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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 03 Page i CONTENTS Vol 18 No 3 December 2019 Editorial Jas Barley Chair of the Editorial Board ix Increased incidence of spontaneous seizures in laboratory mice in an IVC environment Anna Morgunowicz Chelsea Cavanagh Sofya Nikitochkina and Julie Keeble 159 Non invasive extraction of Cnidarian venom through the use of autotomised tentacles Phillip Robinson Steven Trim and Carol Trim 167 RSPCA workshop Congress 2019 Everyday 3Rs for Animal Technologists Penny Hawkins and Juliet Dukes 174 PAPER SUMMARY TRANSLATIONS 179 TECH 2 TECH Refining handling for small laboratory fishes Chloe Stevens 191 Sperm cryopreservation and in vitro fertilisation in Zebrafish facilities at King s College London Dimitra Mantzorou Thom Berriman Will Havelange Jacqueline Glover Sam Berry and Bruno Correia da Silva POSTER PRESENTATIONS Darwin s fishes keeping Malawi Cichlids Jennifer Bartley 194 199 Stiff as a board measuring rigor mortis in Zebrafish Karen Dunford Jenna Hakkesteef and Carole Wilson 203 Why Zebrafish Rhiannon Davies Green and Carole Wilson 206 You are what you eat Elise Hitchcock Carole Wilson Paul Barwood and Visila Moiche 209 A technologist led approach to altering the culture of care regarding blood sampling Nicholas Kaye 213 Variety is the spice of life enrichment in our wildtype colony CD1 B6CBAF1 C57BL6J Davie Black and Kyle Davies 217 MR and CT compatible electrical heating system for mouse imaging Stuart Gilchrist Veerle Kersemans Philip Allen Ana Gomes Sheena Wallington Paul Kinchesh and Sean Smart 219 Food for thought the development of drug loaded diets improve both science and welfare Alison Ritchie Pamela Collier Phil Clare and Anna Grabowska 222 Instructions to Authors 225 i

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 03 Page ii IAT REPRESENTATIVES OFFICERS President Dr Robin Lovell Badge CBE FRS Immediate Past President Professor Sir Richard Gardner MA PhD FRSB FIAT Hon FRS Vice Presidents Senga Allan MIAT RAnTech David Anderson MRCVS Stephen Barnett BA MSc FIAT Hon CBiol FRSB RAnTech Miles Carroll PhD Brian Cass CBE Paul Flecknell MA Vet MB PhD DLAS DipLECVA MRCVS FIAT Hon Penny Hawkins PhD BSc Wendy Jarrett MA Judy MacArthur Clark CBE BVMS DLAS FRSB DVMS h c DipECLAM FRAgS DipACLAM MRCVS Fiona McEwen BSc BVM S MSc MRCVS Tim Morris BVetMed PhD DipACLAM DipECLAM CBiol FRSB CertLAS MRCVS Jos Orellana BVSc MSc Clive Page OBE PhD BSc Jan Bas Prins PhD MSc Vicky Robinson CBE BSc PhD Paul Sanders MIAT RAnTech David Spillane FIAT Gail Thompson RLATG Robert Weichbrod PhD RLATG Life Members 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 Honorary Members Mark Gardiner MIAT RAnTech Andy Jackson MIAT Sarah Lane MSc FIAT Brian Lowe MSc FIAT RAnTech Sue McHugh BSc FIAT Norman Mortell BA Hons MIAT RAnTech Terry Priest MBE FIAT RAnTech Trevor Richards BEM MIAT David Spillane FIAT Wendy Steel BSc Hons FIAT Pete Willan DMS FInstLM MIAT Members of Council Ken Applebee Matthew Bilton Kally Booth Charlie Chambers Steven Cubitt Simon Cumming Haley Daniels Glyn Fisher Nicky Gent Alan Graham Nathan Hill Linda Horan Sam Jameson Elaine Kirkum Adele Kitching Theresa Langford Sylvie Mehigan Steve Owen Alan Palmer Allan Thornhill John Waters Lynda Westall Carole Wilson Adrian Woodhouse Council Officers Chair Linda Horan BSc Hons MIAT RAnTech Vice Chair Glyn Fisher FIAT RAnTech Honorary Secretary Simon Cumming BSc FIAT RAnTech Treasurer Glyn Fisher FIAT RAnTech Assistant Treasurer Charlie Chambers MIAT RAnTech Chair of Board of Educational Policy Steven Cubitt MSc FIAT RAnTech Chair of Board of Moderators Haley Daniels MBA MSc MIAT RAnTech CIPD Chair Registration Accreditation Board Ken Applebee OBE FIAT CBiol FRSB RAnTech ATW Editor Jas Barley MSc FIAT RAnTech Bulletin Editor Carole Wilson BSc MIAT ATW Bulletin Editorial Board Jas Barley Chair Matthew Bilton Nicky Gent Patrick Hayes Elaine Kirkum Carole Wilson Lynda Westall Branch Liaison Officer 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 Matthew Bilton Kally Booth Lois Byrom Simon Cumming Nicky Gent Sylvie Mehigan John Waters Board of Educational Policy Steven Cubitt Chair Steven Cubitt Secretary Adele Kitching Board of Moderators Haley Daniels Chair Simon Cumming Cathy Godfrey Moderators Anthony Iglesias Theresa Langford Jenny Parks Sarah Reed Communications Group Adrian Woodhouse Chair Nathan Hill Elaine Kirkum Teresa Langford Sylvie Mehigan Allan Thornhill Lynda Westall CPD Officer Charlie Chambers Registration and Accreditation Board Ken Applebee Chair Glyn Fisher Secretary Charlie Chambers John Gregory Cathy Godfrey Kathy Ryder Home Office Stuart Stevenson Observer Ngaire Dennison LAVA Congress Committee Alan Graham Chair Haley Daniels Linda Horan Adele Kitching Allan Thornhill John Waters Diversity Officer Haley Daniels MBA MSc MIAT RAnTech CIPD UK Biosciences ASG Representative Home Office Steve Owen Charlie Chambers