCLC but positive results have been very limited 1 One of the reasons for the slow improvement in SCLC clinical care is that patient derived preclinical models are difficult to generate mainly because of limited access to fresh tumour tissue for research purpose To overcome this problem we recently generated patient derived mouse models from the circulating tumour cells of ES SCLC patients 2 21

Andrew Blake Tribute Award Winning Entry In Hodgkinson et al we described the processing of SCLC patient blood samples to enrich circulating tumour cells CTCs to produce tumours in mice for experimental use 2 These tumours or Circulating Tumour Cell Derived Explant CDX models accurately recapitulate the human disease and mimic the patient response to treatment in clinic CDX models have been successfully passaged and maintained growth characteristics however passaging has involved an invasive surgical procedure implant so from one tumour of 1000mm we are able to get 20 pieces and therefore 20 implants Disaggregation of a tumour of a similar size generates 85 million cells When using disaggregated cells we implant 100 000 cells per mouse allowing us to potentially implant 850 mice from one donor tumour Figure 3 120 Method CDX tumours were grown on the right flank of 8 16 week old female NOD Cg Prkdcscid Il2rg tm1Wjl SzJ NSG mice and removed at 800mm Tumours were enzymatically digested with a Human Dissociation Kit Milteyi Biotech and mechanically dissociated using a GentleMACS dissociator Miltenyi Biotech The dissociated tumours were incubated at 37 C for two periods of 30 minutes strained through a 70 m filter and centrifuged at 250g for 5 minutes The cell pellet was run through a negative selection column using mouse and dead cell beads Miltenyi Biotech The resulting cell pellet was either mixed with 50 50 Matrigel Cornig and implanted subcutaneously on the flank of NSG mice or cryopreserved Figure 1 80 60 40 20 0 Fresh Cells Cryopreserved Cells Fresh Pieces Figure 2 Time to enrolment size 200mm post implantation of fresh frozen cells or surgically implanted tumour fragments 900 Number of Implants Per Tumour To reduce the invasiveness of this procedure we decided to trial a tumour disaggregation protocol This protocol was already being utilised for ex vivo research purposes 3 Days post Implantation 100 Mice are anaesthetised and given a small dose of analgesia before a small incision is made on the flank to enable the implantation of a 3x3 mm tumour piece The mice are then surgically clipped before being allowed to recover 800 700 600 500 400 300 200 100 0 Disaggregated Cells Tumour Pieces Figure 3 Example of total number of implants per tumour Discussion The use of the tumour disaggregation protocol has significantly improved the passaging of CDX models reduced the number of donor and recipient mice required for studies and refined tumour passage Figure 1 Dissociation of CDX tumours for implant or cryopreservation Results Tumours have been successfully generated from both fresh and frozen disaggregated cells and the average growth time to 200mm decreased when using disaggregated cells compared to tumour pieces Figure 2 Furthermore the time to reach 200mm was much less variable when using dissociated cells instead of fragments The number of possible implants per tumour increases considerably when passaging tumour pieces we cut the tumour into 3mm chunks for 22 Tumour disaggregation reduces the number of recipient mice required for a study as tumours derived from disaggregated cells reach enrolment time at a more uniform time implanting tumour fragments requires more mice to ensure a sufficient number of tumours of similar size at the same time It is possible that this is due to the more homogeneous implant of tumour cells tumour fragments are likely to contain different amounts of non viable tumour tissue such as necrotic tissue The protocol also reduces the number of donor mice required for a large scale study as one tumour produces enough cells to implant large numbers of mice

Andrew Blake Tribute Award Winning Entry Subcutaneous implant of tumour cells eliminates the need for invasive surgery carried out under general anaesthesia reducing the severity of the procedure and improving the welfare of the mice Surgical passage requires adequate peri and post operative care of the mice providing pain relief when necessary and close monitoring of the animals to ensure full recovery from the anaesthesia Tumour disaggregation also allows depletion of dead cells and contaminating mouse stromal cells to ensure a more homogeneous tumour cell suspension for implant This subsequently reduces intertumour heterogeneity and improves the quality of the experiment References 1 2 3 4 Koinis F Kotsakis A and Georgoulias V 2016 Small cell lung cancer SCLC no treatment advances in recent years Trans Lung Cancer Res 5 1 39 50 Hodgkinson C L Morrow C J Li Y et al 2014 Tumorigenicity and genetic profiling of circulating tumor cells in small cell lung cancer Nat Med 20 8 897 903 Potter D S Galvin M Brown S et al 2016 Inhibition of PI3K BMX Cell Survival Pathway Sensitizes to BH3 mimetics in SCLC Mol Cancer Ther 15 6 Lallo A Schenk M Frese F et al 2017 Circulating Tumor Cells and CDX Models as a Tool for Preclinical Drug Development Transl Lung Cancer Res 6 4 397 408 Tumour disaggregation and subsequent ex vivo culture could also be used as a potential replacement for in vivo studies 4 Disaggregated CDX cells have been cultured in vitro and accurately recapitulate the genotypic phenotypic and pharmacological aspects of the donor CDX tumour Lallo et al submitted suggesting that ex vivo culturing of CDX cells can be used to screen several compounds and may provide an insight into the mouse CDX sensitivity Therefore we will be able to reduce the number of mice used for pharmacologic studies focussing only on those studies that looked promising in vitro More studies are being carried out and testing of direct culture of CTCs from the patients is under evaluation Taken together these data open a new window in the study of SCLC and will help reduce the number of mice used to investigate this disease Conclusion The tumour disaggregation protocol is the enzymatic digestion of CDX tumours for subcutaneous cell implant This reduces the number of mice required as donor and recipient animals and refines tumour passage by eliminating invasive surgery The removal of both dead cells and mouse cells using the negative selection column results in implantation of tumour cells only improving the success rate of tumour growth and their homogeneity Cell cultures of disaggregated CDX models recapitulate in vivo drug studies and therefore could be used as a possible replacement for in vivo studies in the future Acknowledgements I would like to thank Professor Caroline Dive and Dr Kris Frese for their continued support and the Clinical and Experimental Pharmacology in vivo team for their hard work I would also like to thank Alice Lallo for providing the cell culture data and for her constructive criticism of this manuscript 23

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April 2018 Animal Technology and Welfare TECH 2 TECH The following articles are based on presentations made as part of the Applied Learning and Developmental Skills Module of the Higher Education Programme The unit is specifically designed to develop skills in reflection research and the development of effective plans that enhance the advancement and delivery of learning at both an individual and organisational level It provides useful skills knowledge and behaviours for those wanting to advance their own careers and for those who support the progress of other people It would be useful for existing or prospective NTCOs NACWOs and NIOs Delivered by the College of Laboratory Animal Science and Technology CLAST it is an IAT Qualification http www clast org uk Selective breeding of dogs KERRY LAVIN THOMSON Central Bioresearch Services University of Edinburgh Hugh Robson Building George Square Edinburgh EH8 8XD Correspondence kerry lavin thomson ed ac uk Based on a presentation prepared for the Applied Learning and Developmental Skills Module of the Higher Education Programme Introduction From an early age I have owned and been owned by many dogs Most of these have been what we describe as pedigree dogs including German Shepherds Rottweilers Labradors and Yorkshire Terriers but also a number of cross breeds mongrels Over the years I have noticed that breeds have changed with not only dogs going in and out of fashion but with also many breeds I am familiar with experiencing health problems that 30 years ago did not exist to the extent they do now used terms of mongrel cross breed and even Heinz 57 Along with pedigree dogs these designer breeds are sold to owners at extremely high prices with no consideration of the problems that such uninformed breeding may result in Coming from a laboratory animal background it is extremely worrying that the care and attention we pay to breeding the animals in our care is not applied to the breeding of pet species Some of these problems are due to poor diet and exercise and inexpert care by their owners but many are due to selective breeding and a poor understanding of genetics What is a pedigree There is currently a craze for so called Designer breeds which is a euphemism for the more commonly If the Sire and Dam were both Labradors all the offspring would also be Labradors A pedigree dog is a dog that has been born to parents of the same breed 25

Tech 2 Tech There are no restrictions at what age you can start using a Sire or how many litters he can father The Kennel Club does not keep a record for available studs When picking a Sire and Dam for breeding the only restriction in place regarding inbreeding is no litters born through father daughter mother son or brothersister can be registered although the Kennel Club do allow half brother sister matings to be registered Figure 1 Ellie 13 year old Yellow Labrador Retriever The Kennel Club The Kennel Club is an organisation that breeders use to register litters It was founded in 1881 in England All responsible dog breeders will register litters with the Kennel Club and this can then be used to work out the puppy s family tree If you want to register a litter with the Kennel Club both Sire and Dam need to be registered They have strict rules set in place regarding how often and how many litters a bitch can have that can be registered The Kennel Club has introduced a new free online ser vice called mate select which gives you the predicted inbreeding coefficient percentage before a mating takes place This is being used to help reduce inbreeding It works by selecting the potential Sire and Dam and then you are given a percentage to how likely inherited defects are to be passed on to the offspring The Kennel Club have an annual percentage per breed and they only recommend breeding with potential dogs who get a percentage lower than the annual average consequently the lower the better Of course this is only a measure of risk and does not guarantee that puppies produced will or will not have any health related issues Breed standards The Kennel Club sets standards that all breeders must tr y to achieve when breeding dogs These are important when showing dogs as this is what a judge will be looking for in the ring Breeding restrictions A litter may only be registered if the Dam is over 12 months old at time of mating and under 8 years old at time of whelping parturition Exceptions for dogs over 8 years old are allowed if a form is completed and signed by a Veterinary Surgeon certifying that the animal is in good health Currently the Dam can only have 4 registered litters although previously 6 litters were permitted If a Dam has had two caesarean sections then a third litter born by caesarean would not be allowed to be registered Under the Breeding and Sale of Dogs Welfare Act 1999 a breeder who has more than 5 litters in a 12 month period requires a breeding licence from the local council in order to register any litters after the fourth litter is registered A local authority may also request you apply for a breeding licence if you breed under 5 litters a year if they suspect you are breeding for financial gain This means that ANY person can breed dogs and register them without any requirement that the parent animals undergo the recommended health tests as these are purely advisory 26 These standards have changed through the years which has sometimes been detrimental to the breed Your regular member of the public dog owner will not be bothered if the dog is not 22 inches at the withers or if it has double curl in the tail so why does it matter to the Kennel Club and why has it changed these rules N B The withers is the ridge between the shoulder blades of an animal typically a quadruped In many species it is the tallest point of the body and in horses and dogs it is the standard place to measure the animal s height Breeders have decided that certain breeds look better smaller or with a squashed nose and bulging eyes It is my belief that the organisation who sets these standards are as much to blame as the back street breeder or puppy farmer who does not screen for genetic defects Breeds with well documented genetic defects The German Shepherd It was thought that the German Shepherd would look

Tech 2 Tech better if it had a sloped back and smaller back legs This was justified by the breeders who maintained it would look and stand better in the show ring They are also up to 30lbs heavier than 100 years ago which puts extra strain on the legs What has actually happened is they have developed a higher chance on getting Hip Dysplasia which is where the ball of the hip does not fit in to the socket correctly This is a very painful condition which often results in expensive invasive surgery or euthanasia They also have a higher chance of back problems as the slope can be so exaggerated it almost bends Figure 4 Sloped back German Shepherd The British Bulldog These dogs are known as Brachycephalic which means short broad head and a squashed nose As the years have passed this has become more exaggerated resulting in breathing problems body temperature control regulations because they cannot pant like a Figure 2 Comparison of the structure of a normal dog hip with one exhibiting dysplasia Which is better Figure 3 or Figure 4 Figure 5 Original British Bulldog Figure 3 Straight back German Shepherd OR Figure 6 Modern British Bulldog 27

Tech 2 Tech normal dog They also have bad jaw dental problems making it difficult to eat Other breeds like this are Pugs and Boxers In addition they have been bred to have more folds in the face and head which results in dermatitis and eye infections Because they are a lot smaller now they have a smaller vagina and pelvic opening and due to the pups having bigger broader heads Dams often need caesarean sections to deliver live pups What is a designer breed A Designer breed is the crossing of two different breeds In the past these were known as cross breeds or mongrels The only difference from the past to 2018 is the cost of a puppy and the names they give these Breeds The first cross breed to be given a name was the Labradoodle first bred by a man in Australia called Wally Conrad who wanted to get a low shedding dog to use as an assistance dog Although he achieved this he regrets doing so and feels responsible for creating these Frankenstein breeds Figure 8 Chocolate poodle puppy Examples of designer dogs Pedigree cross Designer name Poodle x Cocker Spaniel Cockapoo Jack Russell x Chihuahua Jackahuahua Yorkshire Terrier x Poodle Yorkiepoo Cocker Spaniel x Springer Spaniel Sprocker Bulldog x Shih Tzu Bullshit What s next Figure 7 Harvey 12 year old German Shepherd x Collie Poodles are considered to be hypoallergenic although this is not always the case and because of this people often re sell their pup once they notice it shedding hair This has encouraged people to cross any other breed of dog with a poodle and for people to charge anything up to 800 for them It is a common fallacy that a Designer breed is healthier than a pedigree animal but this is not the case as pup could inherit genetic faults from either or both of the background breeds they are developed from 28 References Kennel Club Pictures from Caen Elegans Science and Dogs Picture Channel 4 Pedigree Dogs Exposed

April 2018 Animal Technology and Welfare Can technologists benefit more from inter facility working personal experiences from an in vivo study during my role as a visiting worker ASHA KONDE Breast Cancer Now Toby Robins Breast Cancer Research Centre The Institute of Cancer Research 237 Fulham Road London SW3 6BJ Correspondence asha konde icr ac uk Based on an applied learning and development skills for science module presentation as part of a Higher Education programme Background In recent years there has been a rise in research collaborations due to the need to access facilities such as imaging as well as merging of facilities It is therefore likely that opportunities for inter facility working will increase At the end of 2016 the team leader of the Gene Function Laboratory within the Institute of Cancer Research where I work called for a face to face meeting where he suggested that there would be benefits for our team in terms of additional skills if I spent some time with the Breast Cancer Now BCN in vivo team at King s College London Naturally my immediate thoughts were in regard to changes in travel time childcare arrangements and destabilising my work structure Usually I like a challenge and many people who deal with laboratory animals on a daily basis tend to crave for opportunities with challenge but this time I was not sure At this meeting it was also mentioned that I would have as much time as I needed to prepare for this move This had a calming effect and enabled me to develop a positive attitude and start to embrace the challenge Getting ready As with almost everything in our modern life there is documentation to complete but you can contribute to make the process less complicated A lot of documentation will be electronic complete any forms you will be given as soon as possible respond to emails in time and keep your appointments Take advantage of people you may already know in the industry at the secondary establishment They know how the system works and can be very helpful Until a few years ago the process of having your licence at a secondary placement could be a difficult hurdle to overcome nowadays it is now much easier and quicker The NTCO at my primary site and the NTCO at the secondary site supported me with my application for secondary availability for my personal licence and it was achieved very quickly compared to in the past If possible visit the facility and see how things work and ask about timescales for inductions Having spoken with other technologists it will help to remember that nearly all establishments have different systems in place so some part of the process may be slower than you would like but some will be faster than your primary establishment Will you learn Working at a different facility opens the mind up quite quickly if only due to rapidly having to learn to negotiate a new maze of corridors Looking at andragogy adult learning one of the popular theories is Vygosky s theory which emphasises the role of social interactions in cognitive development Let s explore the terms in Vygosky s theory 1 Zone of proximal development ZPD the distance between the actual development level as determined by independent problem solving and the level of potential development as determined through problem solving under guidance or in collaboration with more capable peers It is true that Technologists would 29

Tech 2 Tech rapidly learn an in vivo procedure in other words they are in the ZPD Scaffolding temporary framework that is put up for support and access to meaning 2 More knowledgeable other MKO skilled instructor or colleague at a particular task In this case I was in the ZPD and spending time at a different facility helped me quickly pick the following Trocar implantation surgical implant of tumour pieces as opposed to an incision which is a more refined way of performing the technique Tumour dissociation Cleared fat pad surgery Viably freezing tumour samples Surgical wound closure with wound clips half surgery time compared to sutures Management of animal welfare situations for occurrences that I had prior exposure to and experience of Digital calliper direct entries on to the database which is faster and minimises the amount of materials crossing the barrier A closer look at one of the first skills attained Patient Derived Xenografts PDXs implanted by cleared MFP surgery PDXs are more representative of the real life cancer situation compared to cells grown in vitro For breast cancers the tumour material is typically dissociated and implanted surgically into the mammary fat pad orthotopic model On the applied learning and development skills for science module we have been introduced to Learning theories Tumour disassociation in vitro culture Vygotsky s theory stresses the fundamental role of social interaction in the development of cognition 1 Vygosky s Cognitive theory to make it clearer Zone of proximal development ZPD Many times technicians are not far off learning a procedure Patient Tumour sample Single cells Equipment for Cleared Mammary fat pad MFP surgery for PDX models Scaffolding temporary framework that is put up for support and access to meaning 2 More knowledgeable other MKO skilled instructor or colleague at a particular task Learner L some someinstances instances Learner L surgical Trocar Trocar implantation implantation surgical implant chunks asas implant of of tumour tumour chunks opposed opposedtotoa acut cut Tumour Tumour dissociation dissociation pad surgery surgery Cleared Cleared fat fat pad freezing material Viably Viably freezing material Already successfully atat already tested tested successfully ICR ICRononmy myown own Learninghow howto to learn Learning learn e g e g Webinars diluentandand Webinars diluent drugdrug preparations preparations injections Intravenous Intravenous injections ZPD ZPD MKO MKO Scaffold Scaffold other otherinstances instances Oral gavage training Team staff Oral gavage training Team staff and BSU at Kings and BSU at kings Surgical wound closure with Surgical wound closure with wound clips half time wound clips half time compared to stitches compared to stiches Management of animal welfare Management of animal situations welfare situations Digital caliper direct entries on Digital caliper direct to the database Figure 2 Anaesthesia Precise control of delivery is essential and the Recover y suite essential maintained at 38 5 C 3 entries on to the database Figure 1 The figure above summarises the areas as MKO others as a learner and how colleagues support each other while in ZPD for different aspects The interesting part is that for some techniques you will be the MKO and teach others In this scenario Oral gavage training Team staff and newer BSU technicians who were not previously signed off 30 Figure 3 Surgery Hood Every effort is made to be as aseptic as possible Surgery is a regulated procedure ASPA 1986 4

Tech 2 Tech Discussion and conclusions Many skills in animal technology are practical and acquired at work working at another facility will enable you to to learn a lot of skills from projects at a secondary site Time spent at a different facility does offer a unique oppor tunity not only for an individual but an organisation to give and receive at the same time In this case no training costs were incurred as the staff trained each other Animal Technologists adapt to the repetitive nature of laboratory work but can benefit from opportunities such as staff exchange which provide a change of work environment techniques and networking opportunities If an opportunity presents itself do not be quick to say No consider if you can make it work Acknowledgements Professor Chris Lord Dr Rachel Brough Gene Function Team ICR Dr Rebecca Marlow Erika Francesch Domenech Daniel Larcombe Young Breast Cancer Now In vivo team King s College London References 1 2 3 4 Vygotsky L S 1978 Mind in society The development of higher psychological processes Cambridge MA Harvard University Press Wood D Bruner J and Ross G 1976 The role of tutoring in problem solving Journal of Child Psychology and Child Psychiatry 17 89 100 Wolfensohn S and Lloyd M 2003 Handbook of Laboratory Animal Management and Welfare Third edition Oxford Blackwell Publishing Ltd Pg 112 146 147 Animals Scientific Procedures Act 1986 Pg 2 Retrieved from https www legislation gov uk ukpga 1986 14 17 10 2017 31

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April 2018 Animal Technology and Welfare Identification in Xenopus laevis CHERYL YALDEN King s College London Guy s Campus New Hunt s House London SE1 1UL Correspondence cheryl yalden kcl ac uk Based on Applied Learning and Development Skills for Science module presentation as part of a Higher Education programme Xenopus laevis African clawed frog Xenopus laevis also known as the xenopus African clawed toad African claw toed frog or the platanna is a species of African aquatic frog of the Pipidae family Its name is derived from the three short claws on each hind foot which it uses to tear apart its food The word Xenopus means strange foot and laevis means smooth The species is found throughout much of Sub Saharan Africa Nigeria and Sudan to South Africa and in isolated introduced populations in North America South America and Europe In biomedical research it is used for Egg harvesting and oocyte collection Studying embryonic development Adaptation under environmental stress and ageing Why used Tolerate extensive manipulation Large eggs of approximately 1 2mm compared to approximately 0 09mm of mouse oocyte Large quantities Brood size of 2000 3000 eggs at one time The embryos develop externally which allows for manipulation before or just after fertilisation Rapid growth allows for experimental effect to be determined quickly Production stimulated by injection of hormones e g human chorionic gonadotropin or luteinising hormone Natural mating affects when and how many eggs are produced Hormone injections overcome this meaning continually high quantities of eggs of high quality can be collected all year round Xenopus laevis were used as early human pregnancy tests in the 1940s where a sample of urine was injected into the frog and if the patient was pregnant this would stimulate production and release of eggs from the frog Figure 1 Xenopus laevis showing site of hormone injection Implementation of the 3Rs Constant breeding and egg production exer ts physiological pressure on the frogs In nature the frogs would mate 3 or 4 times a year The RSPCA suggests that it is better to use a larger number of animals less frequently in order to reduce individual levels of suffering incorporating the 3Rs 1 Rest periods of 2 4 months are suggested Long laying lifespan is achieved if animals remain healthy and suitable rest periods are utilised Importance of identification To ensure animals are rested thus applying the 3Rs and improving general health Surgery required for oocyte collection in the UK usually restricted to 2 occasions2 33