Alan Palmer IAT OFFICERS MAY BE CONTACTED VIA IAT Administrator admin iat org uk OR VIA THE IAT WEBSITE AT www iat org uk OR THE REGISTERED OFFICE 5 South Parade Summertown Oxford OX2 7JL Advertisement Managers PRC Associates Ltd Email mail prcassoc co uk Although every effort is made to ensure that no inaccurate or misleading data opinion or statement appear in the journal the Institute of Animal Technology wish to expound that the data and opinions appearing in the articles poster presentations and advertisements in ATW are the responsibility of the contributor and advertiser concerned Accordingly the IAT Editor and their agents accept no liability whatsoever for the consequences of any such inaccurate or misleading data opinion statement or advertisement being published Furthermore the opinions expressed in the journal do not necessarily reflect those of the Editor or the Institute of Animal Technology 2019 Institute of Animal Technology All rights reserved No part of this publication may be reproduced without permission from the publisher BRANCH SECRETARIES 2019 Cambridge Edinburgh Hertfordshire Essex Huntingdon Suffolk Norfolk Ireland London Midlands North East England North West Oxford Surrey Hampshire Sussex West Middlesex Wales West West of Scotland ii Sarah Shorne Janice Young Joanna Cruden Jo Martin Lisa Watson Rebecca Towns Ian Fielding Rachel Sandy and Joanne Bland Nicky Windows April Shipton Francesca Whitmore Josefine Woodley Rhys Perry Linda Horan cambridgebranch iat org uk edinburghbranch iat org uk hertsessexbranch iat org uk hssbranch iat org uk irelandbranch iat org uk londonbranch iat org uk midlandsbranch iat org uk northeastbranch iat org uk cheshirebranch iat org uk oxfordbranch iat org uk shsbranch iat org uk westmiddxbranch iat org uk waleswestbranch iat org uk westscotlandbranch iat org uk

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page ix December 2019 Animal Technology and Welfare THE INSTITUTE OF ANIMAL TECHNOLOGY ETHICAL STATEMENT In the conduct of their Professional duties Animal Technologists have a moral and legal obligation at all times to promote and safeguard the welfare of animals in their care recognising that good laboratory animal welfare is an essential component of good laboratory animal technology and science The Institute recognises and supports the application of the principles of the 3Rs Replacement Reduction Refinement in all areas of animal research Editorial Jas Barley Chair of the Editorial Board This is the last hard copy of Animal Technology and Welfare ATW that you will receive No you have not been struck off the membership or anything similarly drastic it is due to the Institute taking the decision to no longer print the Journal but to go fully electronic Not only this but also to move to Open Access Publishing to meet the requirements of Plan S to which most of the major funding agencies are signatories ATW will have its own website www atwjournal com and both current and back copies will be available to read and download as desired Please ensure that the Institute has your email address so that we can notify you when a new issue is published The Institute has arrived at this decision due to many factors on top of Plan S Primarily an open access journal will enable us to reach a far larger audience which given the welfare developments our authors have contributed to must be a good thing Obviously an electronic issue is much greener saving in addition to the impact on the environment of the paper used will remove all those annoying plastic envelopes which obviously benefits the environment Although the Institute has been happy to bear the loss the Journal has made in the past seeing it a service to members postage in particular is steadily increasing and the loss of income from subscriptions due to the move to Open Access will be offset the savings made will enable the Institute to divert income to other projects which will in turn benefit you the members One important thing is that ATW will continue not to charge authors to publish their work an important factor for encouraging the publication of work that may not be published elsewhere However the Institute is adamant that ATW will remain the voice of Animal Technologists giving our members and others the opportunity to publish suitable articles both formal peer reviewed papers posters and technical notes Tech 2 Tech articles in addition to any other material the Editorial Board feels will benefit members Formal papers many of which arise from each year s IAT Congress will continue to be eligible for the Marjorie Whittingham Sandiford Journal Article prize and the Animals in Science Education Trust AS ET will hopefully continue to award a prize for the best Tech 2 Tech article published each year Although I will miss the satisfaction of a hard copy of Animal Technology and Welfare landing on my doormat and seeing that the considerable preparation of each issue has produced