Tech 2 Tech minimum of 12 weeks between each event Most humane method to be utilised least pain and distress long lasting reduce secondary health problems quick and easy economic Use of markings for identification purposes Figure 4 Albino Xenopus laevis with no markings making it almost impossible to distinguish Figure 2 Xenopus with very different markings making it easy to distinguish Tagging as a means of identification Pros 1 2 3 4 Non invasive Little handling Inexpensive Pattern of markings remain consistent throughout life 5 Useful for small numbers of animals Figure 3 Xenopus with similar markings making it harder to distinguish Figure 5 Webbing of hind foot where tag is placed Cons 1 2 3 4 5 34 Time consuming with large numbers of animals Skin colour can change Variation in markings Individual interpretation and eyesight Young and albinos show little markings Pros 1 Quick to establish 2 Allows for individual marking of large numbers of animals 3 Once administered no further equipment required

Tech 2 Tech 6 Long lasting 7 Can be re used Figure 6 Tag applicator tool Cons 1 2 3 4 5 Feet web very delicate with many blood vessels Can be removed accidentally by other frogs Hard to read Requires a lot of handling to read Animals require removal from water Figure 9 Microchip scanning and reading device Cons Figure 7 Style of tags used 3mm available in 5 colours and up to 999 numbers from Vet Tech Solutions 3 Microchipping 4 An 8mm x 1 4mm microchip from pet id microchips was used to identify Xenopus laevis frogs 1 2 3 4 5 Invasive Application requires handling Requires scanning and reading device Relatively expensive Too big to be used in smaller species Xenopus tropicalis 6 Frogs require removal from water Pros Summary 1 2 3 4 5 Involved invasive procedures and animals undergo physiological stress Welfare and legal reasons require resting periods and therefore a need for individual identification Most humane method of identification suitable must be utilised Natural markings most humane but low reliability Use of tags allows larger numbers to be identified but reduced longevity Use of microchips most reliable individual identification but stressful and invasive to apply Combination and fur ther development of techniques Permanently encoded numbers Low incidence of secondary infections at wound site ID easy to read Scanning requires little handling Large numbers of animals can be individually identified References 1 2 3 Figure 8 Microchip applicator Reed B T 2005 Guidance on the housing and care of the African clawed frog Xenopus laevis Research Animals Department RSPCA Home Office 2014 Home office guidance on the operation of the animals Scientific Procedures Act 1986 5 19 use and re use of protected animals Crown Copyright VetTech Solutions Ltd Product information small animal ear tags Available from www vet tech co uk laboratorysupplies identification ear tags small coloured animal 35

Tech 2 Tech 4 5 6 7 8 9 36 ear tags small colour ed animal ear tags rat aspx Accessed 17 October 2017 Mrozek M et al 1995 Microchip implant system used for animal identification in laboratory rabbits guinea pigs woodchucks and amphibians Laboratory Animals Vol 29 339 344 Xenbase 2017 Introduction to Xenopus the frog model Available from http xenbase org anatomy intro do Accessed 17 October 2017 Newman S 2017 Embryology Personal communication Sturges G 2017 Identification Personal communication Woodley S 2017 Xenopus use Personal communication Price C 2017 Presentation Style Personal communication

April 2018 Animal Technology and Welfare AS ET CONGRESS BURSARY COMPETITION ESSAYS The Animals in Science Education Trust AS ET is a charity registered under UK Charity law with the objective of advancing education and promoting excellence in the care and welfare of animals used in science The essays included in this issue were amongst those submitted for the third AS ET Congress Bursary Competition to attend the 2018 IAT Congress The intention of the bursary is to encourage junior animal technical staff to extend their knowledge and experience by attending the largest animal technology meeting in the UK Applications were invited from animal technical staff based in the UK or the Republic of Ireland who were awarded the IAT Level 2 Diploma in 2014 2015 2016 or 2017 The successful applicant is selected on the basis of a 1000 word essay on a prescribed subject The 2017 subject was How would you convince members of the general public that the use of animals in medical research was justifiable How would you convince members of the general public that the use of animals in medical research was justifiable JOBIE BROADHURST University of Nottingham Bio Security Unit Medical School Queens Medical Centre Derby Road Nottingham NG7 2UH Correspondence nbzjb nottingham ac uk Winner of the 2017 AS ET Congress Bursary Essay Competition I do not agree with animal testing but I do understand the need I am often asked what I do for a living and always get that feeling of dread like swallowing a lump of coal when I have to explain myself Almost every time I have given my true job title I am met with hostility and anger Members of the public already have their minds made up on the subject of animal testing and to them they are the experts in what actually happens I am automatically the devil incarnate because I enable the suffering and pain of animals So how do I get people to become more accepting of what it is I do I fight the point of the need for animal research not whether it is right or wrong When approaching the question without trying to change people s views on the subject they seem more perceptive to listen and learn They don t 37

AS ET Congress Bursary Competition Essays want the statistics you can find on any animal research website but the truth explained with emotion The most common question I have been asked is Why use animals why not use computers or test tubes The simplest answer I can give is we are just not there yet Some experiments can be run in vitro using test tubes and cell cultures or in silico using data programmes but our technology just isn t advanced enough to give the full picture You have to consider that we are advancing we can replicate some of the functions and pathways of an organ on a biochip This is huge but it cannot tell us how a disease progresses through the body or how a drug can affect brain function or hormone production We need to run these tests on animals in order to see the full picture and learn everything we can about a pathogen so we can create a drug then we need to be sure that it only affects the pathogen and does not damage body function before it can be progressed to human trials This often leads to other questions So why not test on criminals Well criminals may have forfeited their rights when they broke the law but they are still human and it is still ethically and morally wrong However it is not just this We do not know the full history of a human what disease they are genetically prone to what life experiences have shaped their mind or even how their diet has changed their body So this is when we turn to animals that share our DNA but I don t mean great apes It is illegal to use them for research in the UK I mean rodents Mice share 95 of our DNA and their anatomy is pretty similar Fair enough data will never be 100 true to what we can expect to see in humans but it is the closest thing we have and mice have brought research on in leaps and bounds we even use mouse DNA in human medicine Herceptin is a mouse protein that has proven results in reducing breast cancer the study of Tamoxifen in mice has also made great strides in reducing the danger of breast cancer When people see we do actually need animals they begin their assault from a different angle But you are causing undue pain and suffering to the animals where does it stop Trying to explain all the legislation and regulations can give anyone a headache so I will try and keep this simple For an establishment to hold animals for research it must have a licence stating what species can be kept where and for how long If a building or a room does not come under the license then animals for the purpose of research cannot be kept there Then each set of studies has to have a project licence each of these licenses have to state exactly what is being done to the animals when and the worst possible effects the animals may suffer Researchers also have to prove they have done as much as possible to meet the 3Rs Replacement Reduction and Refinement A project license is only 38 granted if there is absolutely no alternative Once a project licence is granted they are monitored thoroughly any deviation from the project details results in severe consequences Even the staff like myself are upheld to specific regulations and codes of practice we have a legal responsibility to ensure the wellbeing of the animals If any animal suffers unnecessary harm and we do nothing to prevent or stop it we could not only lose our jobs but face large fines and even imprisonment In order to stick to these regulations I have a duty to end any pain and or distress that an animal may be suffering beyond that authorised in the license sadly most of the time this means culling the animal So why do you do it do you enjoy it Well the answer is not that black and white do I enjoy animals being used for research definitely not In an ideal world I would be made redundant because there was no longer a need for people to do my job However until this time comes I would rather people like me with a genuine passion and love of animals are the people to provide their care My job brings me a lot of joy as I have the opportunity to raise these animals and provide them with the best quality of life I can no matter the length of life We as Technologists do bond with the animals because of a genuine love for animals it is often unavoidable and does make the job difficult When a study ends or an animal is suffering we must humanely end their life This is partly because these animals would not sur vive in the wild Ever y Technologist is trained to do this as many training sessions as required until they are deemed competent I do not enjoy the thought of having to cull an animal I have seen grow but I do find peace in knowing that if that animal was suffering I could end it swiftly and as painlessly as possible Like you I am human and it does affect me deeply Any day an animal has to be culled is a bad day for me but one day this will no longer be an issue because animals will no longer be needed If you are still not convinced that there is a vital need to use animals in advancing medicine and medical techniques you do not have to take my word Many research institutions have signed a Concordat on Openness meaning you can ask as many questions as you can think of and they will answer as best they can Some facilities may even be willing to arrange a tour so you can see what goes on and what we do Animal research and Technologists have nothing to hide You may think that what we do is not ethically or morally right but for now it is an unfortunate need and maybe if we are lucky enough in our lifetime we may see the end of animal testing and the dawn of in vitro and in silico testing

April 2018 Animal Technology and Welfare How would you convince members of the general public that the use of animals in medical research was justifiable GAVIN SKEELS The Sainsbury Wellcome Centre University College London 25 Howland Street Fitzrovia London W1T 4JG Correspondence g skeels ucl ac uk 2017 AS ET Congress Bursary Essay Competition entry The use of animals for research purposes has been a controversial subject for many years with varying support from the public A recent poll of US adults showed 50 opposition and 47 support for animal research It was also found that those who had a highlevel education or understood general science were more likely to support the use of animals 1 Despite this animals have helped with many medical breakthroughs through the past century from the discover y of insulin in dogs in the 1920s to developments of Ebola virus vaccines using monkeys within the last decade 2 3 The benefit that comes from using animals in medical research needs to be highlighted along with the level of welfare that is expected and achieved in order to educate the general public and allow them to have a greater knowledge base with which to make a decision on support In the UK knowledge on how and what animals are used for in research is sporadic A 2013 poll found that only 37 of people knew that cosmetic testing was illegal with the rest believing it was legal or were not sure 4 Similarly the information available to the public through a general search engine is often biased and from organisations that campaign against animal research misconceptions such as animals living inside barren cages languishing in pain and that many animals die when they are still babies due to breeding 5 6 This means that more information needs to be made available to the public as the Concordat of Openness on Animal Research outlines of which 72 organisations have signed 7 This has already begun with some facilities who have allowed cameras to film virtual tours and videos of the environment daily activities and why the animals are used 8 This is an important step towards providing factual information as a common argument against animal research is the compromise to animal welfare 9 With further planning and organisation however the step can be made to allow the public entry into facilities This can be done in two stages the first would be approaching colleges and universities with propositions for talks and tours students from the ages of 16 to 21 will be currently studying for their future careers and ambitions and by offering the opportunity to seeing with their own eyes what a facility is like they can not only further understand animal research and its necessity but also for those interested in science can see a possible career path and be guided to look into organisations such as the Institute of Animal Technology IAT for more information The next stage would be to invite adults for tours This could be achieved in several ways Firstly the students can be asked to discuss their tour with family members such as parents and older siblings These family members could then be invited for tours where they would also see day to day activities and ask any questions Tours could also be advertised through organisations such as the IAT National Centre for the 3Rs NC3Rs and Understanding Animal Research UAR and through social media As the tours are voluntary those who go would be willing to at least see what a facility is like The groups would need to be kept small around 6 8 people per tour This allows for the tour guide to provide more attention to each person and to answer questions in more detail It also means that health and safety would be more easily adhered to Larger groups would mean a greater risk of someone wandering off or getting lost This could endanger that person but also endanger the animals and staff if they were to go somewhere or do something they should not A small group of less than 10 will make it easier for the guide 39

AS ET Congress Bursary Competition Essays to keep track of each person and count the group at each checkpoint which can be determined by individual facilities 5 Along with showing the public daily activities discussing the medical and scientific breakthroughs that are being achieved in present day that will help animal and human life will also increase understanding of why we use animals in the first place Finally at the beginning of the tour a questionnaire could be given to the guests which will ask what their current knowledge of animal research is as well as their opinions of research in general The tour can then commence discussion time given at the end and the same questionnaire can be given again The questionnaires would be anonymised in order for people to feel comfortable in giving their actual opinion before and after the tour These opinions and any differences between them before and after if any can be used to determine how successful the tours are whether or not they are allowing people to become more knowledgeable and if support of animal research has changed at all If knowledge has increased and if people are more inclined to further think about their stance on animal research then it can be considered successful the industr y has given their true information over and it is the individual person s right to use that information how they wish Great steps have been taken to provide the public with more information and facts about the industry It can now be time to apply further steps and get the public more personally involved it is a good start talking to them and showing them images videos of husbandry and staff routines as the IAT and UAR have done but to allow them to physically see the industry activities and standards will help them to understand how important health and welfare is as well as the specific type of research that is allowed in the UK and prevent the belief that talks or videos have been edited to look better and hide problems They can see for themselves the work Technologists and researchers put into caring for their animals References 1 2 3 4 40 Funk C and Rainie L 2015 Chapter 7 Opinion about the use of animals in research Online Available at http www pewinternet org 2015 07 01 chapter 7opinion about the use of animals in research Accessed 08 08 2017 Scrase R 2014 A breakthrough treatment for Ebola Online Available at http www understanding animalresearch org uk news staff blog a breakthroughtreatment ebola Accessed 08 08 2017 Understanding Animal Research UAR 2014 Insulin for diabetes Online Available at http www understandinganimalresearch org uk why humanhealth insulin for diabetes Accessed 08 08 2017 UAR News Team 2013 Two thirds of the British public are unaware animal cosmetic testing is illegal in the UK 6 7 8 9 Online Available at http www understanding animalresearch org uk news communications media two thirds of the british public are unaware animalcosmetic testing is illegal in uk Accessed 08 08 2017 Cruelty Free International no date Types of animal testing Online Available at https www crueltyfree international org why we do it types animal testing Accessed 08 08 2017 PETA no date Animal Testing 101 Online Available at https www peta org issues animals usedfor experimentation animal testing 101 Accessed 08 08 2017 BBSRC no date 72 organisations sign Concordat on Openness on Animal Reseach in the UK Online Available at http www bbsrc ac uk news policy 2014 140514 n concordat openess animal reseach uk Accessed 08 08 2017 Lab Animal Tour no date 360 Laboratory Animal Tours Online Available at http www labanimaltour org Accessed 08 08 2017 Knight S Vrij A Bard K and Brandon D 2009 Science versus human welfare Understanding attitudes toward animal use Journal of Social Issues 65 3 pp 463 483

April 2018 Animal Technology and Welfare How would you convince members of the general public that the use of animals in medical research was justifiable JUSTYNA BARRATT c o Institute of Animal Technology 5 South Parade Summertown Oxford OX2 7JL Correspondence atweditor iat org uk 2017 AS ET Congress Bursary Essay Competition entry Animals are used in research when there is a need to find out what happens in the whole living body which is far more complex than the sum of its parts It is difficult and in most cases simply not yet possible to replace the use of living animals in research with alternative methods Adding to scientific knowledge through basic biological research helps us understand how living things work and apply that understanding for the benefit of both humans and animals Animals have been used for testing and developing new ways for disease treatments Without animal experiments diseases like heart bypass surgery organ transplants cervical cancer and vaccines for polio would not have been developed Using animals as experiments helps develop different treatments and also helps find safer ways for treatments Animal testing is the only accurate way of learning about the human body without harming the human Although many might argue that scientists are putting the lives of animals at risk by testing on them but is animal life valued more than the human life Thinking emotionally there might be some guilt for harming the lives of animals but thinking about how many human lives have been saved from animal testing makes all animal testing worthwhile Say a loved one has been diagnosed with a once fatal disease it may be because of animal research that medicine can help them live If I had to choose between the life of a dog and the life of my brother child niece nephew or any person I knew I would pick the person It can be demonstrated that animals can be helped by using drugs medicines developed through research For example with the help of a medicine a cow who gets sick some disease can be cured and made to produce 20 litres of milk a day Similarly a lion suffering with a disease can also be cured So the development of such research doesn t only help us it gives life back to animals suffering with any problems as well There are incurable diseases out there for example Alzheimer s multiple sclerosis and schizophrenia that shatter lives If we are to have any hope of treating such conditions medical research needs every tool at its disposal For everyone s sake that must include animals For medical research to progress and advance testing is essential Human life is more sacred than animal life and the effects of human death is felt more keenly and for longer periods of time as opposed to animals However testing should not be done cruelly and every care should be taken to cause the animal as little pain as possible Successful testing may result in the death of many animals however the number of people who will benefit from the medicine is countless I am an animal lover but I also like my own species and appreciate the work being done to improve our lives There are also animal rights acts put in place that set regulations for an animal s cage amounts of food scheduled outings and regular inspections from veterinarians A lot of people against animals in research keep saying animals are important then asking a question like How would you feel if you or your sibling was taken to be tested on This question is illogical and irrelevant because we re not animals Animals might have feelings but in most cases animals who are being tested on are raised for that specific procedure Taking an animal off the street and performing a test to find a cure won t work unless that animal has that disease Many scientists are trained to give that animal the disease through a medical procedure then give the antibiotic in search of curing that disease Even if that sounds inhumane to you the previous question can be shifted to say again How would you feel if you or your sibling developed a disease that didn t have a cure because one hadn t been discovered due to not being able to research and test on animals 41

AS ET Congress Bursary Competition Essays No one chooses to use animals where there is no need It gives no one any pleasure and it is time consuming expensive and quite rightly subject to layers of regulation Yet it is still the best way of finding out what causes disease and of knowing whether new treatments will be safe and effective Medical researchers are not a bunch of scalpel wielding lunatics Those I know are compassionate humane people who carry out their work with great caution and consideration and with ever y effort to minimise suffering We all hope for a day when animal research is no longer needed but until then it is vital 42

April 2018 Animal Technology and Welfare POSTER PRESENTATIONS Originally presented at IAT Congress 2017 The introduction of a cable tie swing and its impact on animal welfare LAURA DEAN 1 JACQUI SWAN 1 NOELIA LOPEZ SALESANSKY 2 SIMON POUCHER1 and LISA DOAR1 1 2 Biological Resource Unit Cancer Research UK Manchester Institute The University of Manchester Wilmslow Road Manchester M20 4BX The Royal Veterinary College Royal College Street London NW1 0TU Correspondence laura dean cruk manchester ac uk Abstract Common enrichment for mice is usually restricted to the floor and easily damaged leading to frequent replacement We wanted to refine the enrichment we provide to overcome these limitations We have been studying the behaviour of NOD CgPrkdcIl2rg Szj NSG mice towards a swing made from plastic cable ties This enhances the 3 dimensional space which is an advantage for animals that naturally like to climb The cable ties are cost effective easy to assemble and autoclavable making them safe for re use A cohort of 16 6 8 week old mice was used including both sexes After acclimatisation the swing was introduced and the mice monitored over 4 weeks using a behavioural ethogram Results showed they interacted with the swing 40 of the time suggesting it is indeed beneficial Some data was collected for 43

Poster Presentations C57Bl 6JOlaHsd Bl 6 mice which showed that this strain did not interact much with the swing indicating strong strain differences Interaction Behaviour observed Description of behaviour Sniffing Standing near to or or under the swing and sniffing it or in its general direction Touching Placing one or more limbs on the swing Hanging on When the mouse is fully suspended on the swing for a period of time With the cable tie Chewing Nibbling or biting at the cable ties Digging Burrowing into or kicking up the bedding Within the cage Methodology A cohort of 16 NOD Cg PrkdcIl2rg Szj NSG mice were used 8 males and 8 females at 6 8 weeks of age housed in same sex pairs One mouse from each same sex pair was shaved around the lower back for identification After a week acclimatisation a cable tie swing was introduced to the cage as a novel object hung in the same position and height throughout all the cages Eating Drinking Interacting with the food hopper or water bottle Grooming Cleaning licking or preening themselves or allogrooming Climbing Hanging upside down on the cage lid by one or more limbs Other Enrichment Engaging with enrichment other than the cable tie swing This includes Sizzlenest Fun Tunnels or Aspen Blocks Figure 1 The specific behaviours monitored for during the Ethogram behavioural observations Prior to monitoring the cages were removed from the IVC rack placed into a LAF Cabinet without the actual use of the cabinet flow or light and allowed to acclimatise to the disturbance for 3 minutes the mice were then monitored for 3 minutes per cage Daily observations were carried out for the first 5 days including twice a day for the first day and then twice a week for the following 3 weeks using a behavioural ethogram See Figure 1 The daily observations were carried out within the same time frame in the afternoon to maintain consistency Figure 2 Proportion of time spent interacting with the cable tie vs other behaviour for the NSG mice The same obser vers were used throughout the investigation to monitor one mouse each per 3 minute period This was alternated per observation to avoid observer bias Using the same methodology we also collected some limited data from a cohort of 20 C57Bl 6JOlaHsd C57 mice 10 males and 10 females at 6 8 weeks of age housed in same sex pairs We revisited the investigation on Day 40 44 Figure 3 Total number of behaviours recorded for the C57 mice throughout the study

Poster Presentations Using tunnel Using Chewstick Using Sizzlenest mice Figures 3 and 4 We can also see that even after 40 days they still use the cable tie very little Figure 5 NSG mice show an increase in hanging behaviour as they become used to the cable tie and they continue to use it over time Figure 6 Climbing Grooming Eating Drinking Digging Chewing Hanging on Day 3 of the NSG data is very interesting because they were cleaned out in the morning which caused a marked reduction in activity during the observations This is shown by the black circle on Figures 4 and 6 Sniffing Touching Figure 4 Total number of behaviours recorded for the NSG mice throughout the study One female NSG mouse was culled between day 25 and day 40 because it was unwell so please note that the day 40 data had one less mouse Further research The amount of climbing behaviour shown by the NSGs is quite high it would be interesting to test if the presence of a swing increases climbing behaviour Chewing Hanging on Sniffing Touching Figure 5 Total number of cable tie specific interactions recorded for the C57 mice throughout the study It was noted by the observers that the C57s showed aversive behaviour and kept to the edge of the cage when they did move around Repositioning the tie to the edge of the cage may encourage greater use so this would also be interesting to investigate Reference 1 Code of Practice for the Housing and Care of Animals Bred Supplied or Used for Scientific Purposes 2014 UK Crown Copyright Chewing Hanging on Sniffing Touching Figure 6 Total number of cable tie specific interactions recorded for the NSG mice throughout the study Results and conclusions For the NSGs there was no significant difference observed between the sexes in the number of cable tie associated interactions p 0 879 C57 data is limited compared to the NSG data however again there does not appear to be a sex difference Overall the NSG mice were found to interact with the cable tie swing for an average of 40 54 of the observed time period Figure 2 The activity of the NSG mice for all behaviours throughout the study is much greater than for the C57 45