an attractive issue I am excited about the changes and feel that these will provide exciting opportunities for authors both current and future and of course the IAT 2020 is the 70th anniversary of the formation of what became the Institute of Animal Technology IAT Animal Technology and Welfare will reflect the developments that the Institute and our members have achieved over the years Taking the theme of Making a Difference we will be including some material from the past alongside the latest developments in animal technology and the welfare of the animals in our care This is an ideal opportunity for authors to publish their latest work if you would like to see your work published please do not hesitate to contact me The Editorial Board are happy to work with new authors to produce an article suitable for publication This last hard copy issue contains three formal papers of which the RSPCA workshop paper by Penny Hawkins and Juliet Dukes and the Non Invasive Extraction of Cuidarian Venom by Phillip Robinson both arise from Congress 2019 presentations The third formal paper by Anna Morgunowicz and the team at University College London is based on a platform presentation from the West Middlesex Technician and Trade Day in February 2019 The Editorial Board will continue to strive to include papers and articles based on presentations given at various IAT meetings so if you cannot attend a meeting you will not necessarily miss out on learning something new although that does rely on the co operation of presenters and your willingness to learn Several of the other articles and posters take an aquatic theme with contributions from the RSPCA and staff at the University of Cambridge University College London and King s College London Topics covered include the refinement of fish handling Zebrafish Sperm cryopreservation and In Vitro Fertilisation Malawian Cichlids and three Congress posters on Zebrafish I would like to take this opportunity to thank the staff at Warwick Printing Company for all their help as they not only typeset and print ATW as it is now they have also acted as mailing agents for enabling the Journal to be delivered to members Jon Clucas in particular who has to deal with my continual missing of deadlines We will continue our association with Warwick and I hope to improve my timekeeping E atweditor iat org uk ix

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 December 2019 14 04 Page 159 Animal Technology and Welfare Increased incidence of spontaneous seizures in laboratory mice in an IVC environment ANNA MORGUNOWICZ CHELSEA CAVANAGH SOFYA NIKITOCHKINA and JULIE KEEBLE Biological Services King s College London Maurice Wohl Clinical Neuroscience Institute 5 Cutcombe Road Denmark Hill London SE5 9RX Correspondence anna morgunowicz kcl ac uk Winner of the IAT Congress First Time Presenter award sponsored by Marshall Bioresources Abstract Over the past decade there has been an enormous shift away from the use of open top rodent cages to the use of individually ventilated cages IVCs This has brought with it many benefits including better control of health status and minimisation of contamination of immunocompromised animals At King s College London KCL the transfer of mice from open top cages to IVCs has coincided with an apparent increased incidence of spontaneous seizures in mice The majority of seizures were observed when the cage was opened in a change station and it has therefore been proposed that a significant change in environmental stimuli may contribute to the incidence of seizures This study aimed to record the incidence of seizures in mice following their transfer to IVCs and investigate the environmental differences between the inside and outside of IVCs in particular noise levels This study highlights the increase in spontaneous seizure incidence in mice in IVCs and the need to investigate the impact of environment on seizures amongst laboratory animals in order to ensure the highest level of animal welfare maintained Key words mice seizures IVC Introduction A seizure is a sudden uncontrolled electrical disturbance in the brain which can be spontaneous or disease related Unexpected changes to electrical activity in the brain can induce jerks shaking and unconsciousness due to messaging conductivity being disturbed resulting in an epileptic seizure There are different types of seizure for example audiogenic which is caused by sound photogenic caused by light olfactory seizure caused by smell and many others 1 2 First recorded seizures in laborator y mice were reported in the 1930s 1940s Audiogenic seizures were noted in rodents by Mirsky in 1943 3 Furthermore around that time there were first reports of seizuresusceptible and seizure resistant strains Researchers also identified that not only sound but also frequency modulation rhythm and vibration might cause seizures in mice Even at this early time C57BL and DBA strains purchased from Jackson Laboratory