Animal Technology and Welfare April 2018 Target training pigs within an isolation unit a pilot study LOUISE CARDER The Pirbright Institute Ash Road Pirbright Woking GU24 0NF Correspondence louise carder pirbright ac uk Concept Within this high containment environment we have started to undertake target training using positive reinforcement in pigs with an aim to improving the welfare of the animals housed in our facility and thus the quality of the research conducted Target training is getting an animal to focus on or produce an action towards a target This can be anything that is big enough for them to see and put their nose on Positive reinforcement is increasing desired behaviours with a reward usually food after they have been performed The reward needs to be a food they are keen on and something they do not get other than for training For this I used grapes Introduction Training is an important tool which can be used to reduce fear and distress which may occur as a result of catching restraining and carrying out out regulated procedures on protected animals In non human primates training vastly increases voluntar y cooperation and is recommended in many guidelines 1 The act of the training itself also functions as a great source of enrichment even before it is completed as well as building up trust with the animal care staff and scientists Figure 1 Target stick used Methods The Pirbright Institute undertakes research aimed at preventing and controlling exotic viral diseases of livestock As such we undertake much of our large animal work in high containment animal isolation units 46 I followed the method below 1 Acclimatise the pigs to the trainer by sitting in the pen and getting them used to you 2 Once the animals are approaching on a regular basis then reward when they come over and stay near

Poster Presentations 3 The pigs can now be introduced to the target If an animal approaches the target and actively touches it with their nose then a reward is given Figure 2 Touch Figure 3 Reward 4 Once the group of pigs are familiar with the target repeat step 3 with individuals until they actively seek a food reward after they touch the target 5 The target can then be moved into desired locations where the trainer needs the animal to go Just remember the target is not a lure and rewards must continue to be given when desired behaviour is achieved 6 After these steps have been achieved the target can be used in combination with items such as the weigh crate or a swab to make these tasks easier to accomplish Rewards should still be given in the same way but they can also be given after the full set of behaviours has been reached Figure 5 Pig guided further into the crate rewarded and then the gate at the back is closed Application This training approach has been successfully implemented in order to make pigs move into a weigh crate The process is shown below Figure 6 Once weighed the pig can be guided out and rewarded once more Once the pigs get used to this they can be rewarded less Future Following the success in this pilot study we will Continue current training programme with a long term pig study to facilitate future procedures Collaborate with other institutes for restraint training of pigs Implement target training in other species such as cattle Acknowledgements Thanks to Linda Dixon for allowing me to train her long term pig experiment and the rest of Animal Services for the encouragement and guidance throughout Reference 1 Figure 4 Targeted pig guided into weigh crate and rewarded once reached the target Prescott M J and Buchanan Smith H M 2003 Training nonhuman primates using positive reinforcement techniques J Appl Anim Welf Sci 2003 6 3 157 61 47

Animal Technology and Welfare April 2018 Improving safety assessment studies by the introduction of a refined ECG vest for dogs PETER FISHER IBPS GlaxoSmithKline Park Road Ware Hertfordshire SG12 0DP Correspondence peter e fisher gsk com Introduction EMKA has become a common non invasive method of capturing the Cardiovascular CV effects from a compound given to laboratory dogs This is achieved by placing the dog in a modified vest and jacket that holds a transmitter attached to Electrocardiogram ECG pads placed on the dog s chest for between 22 24 hours All dogs are acclimatised to vests and jackets used for ECG data capture prior to allocation to study A high number of dogs were able to gain access to ECG leads and chew through them causing a loss of vital data capture Dogs that chewed through the leads on a pretreatment day would need to have a repeat capture requiring the dogs to be re jacketed for up to 22 24 hours The wearing of a jacket for ECG capture is challenging for the dog This coupled with being singly housed can cause additional impact on the dog s welfare Refinements for a new vest to help prevent repeat capture was initiated Refined vest proposal was discussed with the Lomir representative and a prototype was made The new vest was designed with a channel to chase the leads through The channel was designed to keep the leads together and to not be accessible to the dogs This was achieved by running the channel in parallel to the side of the dog and directly to the receiver that is placed in the jacket as seen in Figure 2 There is also a padded area on the inside of the vest to prevent any leads from hanging loose if the ECG pads were to detach from the chest This padded area also makes it more comfortable for the dogs from the cluster of leads as seen in Figure 3 An already existing study dog was selected that was known to have previously chewed through leads whilst having ECG data captured to trial the new prototype vest After a full 22 hrs data capture all the leads were intact and there were no signs of damage to the vest and therefore this encouraged us to invest further in the refined jacket Further vests were commissioned that allowed us to carry out a trial on more dogs with the aim that with continued success the refined vest design would be used on future studies using ECGs Figure 1 Chewed leads from an ECG capture day from original jacket and vest Method The previous vest allowed for the ECG leads to become accessible to the dogs as they were loose within the vest This enabled the dogs to manipulate the leads out of the vest and chew them 48 Figure 2 Shows the added channel that runs along the side of the vest

Poster Presentations Objectives The main objective was to prevent dogs chewing the leads avoiding the need to repeat a full 22 hrs pretreatment data capture By preventing repeat data capture the dogs are only subjected to wearing jackets for the minimal scheduled ECG data capture sessions within the study and averted the need for additional single housing all of which can be detrimental to the welfare of the dog Singly housing can be challenging for dogs along with wearing the jacket which restricts dogs from exhibiting more natural behaviours Any refinements that can be made to minimise the exposure to either of these procedures is a benefit to the dog To enhance the jacket design to simplify the application of the vest and incorporate the leads within the vest itself If the refined vest prevents leads being chewed this will benefit study data capture as any post dose capture is unable to be repeated within our repeat dosing safety studies and therefore it is vital that we are able to capture quality post dose data the objectives set Unfortunately there were a couple of dogs that needed repeats but these were due to ECG data showing possible heart effects such as bundle branch blocker An additional benefit to the redesign of the jacket is its ease of use for the user as it is far easier to manipulate the leads which makes the whole process of placing a dog in a jacket a lot simpler Conclusion Due to the positive results of the new vest minimising ECG Data repeats these have now replaced the old jackets This is a 3Rs refinement by reducing potential stress to the dog The redesign of the vest has minimised the need for repeat ECG recording and averted the need for further single housing All animal studies were ethically reviewed and carried out in accordance with Animals Scientific Procedures Act 1986 and the GSK Policy on the Care Welfare and Treatment of Animals Acknowledgements Figure 3 Shows the padded area where the leads come through the jacket via the channel Chas Bailey IBPS GSK Ware UK Rachael Lincoln MSD Safety Pharmacology Ware UK Results After trialling these vests in excess of 100 dogs and to date we have had no repeat studies due to dogs chewing leads The vest was a success and achieved Figure 4 Side profiles of the new vest with the leads through the new channel 49

Animal Technology and Welfare April 2018 A method for repeated dosing of juvenile mice via oral gavage PETER PACCAGNINI Sequani Ltd Bromyard Road Ledbury Herefordshire HR8 1LH Correspondence peter paccagnini sequani com Abstract Whilst the rat is the most common rodent used in juvenile toxicity studies mice are sometimes a more suitable model Challenges in dosing mice via oral gavage are considerable due to their smaller size and being less co operative than rats The objective of this study was to develop an effective method for repeat dosing mice from Post Natal Day PND 14 of age via oral gavage Animals were dosed either at PND 21 or PND 14 for 5 days using two types of dosing equipment Background Changes in the regulations over the last decade have been designed to encourage development of paediatric medicines resulting in the need to perform juvenile toxicity studies if a potential compound is likely to be administered to children 1 2 The Sprague Dawley rat is the most common species used however in some cases the mouse may be a more relevant species due to metabolic differences or differences in expression of the pharmacological target Dosing juvenile mice can be challenging due to their smaller size and their less compliant nature when compared to rats Whilst there are many reports of juvenile mice being dosed from young ages there were very few reporting successful repeat dosing methods Methodology Study Design Prior to repeat dosing appropriate aged CD1 mouse litters PND 14 and 21 were obtained from Charles River UK The pups in the litter were euthanised by an appropriate Schedule 1 method Immediately post mortem a dosing cannula was inserted into the oesophagus Two types of cannula were tested a steel 6 30 feeding tube with a metal ball on the end external diameter of the ball 0 55mm and a plastic 22G feeding tube external diameter 0 65mm Whilst the procedure was successful at PND 21 with almost all cadavers successfully sham dosed at PND 14 less success was achieved This was thought to be due to the lack of a swallowing reflex and less tensile strength in the younger pups oesophagus postmortem It was decided to continue with the second phase where seven standardised litters of 5 male and 5 female live pups would be dosed with aqueous carboxymethylcellulose at a constant dose volume of 5mL kg once daily via oral gavage from PND 21 25 In addition 2 litters containing 5 male and 5 female animals at the same age PND 21 25 were kept undosed Bodyweight and food consumption were recorded daily on all animals to serve as an indicator of the animals wellbeing Upon the successful completion of phase 2 a further 4 litters were obtained for oral gavage evaluation from PND 14 18 using only the metal 6 30 feeding tube Two litters of the same age animals PND 14 18 were maintained un dosed to act as controls The metal feeding tube was selected due to concerns over the younger pups biting the plastic tubes and increasing the possibility of trauma miss dosing From arrival all animals on this study were fed VRF1 diet manufactured by SDS and mains tap water were freely available All animals were housed in solid floor cages with appropriate environmental enrichment The environment was maintained between 19 23 C and between 40 70 humidity in a 12 hr light dark cycle Figure 1 PND 14 CD1 mouse with 6 30 metal feeding tube 50 All animal work was conducted under authority of a

Poster Presentations Project License in compliance with the Animals Scientific Procedures Act 1986 as amended Upon completion all dosed animals were euthanised via an appropriate Schedule 1 method and subjected to a macroscopic examination The oesophagus was examined microscopically to investigate any potential trauma caused by the dosing procedure Results All animals were dosed by gently introducing the relevant feeding tube gently into the mouth of the animal past the tongue and down the oesophagus Unlike with other types of gavage dosing the feeding tube was not introduced all the way into the animals stomachs instead an estimation was made as to the point where the oesophagus ceased to benefit from the support of the trachea and the tube was introduced this far All animals were successfully dosed from PND 21 25 and retained for 3 days un dosed with no adverse clinical signs and all gained weight steadily see Figure 3 There appeared to be no significant difference in food consumption between the treated and untreated animals In addition there appeared to be little or no difference between the two types metal or plastic of feeding tube used At PND 14 one male animal was euthanised due to poor clinical condition immediately post dose At necropsy however no direct evidence of dosing trauma could be found All other animals were successfully dosed from PND 14 18 and retained un dosed for 3 days with no adverse clinical signs and all gained weight steadily see Figure 4 Food consumption was not measured at this age as the animals were still feeding from the dam The oesophagus of all animals was examined microscopically and whilst a slight thinning of the oesophageal wall was observed in treated animals this appeared to be well tolerated by the animals Figure 4 Mean bodyweight animals dosed PND 14 21 Discussion and conclusion Except for one male animal at PND 14 all animals were successfully dosed at all ages with both plastic where applicable and metal feeding tubes Bodyweight and food consumption data where applicable appeared to be normal when compared to untreated animals As a result of this study further work has been performed where animals were dosed from PND 22 to 42 where no dosing related effects were observed References 1 Figure 2 PND 21 CD1 mouse with 22g plastic feeding tube 2 United States Food and Drug Administration FDA Guidance document Non clinical safety evaluation of paediatric drug products February 2006 European Medicines Agency EMEA Committee for Human Medicinal Products CHMP Guideline on the need for nonclinical testing in juvenile animals of pharmaceuticals for paediatric indications January 2008 Figure 3 Mean bodyweight animals dosed PND 21 25 51

Animal Technology and Welfare April 2018 A barrier breakdown with potential human health implications while not affecting animal welfare SARAH TAMASAUSKAS SOPHIE CARTWRIGHT and STUART STEVENSON Agenda Life Sciences PO Box 24 Hull HU12 8YJ Correspondence tamasauskas_sarah network lilly com Background Citrobacter freundii is a human pathogen that is routinely carried within the human intestine and is not usually a concern to healthy individuals It is not routinely found in rodents and is not routinely screened for While performing our routine health screening results showed that we had a positive test for Citrobacter freundii This positive result was initially thought to have been brought in from a member of the staff but upon further investigations it was seen to be bought in from an outside animal source Measures have been tightly put in place to prevent the spread of the infection to the other animals within the facility These include a quarantine area autoclaving of all cages within the area and the treatment or animals that are due to stay within the unit for longer than 1 week was concern that a breakdown of the barrier had occurred The pathogen is a concern to personnel who may have a weakened immune system and may be more susceptible to infections Due to the research at the facility involving mostly behavioural experiments there was a concern from the scientists regarding the effect of the breakdown on their experimental data Discovery Citrobacter freundii was found during our routine sentinel screening programme Initially we thought that the breakdown had occurred within our own facility During our investigations we checked that the cage wash autoclave and hydrogen peroxide machines were working correctly and at the correct temperature We also ensured that all personnel who entered the facility wore their personnel protective equipment correctly What is Citrobacter freundii Citrobacter is a genus of gram negative coliform bacteria that can be found in the human intestine There are 2 types that we are most concerned about within our facility are C rodentum and C freundii Citrobacter rodentium Affects mice and is routinely screened for by the animal suppliers and by animal units Citrobacter freundii Human pathogen that lives naturally within the gut and is routinely carried by humans We have also found this within our rats Why a concern When the pathogen was first brought to our attention during a routine health screen It was advised by our NVS that quarantine procedures were implemented within the rat area To the manager of the animal facility 52 After several months of treatment and new health screenings showing negative results one of our clean rooms had a positive screen We then began to screen at a batch and cage level using a PCR test that was specific for the organism This helped us establish that the organism was actually very difficult to transmit from cage to cage and that our supplier was the source of the infection

Poster Presentations After all these checks had been performed to ensure there were no equipment or personnel failings our treatment plan was implemented Faeces testing report Jan 17 Sample 10 faeces for C freundii Batch Code BH 17 000037 Lab No 17 334 from the treated animals to determine negative results We also established a protocol that all newly arriving animals from this supplier would be placed into quarantine and treated for 10 days with Baytril As well as our standard sentinel screening we also routinely take faecal samples from the rats to test if C freundii still remains Discussion conclusion This has been a huge learning process to all the facility staff having no previous experience in implementing a quarantine while maintaining the scientific work This is a potentially high hazard organism with a low risk that infection will occur This Citrobacter infection was easy to manage and contain but disruptive to our science Knowing a pathogen is present is great but it is difficult to determine its significance with many var ying opinions from interested parties such as scientists veterinary staff and occupational health staff The supplier is mostly interested in FELASA listed animal pathogens only and not others We feel other organisms may be good early indicators if the barrier and hygiene procedures have not been followed Faeces testing report July 16 Sample 7 faeces for C freundii Batch Code BH 16 000584 Lab No 16 9209 9215 Treatment prevention Citrobacter samples were subjected to an antibiotic sensitivity test at the laborator y These results established that there was a sensitivity to Enrofloxacin After discussions with the NVS a 10 day Baytril treatment was implemented for all the rats to eliminate the Citrobacter Faecal samples were then screened 53

Animal Technology and Welfare April 2018 The importance of high quality health and phenotype information prior to importing new genetically altered GA lines ANNABELLE CRISTI and MELISSA FLORY Charles River UK Ltd Manston Road Margate Kent CT9 4LT Correspondence annabelle cristi crl com Introduction In 2001 the Animal Procedures Committee APC and other organisations including the Joint Working Group on Refinement published reports recommending that information should be collated for all Genetically Altered GA mice regarding their creation breeding husbandry and care Charles River UK Ltd is licensed to provide housing breeding and scientific procedure services for GA mice and rats to various academic and commercial establishments We have created a Genetically Altered Model Information Form GAMIF based on the mouse passpor t recommended by RSPCA GA Passpor t Working Group GAPWG and our Corporate Animal Welfare Group to collect all required information in a standardised format prior to delivery and or creation of new GA strains This poster shows how we use the GAMIF to collect and communicate the information related to phenotype housing husbandry and the colony s health status at origin both internally to Named People and Technicians and externally to our customers Genetically Altered Model Information Form Customer Line Information Form The name of your GAMIF Charles EXAMPLE River PROJECT Project Project Number 17 0236 Your Institution or Company 54 University of Mars

Poster Presentations Planned Date of transfer to CR 23 06 17 Contact Email Dr Aline uni ofmars ma Please propose a Short Name NOD SCID for your line max 8 Chars Your Full Line Name NOD CB17Prkdcscid Nc rCrl Number of Mice to be 2 males 2 sent females Males Fem ales Health Status Animals are Welfare Assessment free of pathogens and opportunist ic organisms Severity Mild historical 18 month data provided Immune Status Severe combined immune deficiency Biosecurity level SOPF SOPF SPF Welfare Issues Thymic lymphomas occur with high frequency life span typically is limited to only 8 5 months under specific pathogen free conditions Breeding HO x HO 55

Poster Presentations Background The SCID Background mutation Endpoint has been transferred onto a nonobese diabetic background Mitigation Laboured Breathing acute acute Laboured breathing body weightloss loss hunched bodyweight hunched posture gaspingmay maybebeseen seen posture gasping with advancedstages stagesofofthymic with advanced lymphoma Those animals thymic lymphoma Those should be monitored closely animals should be monitored and assessed by Veterinarian closely and assessed by Veterinarian Animals should Animals shouldbe beretired retired from breeding and humanely Research Area Cancer research Nature of Prkdc Prkdc Genetic protein protein kinase Modification kinase DNA Expressed DNA activated Transgenic activated catalytic Protein catalytic polypeptide polypeptide Animals Animals homozygous homozygou the sfor for the SCID SCID mutation mutation have have impaired impaired T T and B cell and B cell lymphocyte lymphocyte development developmen The NOD t The NOD background background additionally additionally results in results deficientin deficient natural natural killer NK cell killer NK ce llfunction function Method of Method of Transgenesis Transgenesi s 56 from andofhumanely killed breeding by 7 months age killed by 7 months of age Spontaneo Spontaneous us mutation mutation Likelihood of suffering in transit Not likely under normal conditions Note for Customers Please fill out the form as completely as possible to ensure animals are treated appropriately Thank you Please add any additional information you think is relevant here

Poster Presentations Location For example the rack or isolator number where the animals are kept it should also be visible on the supplied health report following Quarantine 57

Poster Presentations if the customer has requested animals go into one of their existing isolators note the health status of current isolator NVS Date Signature Mice AP 22 Feb 2017 approved to arrive on site Standard HM Schedule Pellets Swabs taken following arrival for PRIA bedding sentinels introduced into isolator and sent for full HM 8 10 weeks later The below is for Charles River Internal Use EXPORTING Likelihood of suffering in transit under normal conditions Not likely Follow Follow standar standard d PPL PPL NVS Comments NVS recomRecommendations recomm Comments Re endatio commendation mendation n and s and pay pay particuparticul lar ar attention attentio to n to Thymic LymphThymic oma Lympho symptma oms sympto and ms and desired desired age of age of retireretireme ment nt 7M 7M 58 If GA animals transferred to scientific establishment outside the United Kingdom A veterinary surgeon confirms that he she is not aware of any reason why these animals might suffer by the fact of being moved to another recognised scientific establishment NVS Date and Signature AP 22 Feb 2017

Poster Presentations Animal welfare One of Charles River s key corporate values states Charles River is committed to the humane care of the research animals we produce and work with in all of our activities Our commitment to animal welfare goes beyond meeting regulatory requirements Instead we seek to implement best practice across business units worldwide It is impor tant to Charles River to meet our commitment of best animal welfare practice when working with GA animals The best way to do this was to produce a quick easy to understand and use form where all the relevant information on the GA line could be collected centrally in a standardised format With each section on the form a question or example is provided to help the researcher understand why and how information is used by Charles River to adjust housing and husbandry care to ensure the highest welfare standards Risk assessment is also performed with regard to transport of customer s GA animals from our site Communication GAMIF forms are readily available to project coordinators Animal Welfare and Ethics Review AWERB Named Animal Care and Welfare Officer NACWO Named Veterinary Surgeon NVS and senior members of staff in the animal rooms Technologists receive relevant information from the GAMIF through our Internet Colony Management system ICM a line the GAMIF and ICM can be updated easily thereby providing the information directly to the care staff and the customer Biosecurity As well as having the knowledge about the welfare of the incoming animals and their phenotype it is important to collect the information regarding their Health Status Understanding housing and husbandr y health monitoring programme recent and historical results from the facility of origin is the essential part of our biosecurity risk assessment Depending on the results of this assessment we adjust the health monitoring paradigm during the quarantine at our facility If the biosecurity risk is deemed to be high animals are sent directly to our rederivation facility Summary The GAMIF form has been in use for nearly two years at Charles River UK Ltd It has greatly improved the way we obtain use and communicate information regarding animals phenotype housing and husbandry and their initial health status It enables us to make rapid decisions regarding in house quarantine vs re derivation and to tailor housing husbandry and quantity and quality of clinical observations to specific strains and or their progeny and feedback this information to the customer When a Technologist scans a RFID tag on an isolator special instructions appears on their screen before they see any assigned tasks in that isolator The information displayed to the Technologist is taken from the GAMIF and entered into ICM by the project coordinator and provides the Technologist with the important welfare phenotype information or specific customer instructions they need for the management of every GA line in the isolator If any unexpected phenotypes clinical signs are seen in 59