and also some albino mice were being tested due to visible seizures 4 In laboratory mice epilepsy can be experimentally induced for the purpose of scientific interest in order to provide cures for human diseases It should be emphasised that in this study neither seizures nor epilepsy were induced and none of the mouse strains have a connection with epilepsy or seizure related experimentation This study is looking at increased number of spontaneous seizures in random mouse strains At the end of 2016 most of our mouse colonies were housed within the Biological Services Unit in the James Black Centre building in South London in either individually ventilated cages IVC systems or traditional open top cages At that time seizures were observed as very rare reoccurrences of single isolated cases typically 4 6 months apart particularly in two animal holding rooms In September 2017 all our animals were moved temporarily to a new building next door the Maurice Wohl Neuroscience building while the James Black Centre building underwent major refurbishment and every mouse was then housed in IVC systems The Maurice Wohl Neuroscience building s Biological Services Unit Wohl BSU is situated in the basement of the building with its cagewash which has a clean and a dirty side one surgery room two procedure rooms 159

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 160 Increased incidence of spontaneous seizures in laboratory mice in an IVC environment and fifteen animal holding rooms which house primarily breeding and stock mice Breeding is not performed in four of the animal rooms as one holds rats in open top cages immunocompromised mice the third room acts as a quarantine room and holds all delivered mice from external suppliers or animals moved from other King s College campuses and the fourth room holds animals recovering after surgical procedures After relocation to the Wohl BSU with all the mice now being housed in IVCs the number of seizures observed in mice increased In November 2017 epileptic fits were mainly observed in mice in two of the animal holding rooms which corresponded to the animal holding rooms with obser ved seizures before relocation Soon after spontaneous seizures were noticed across the remainder of the Wohl BSU without discriminating between mouse strains Seizures were recorded from January 2018 until the present time The peak in recorded spontaneous seizure animals was in April 2018 when the present study began To investigate these seizures in more detail seizure report sheets were created and employed in every animal room Additional investigations included monitoring genetic predisposition for the attacks and measuring noise levels in the animal rooms In summary this study was carried out to address concerns in relation to spontaneous seizures in laboratory mice after transfer from traditional open top cages to IVCs in order to improve animal welfare by identifying the causes of spontaneous seizures in mice and reducing incidence of these attacks which can cause brain disturbances changes in behaviour and movement and alteration of consciousness A further aim was to reduce the number of animals used for experimental procedures by eliminating false results or the need to increase sample size due to seizure activities This study emphasises the importance of training for Animal Technologists and others in noticing and communicating spontaneous seizure activity in mice early which is crucial for animal wellbeing and the successful completion of the experiment Materials and methods Animal husbandry In the Wohl BSU all mice are kept in individually ventilated cages which are GM500 Mouse IVC Green SelfSeal Line supply by Tecniplast Each room holds four racks of 60 IVC cages connected to either easy or smart flow air handling units set up at 25 air exhaust and 75 air supply The individual cages are serviced within three different type of change station units CS5 Evo Plus CS5 Evo GP ARIA CS48 This study was performed primarily on animals under breeding and maintenance Project Licence PPL protocols held under the Animals Scientific Procedures 160 Act 1986 ASPA and approximately 10 of animals under experimental procedures data not published licences also held under ASPA Over 90 of the mice included in this study were on a C57BL background of which 45 were C57BL 6 Charles River and 45 were C57BL 6J Envigo with 10 of all studied mice were of unknown genetic background There was no control group as each animal was studied on an individual basis All age stages were taken into consideration and ranged from 3 weeks old to approximately 24 months of age including ageing studies Animals were only single housed for the welfare reasons Primarily the study included different numbers of mice in one cage with 5 animals being the maximum Mice had ad libitum access to LabDiet 5053 and reversed osmosis RO water throughout the study Each cage contained Lignocel select bedding aspen wood product