Animal Technology and Welfare April 2018 The life of Birdie hand rearing for comparative cognition studies SAM MELVIN Sub department of Animal Behaviour University of Cambridge High Street Madingley Cambridge CB23 8AA Correspondence ms2125 cam ac uk Background and research Avian research continues to be crucial to the development of many areas of biology such as ecology sociobiology neurobiology and ethology Importing Western Scrub Jays Aphelocoma californica has been stopped since July 1st 2007 throughout the EU because of concerns for human and animal health Importing small numbers of wild birds in the EU by zoos and some pet owners will still be allowed This makes it difficult for Cambridge University researchers as the birds need to be quarantined Therefore breeding in captivity is currently the only realistic option for the future of comparative cognition studies in this species Future planning depends on common neuropsychological processes This has been evident in cases of both brain damaged and healthy humans Experimental studies have shown a similar ability in Western Scrub Jays who store and recover food caches in the wild Researchers at Cambridge University are exploring whether members of the corvid family can plan and provide for future needs The Animal Technologist s role was to hand raise and care for a chick every day This involved taking the bird home every night preparing and providing feeds every 15 30 minutes checking faeces and giving physio to its weak legs Wild Scrub Jays Information Most birds have to look after their young from day one as a chick is born naked blind and helpless Both parents bring food to their young until they are ready to leave the nest Monogamous pairs generally feed their young for a while after fledging The male does more of the food gathering and the female does most of the brooding The male also feeds the female while on the nest Each monogamous pair has their own territory and the surviving offspring remain and help with the next breeding season Figure 1 Western Scrub Jay Birdie 60 The average clutch size is 2 to 4 eggs and the incubation time is 14 17 days

Poster Presentations Methods A Californian Scrub Jay was hand reared by Technologists over 3 months The egg was kept in a Brinesea incubator and then raised in a brooder The temperature was lowered slowly in line with feather growth and the bird s ability to regulate its body temperature The diet consisted of blended euthanised new born mice pinkies pinkies with banana and papaya At 3 weeks white mealworms were added Nesting material was provided in the form of surgical swabs then rough plastic scourers for more grip and finally twigs Moved to small cage when pin feathers grew and added perch Moved from small cage to aviary when all juvenile feathers had developed Figure 4 Birdie in the aviary Housed next to Scrub Jay pair in next compartment for visual contact Figure 2 Birdie in initial cage Chart 1 Daily growth rate in grammes Figure 3 Birdie on perch Chart 2 Daily growth rate as percentages 61

Poster Presentations Figure 5 An example of a fertile egg at day 2 Figures 6 and 7 Birdie exhibited splayed legs and foot problems Discussion Birdie had splayed legs and deformed claws which developed in the nest and needed extensive physiotherapy This became a problem when walking and gripping We started off feeding with mealworm guts but this was very time consuming for Technologists Blended pinkies worked better and are high in protein which is important for early development The high protein diet caused constipation so banana was added which improved the form of faeces White mealworms were added to the food for more nourishment Claw stretching exercises were tried but had little effect By day 10 it gained 37 of bodyweight and its food intake increased Eyes opened on day 15 62 Figure 8 An example of a fertile egg at day 9 Improvements for future hand rearing Ideally hatch two or more eggs so that the chicks get moved around in the nest which makes their feet and legs stronger Research hatching times as they can vary with some chicks taking 24 hours and others 10 minutes Locate a large local supplier of pinkies

Poster Presentations Chicks need to be more active in the nest from an earlier age ideally with other chicks and rougher nesting material Start with blended pinkies from a hatchling and then add extras to the diet as the chick grows Introduce the chick juvenile to other birds at a early age so that it gets habituated to them Have more than two people raising the chicks as it is very time consuming for one person Introduce a cage as early as possible to allow the bird to habituate to it Acknowledgements I gratefully acknowledge the assistance of Maggie Dinsdale Sarah Manley and Jenny Bartley Key references Raby C R Alexis D M Dickinson A and Clayton N S 2007 Planning for the future by western scrub jays Nature 445 919 921 Woolfenden G E and Fitzpatrick J W 1996 Florida scrub jay Aphelocoma coerulescens Available https birdsna or g Species Account bna species flsjay introduction Last accessed 10th March 2017 63

Animal Technology and Welfare April 2018 Advances in animal welfare and scientific outcomes of the bile duct cannulated rat model utilised in ADME studies HANS VAN WIJK LEE CROSSMAN GEORGINA ADJIN TETTEY DAWN HAIDA EMILY NEWELL and JOHN KENDRICK Covance Laboratories Ltd Otley Road Harrogate Yorkshire HG3 1PY Correspondence hans vanwijk covance com Introduction Rats are typically the rodent species of choice for investigating the absorption distribution metabolism and excretion of new chemical entities Since rats lack a gall bladder they are an ideal model for investigating continuous biliary excretion and biotransformation in this common toxicological species The design and implementation of a modified tail cuff and cannula system introduced at Covance permits the animal s own bile to be re routed back into the duodenum during the period following surgical cannulation and prior to dose administration This innovation means that animals do not require tethering or single housing during this critical time frame three to five days before initiation of the experimental sample collection It also allows for the continued use of animals with multiple dosing and sampling phases interspaced with respite periods to allow for group housing and complete clearance of the previous test substance administered compared to historic data from animals which were tethered and singly housed completely during the surgical recovery period Methods Rats were anaesthetised using isoflurane in oxygen and were given Carprofen for pain relief along with the antibiotic cefuroxime The bile duct was exposed through a mid line incision and a cannula inserted proximally in the direction of the liver and secured with ligatures The distal end of the cannula was then exteriorised via the tail A second cannula was inserted into the duodenum The two catheters exteriorised from the tail were attached to a port which was protected by a modified tail cuff A U shaped loop was placed in the socket to permit flow of bile from the bile duct to duodenum Following completion of surgery animals were returned to group housing and their health and welfare monitored during the recovery period Figure 1 Study design The sample collection and respite periods were designed to mimic those typically used in a regulatory study biliary investigation using a cross over design including dose administration This was extended to investigate a separate study phase representing a biliary elimination screening design Only animals deemed to be healthy and with a good bile flow were used on each sample collection cycle In all five cycles of sample collections were completed Animal observations bodyweight and excreta output were used as indicators for animal welfare All rats were euthanised 26 days after surgery when the study objectives had been fully met Surgical success rates animal health observations bodyweight losses excretory output and bile flow were 64 Figure 1 Group housed animals that are untethered with the PinPort cannula transferring the animal s own bile between the bile duct and duodenum TM

Poster Presentations Animals received a second dose of analgesia approximately 24 hours after surgery At the start of each phase of the study when bile was to be collected for analysis the U shaped loop was removed from the tail cuff adapter Figure 2 Extension cannulae were fed through a protective flexible coiled stainless steel tube and connected to a stainless steel swivel device and the extensions fitted to the port vacated by the loop The apparatus was designed so that the cannulae for collection and reinfusion could not be switched in error The tube and swivel were designed to allow freedom of movement of the animal whilst retaining the patency of the cannula Artificial bile salts were infused through the duodenal cannula continuously during each study phase Figure 2 The PinPort recirculation phase TM Following surgery animals were group housed for recovery respite periods of up to 96 hours before being connected to exteriorised bile collection infusion lines Animals were then placed singly in metabolism cages so excreta and bile could be collected for periods of up to 96 hours Figure 3 Following removal from the metabolism cage the pin port adapter was placed back into the tail cuff and animals returned to group housing tail cuff in situ during the At the completion of each experimental sample collection phase animals were removed from the metabolism cages and group housed once more Extensions cannulas and swivel devices were removed and a na ve U shaped loop was placed in the tail cuff adapter Figure 3 Tethered singly housed animal in a metabolism cage during the sample collection period Results The surgery success rate using the PinPort apparatus at the completion of the first phase of the investigation TM 65

Poster Presentations 96 hours post dose was 83 which was comparable to historic success rates of 89 In historical studies animals were typically terminated prior to study completion due to either bile flow problems or chewing through the cannulae which prevented collection of subsequent samples In this PinPort model two animals required termination as the cannulae detached from the pin port This issue could be eliminated by the use of an additional suture No instances of chewing cannulae were noted in the pin port model TM Animals involved in the PinPort model were calm and showed no agitation towards the tail cuff post surgery There was evidence of food and water consumption and no indications of inter ference from cage mates observed during post surgery group housing Minor skin scabs on the tail and neck due to localised irritation were observed on two of the animals at completion of the study at 26 days TM Figure 6 Mean bile sample weights g collected from Phases 1 2 and 3 compared to 10 studies using the current bile duct cannulation surgical method Renal faecal and biliar y output were measured Figures 4 5 and 6 respectively Bodyweight changes are presented in Figure 7 Figure 7 Bodyweight growth comparison between nonsurgical animals and animals that had received bile duct cannulation surgery that were either singly housed and tethered or group housed and untethered during the recovery period Figure 4 Mean urine sample weights g collected from Phases 1 2 and 3 compared to 10 studies using the current bile duct cannulation surgical method Dashed lines indicate when animals were housed singly in metabolism cages Non surgical animals Day 0 taken as time when animals were of similar age and weight as surgical animals at the time of surgery Conclusions Following bile duct cannulation of rats using the PinPort apparatus with untethered housing during respite periods the following conclusions have been drawn TM Surgical success in this new surgical model was measured by the number of animals producing acceptable bile flow after a 96 hr recovery period plus a 96 hr sample collection period which was 83 This was comparable to the existing surgical model which has a success rate of 89 Two animals were terminated after completion of the first phase due to issues with internalised bile cannulae and their attachment to the PinPort In future studies this could be prevented by the use of a further suture No examples of chewed cannula the most common reason for early termination in historical studies were noted using this model TM Figure 5 Mean faeces sample weights g collected from Phases 1 2 and 3 compared to 10 studies using the current bile duct cannulation surgical method 0 412 0 745 0 813 0 933 0 695 0 683 66

Poster Presentations Animal clinical observations were typically minor and did not impact upon animal welfare Bodyweight losses 2 were 5 fold lower in PinPort animals compared to animals that were singly housed and tethered during the post surgery recovery period Bodyweight losses 10 in the PinPort model were observed when placing animals in metabolism cages in order to collect bile and excreta Upon removal from the metabolism cages and group housing bodyweight increased rapidly and were linear with growth curves for non surgical animals Maximum weight losses in the historical studies were ca 20 suggesting bodyweight loss is linked to tethering and single housing rather than surgery or analgesia Excretory output and bile flow were acceptable to support investigations into routes and rates of excretion Mean bile flow in pin port animals was elevated in comparison to existing models by as much as 2fold at peak flow ca 22 days after surgery The bile duct cannula remained patent over a 26 day period TM TM Surgical success rate reduction in bodyweight losses comparable animal health observations and acceptable excretory output and bile flow results all suggest that the PinPort model improves animal health and welfare without infringing scientific integrity It also offers the potential to reduce the number of animals undergoing surgery to support metabolism studies On this basis the PinPort model has become the new standard for bile duct cannulation surgery at Covance Harrogate TM TM References 1 2 3 4 Remie R Rensema J W Van Wunnik G H J and Van Dongen J J 1990 Permanent double bile fistula with intact enterohepatic circulation In Manual of microsurgery on the laboratory rat eds R Remie J W Rensema G H J Van Wunnik and J J Van Dongen Elsevier Science Publishers B V Biomedical Division Amsterdam New York Oxford Ch 10 pp 201 213 1990 Van Wijk H Donachie P Mann D L McMahon H and Robb D 2001 A novel bile duct cannulation method with tail cuff exteriorization allowing continuous infusion and enterohepatic recirculation in the unrestrained rat Laboratory Animals 2001 35 325333 Van Wijk H Wirnitzer U and Hartmann E 2016 Nude Female Mice Catheterised with the PinPort in Tail Cuff System Allowing Disconnection of Tether Potential Group Housing in Intermittent Infusion Studies for up to 108 Days The Toxicologist 150 1 493 PS No 3104 2016 Hartmann E Wirnitzer U and van Wijk H Tail CuffInfused Nude Mice Comparison of Continuous versus Intermittent Tethering with the PinPort in Tail Cuff System Including Histopathological Parameters Presented at STP San Diego in print 2016 67

Animal Technology and Welfare April 2018 Are callipers obsolete A novel 3D scanning technology to measure subcutaneous tumour volume ZENA WILSON 2 JUAN DELGADO 1 MICHAEL DAVIES 1 REBECCA WHITELEY 2 JENNIFER HARE 2 AMAR RAHI 2 STEPHEN MARSHALL 2 JARNO RALL 3 ANDREW SMITH 3 STEPHEN ATKINSON 3 ADEALA ZABAIR 3 and JANE KENDREW 1 2 3 Modelling and Simulation DMPK Oncology iMED AstraZeneca 310 Cambridge Science Park Milton Road Cambridge CB4 0FZ Bioscience Oncology iMED AstraZeneca 310 Cambridge Science Park Milton Road Cambridge CB4 0FZ Fuel3D Technologies Inc Unit 2 Douglas Court Seymour Business Park Station Road Chinnor Oxford OX39 4HA Correspondence charlie fuel 3d com Introduction Why do we measure tumour volume To monitor disease progression and response to therapy in Oncology tumour studies in vivo Graph 2 Tumours become more eccentric when growing error is not homogeneously distributed Graph 1 Results show that height is always smaller than width therefore the spheroid shape is almost always overestimating the volume 68 Figure 1 Simulations show that tumours are not spheroids Flatter tumours diverge especially from the canonical spheroid

Poster Presentations Tumour growth is easily measured using tumour callipers to obtain length and width measurement Callipers assume tumours are spheroids with volume of length x width2 How the system works The technology is using a combination of Stereo and Photometric imaging to capture the data 2 Data is captured 1 Rodent and tumour is presented 3 Screen display image quality control by operator G G G G volume length width and height 2D full colour image of tumour 3D full colour image of tumour Animal welfare and 3Rs benefit across oncology in vivo research REDUCE REPLACE REFINE number of animals with other options test to ensure most humane 4 Data processing Image processing method Refinement faster reduction of animal handling tracking tumour volume condition efficient and objective measures for animal welfare morphology independence irregular tumour models will not compromise the studies independence of operator more flexible shifts and less qualified staff 69

Poster Presentations Project plan Proof of concept G 600 tumours G manual segmentation G hardware calibration G prototype design Refinement Prototype finalisation for production Further development and refinement of automatic tumour recognition software complex morphology Full system trials and testing Standard CSV data output to allow easy integration to other systems Resolving issues from Phase 2 Development G 3k tumours G multiple strains sizes cell lines G automation segmentation simple morphology G software calibration G prototype improvement G capture of tumour condition Post launch enhancements Automatic evaluation of tumour condition Smart database that will suggest potential issues with tumour condition Further enhancements to system 70 75 of 1100 automatically recognised tumour length width boundaries are found within 3mm At this stage the algorithms are not fully developed so the performance will improve Results Automatic boundary outline linear measurements Volume estimation Classic calliper measurements spheroid typically overestimate volumes when compared to tumour weight True volume from the scanning system demonstrates greater accuracy and a tighter spread when compared with tumour weight vs callipers especially for small tumours

Poster Presentations Tumour morphology More irregular tumours can be more difficult to detect by the scanner 0 2 can be detected automatically 3 onwards will have to be manually mapped within the software Acknowledgements Thanks to AstraZeneca for the funding of this project Also thanks to AstraZeneca s bioscience senior management for believing in this project and making it possible from a financial point of view as well as all the in vivo scientists at AstraZeneca Nick Moore and Katherine Moyser for their devoted work and help throughout all the experiments Last we acknowledge the employees of Fuel3D for promptly responding to the technical needs of the project Reference Delgado San Martin J A Worthington P and Yates J W T 2014 Non invasive 3D time of flight imaging technique for tumour volume assessment in subcutaneous models Laboratory Animals 2014 0023677214562653 Errors in estimating the tumour weight from scanned dimensions are smaller for regular morphology tumours Symptoms Potential scope for Phase 4 Symptoms such as redness pallor or ulceration can be recalled and fed back to a machine learning algorithm to associate different tumour conditions and their outcomes This is a major refinement in the animal welfare since suffering can be anticipated being able to act about it promptly 71

Animal Technology and Welfare April 2018 BOOK REVIEW Management of Animal Care and Use Programmes in Research Education and Testing SECOND EDITION Edited by Robert H Weichbrod Gail A Heidbrink Thompson and John N Norton Published by CRC Press 2017 SRN 978 1 4987 4844 5 As anyone who knows the three editors would expect this is a superbly crafted volume It is extremely comprehensive divided into nine sections and 36 chapters over 857 pages Sections include Introduction Historical Over view Developing a Collaborative Culture of Caring Compliance Assessment and Assurance Program Management and Stewardship of Resources Physical Plant Environment and Housing Husbandry Animal Health and Care Conclusion Some of the sections hold few surprises to anyone who keeps up to date with developments in facility design planning etc Similarly basic facility management environment and housing and animal husbandry is very much as one would expect although a chapter on water quality and water delivery systems is unusual until one reminds oneself that this book is intended for an international market including countries where municipal water supplies are at best erratic and in some cases non existent In more than sixteen years since the original edition there have been many advances and changes in Biomedical Research This second edition has been expanded to address change and provide a more thorough overview of the current complexities and extensiveness of the area worldwide However many sections chapters introduce novel information previously only found in specialist text books or publications The Introduction and Historical overview of the use of animals both in research and society in general provided in the first section is both useful and interesting especially to anyone who collaborates with internationally based colleagues Starting with the creation of societies for the protection of cruelty to animals for example the Royal Society for the Protection of Animals RSPCA in the UK in 1824 From necessity this is only a summary but for the student of how both the biomedical research the organisation and protection of animals and the human beneficiaries have all developed this is a helpful picture of training legislation etc The chapters are written by individuals with expertise within the biomedical research industry One possible weakness given that it is aimed at an international audience is only five authors are from outside the USA which gives a strong bias to the content Some of the terminology is different from that used in the UK particularly that relating to roles within animal research but it is easily translated so does not pose a problem to those readers more familiar with our own job titles As the title suggests this is very much a book for those involved in animal facility management or those seeking progression to management rather than more junior Animal Technologists 72 The establishment of a Culture of Care has been given increasing prominence in discussions between research establishments legislators and animal welfare organisations in all developed countries and the section of establishing a Culture of Caring reflects

Book Reviews this It discusses organisational responsibilities fostering collaborative notes and responsibilities for members of an Institutional Animal Care and Use Committee IACUC or oversight body Bioethics and animal use in programmes of research teaching and testing behavioural management programmes to promote Laborator y Animal Welfare The section includes very useful statements from different types of institutions and a outline of what a Culture of Care usually includes In addition to these tables outlining CIOMS ICLAS Guiding principles for Biomedical Research involving animals are provided As to be expected in a book for management further chapters cover designing a Culture of Care including the structure of the human animal bond staff empowerment communication training and auditing For those working in international companies with sites around the globe the chapter on harmonisation of international care and use programme should be of interest The adoption of the 3Rs has not been as rapid outside of the UK as it has been with our own community However they are now key components of international regulator y and guidance documents across the globe and there are descriptions of how acceptance of the basic principles has allowed a level of harmonisation of animal care and use practices worldwide within the varying international legal systems In addition to animal welfare human care is also dealt with and the role of the manager is outlined including manager training As well as discussion of the managers role the book also deals with employee composition and the differences in managing contract staff from that of members of permanent staff Recruitment is also covered although obviously differences in employment law between countries exist There is some useful guidance as to ways of encouraging recruiting employees from diverse backgrounds as well as tips on screening questions useful during telephone interviews and the use of modern technology such as Skype Charts showing the different technical and managerial roles are available Occupational safety and health is well covered including a separate chapter on working with experimental hazards Although possibly of less use to those of us working in the developed world useful tables are provided as to appropriate Personal Protective Equipment PPE when working with nonhuman primates as well as lists of zoonotic diseases which would be useful to consider when importing species especially where they have been sourced from non laboratory environments Education and training deals with not only the establishment of training programmes but also with why we need competent training and how we learn and the expectations of a management in the training environment The chapters deals with the importance of training being a topic that the internal oversight board IACUC AWERB etc is involved in The chapter also acknowledges that cultural differences need to be considered not only the fact that English may not be the students first language but also ways of learning may differ Usual reference material include lists of journals related to laboratory animal science and technology as well as contact information for various training groups outside of the major awarding bodies for qualifications associated with the biomedical industry I think a first is achieved in this book in that Fiscal management is addressed From my own experience of facility management other than personnel management financial control was probably the most difficult task to grasp when first achieving managerial status The chapter not only covers planning budgeting control and monitoring it also provides a useful guide to the ever present problem of budget cuts and shortfalls Technology is also touched on as are possible sources of financing Possibly another first in a book of this nature is the chapter on special security acknowledging the risk of both physical security threats from various parties sadly currently not just from animal rights extremists and those of cyber attacks A step by step approach is suggested and includes not only the planning and review stages but table top exercises emergency planning and raising staff awareness Another novel inclusion is the chapter existing and emerging information technology IT Obvious uses of IT are covered such as managing documents ordering animals and other items billing accountancy breeding programmes etc However it is the use of IT for communication and collaboration in the current environment that it is of most interest and includes the use of social media and email Unsurprisingly IT security is dealt with although under the chapter dealing with special security considerations You will appreciate that reviewing such a large volume containing vast amounts of information is difficult and I apologise if I appear to have skimmed over topics There is a wealth of useful tables illustrations and references that the reader will find helpful As I mentioned earlier much of the information reflects the Biomedical Industry within the USA but this does not render the information irrelevant Plenty of information relating to other countries is given and it is useful to be able to compare the situation within our own countries and establishments The editors and authors are to be congratulated on achieving such a high standard of publication and I would recommend that this book becomes a part of everyone s library 73

INDEX TO ADVERTISERS April 2018 AAALAC IAT iv Allentown Inc OBC Avid Plc vi Bell Isolation Systems iii Contec v Datesand IFC IPS Product Supplies Ltd IBC Institute of Animal Technology xiii LBS xiv xv PFI Systems vii Special Diets Services viii Tecniplast UK x Vet Tech Solutions xii