and environmental enrichment consisting of a mouse fun tunnel and a mouse chew stick with a disc of Bed r nest for nesting along with cocoons as soft addition to supplement the nesting as required Recording of seizure and coat state The study commenced in April 2018 due to a major concern about animal welfare related to spontaneous seizures Mice seizure report sheets were prepared discussed with BSU staff and placed in each mouse holding room Each member of staff was educated on how to identify and record seizures Animal Technologists responsible for their rooms recorded spontaneous seizures on a weekly basis as the attacks generally coincided with cages cleaning Cage cleaning in BSU Wohl is performed every week or every second week depending on the number of animals in the cage The IVC cage is taken into a working changing station where the cage lid is opened and animals are transferred into an autoclaved cage base with sawdust then sterilised environmental enrichment is added into the cage or for example when weaning animals are transferred into an autoclaved prepared complete cage with environmental enrichment already in place Normally mouse cage cleaning takes less than one minute However in case of any suspected spontaneous seizure an Animal Technologist paused their work to observe the attack and allow full recovery from it In the case of a severe seizure the fit could last up to 1 minute During the study only one animal died during a seizure the remaining mice fully recovered from their seizures The incidence of seizures was recorded over an eight month period from April when the seizures peaked to November 2018 Recording included severity of seizure number of incidences age of mice sex genotype coat state and cage location on IVC rack Severity of seizures was differentiated as Mild where stiffness and short jerks were observed Moderate having multiple jerks and convulsions

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Increased incidence of spontaneous seizures in laboratory mice in an IVC environment Severe with violent convulsion that could cause an animal to dribble or lose consciousness for a time The coat dribble was added in order to compare the condition of animal The sum of the different body parts score head neck back and tail determined the coat state Smooth and shiny hair indicated a good coat condition and gave score equal 0 0 0 0 0 0 and characterised the animal as well and in good health as expected Slightly fluffy coat with some spiky patches would be moderate coat state with score 0 5 Not groomed fluffy jutting greasy hair would be rated 1 point as bad coat for example head 1 neck 1 back 1 tail 1 giving the highest possible score of 4 and signifying an unwell animal Coat scoring was harmonised across all Animal Technologists with scores and their descriptions and recorded on mice seizure report sheets Additional images were also attached showing a mouse with good coat score 0 and other one with the highest coat score 4 is exemplified in Figure 1 Figure 1 Coat score to seizure severity Axis Y Number of mice Axis X Coat state score Figure 2 Age of mice with spontaneous seizure Noise Image 1 Photograph of mice showing differences in coat condition Current Protocols in Pharmacology 5 65 1 5 65 17 June 2013 Published online June 2013 in Wiley Online Library wileyonlinelibrary com DOI 10 1002 0471141755 ph0565s61 Copyright 2013 John Wiley Sons Inc An example of coat scoring taking inspiration from above photos Body Part Mouse A Mouse B Head 0 1 Neck 0 1 Back 0 1 Abdomen 0 1 Score 0 4 Aware that noise is a big contributing factor for seizures in rodents our study measured noise within the human hearing range 20 Hz 20 kHz and mouse hearing range 1 100 kHz in IVCs Noise levels were also measured with the change station turned on representing the noise difference that animals experience when welfare checks cleaning are being carried out Noise in the presence of vacuuming was also determined Noise was measure in A weighting dBA which is standard measurement and covers frequency range from 20Hz up to 20kHz characteristic for human hearing and also in Z weighting dBZ which is flat frequency between 10Hz and 20kHz 1 5dB and more closely resembles a mouse s hearing While it does not represent mouse hearing per se it lacks the adjustment to human hearing that is present in A weighting The noise was measured in several animal holding rooms that had one of three types of cage changing station A smart phone application Decibel X pro was used following advice from our Named Veterinary Surgeon NVS The measurements were performed 161

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 162 Increased incidence of spontaneous seizures in laboratory mice in an IVC environment outside and inside an IVC cage 1 in the absence of any activity no other people in the room changing station switched off 2 when the changing station was switched on 3 when hoovering the floor and 2 and 3 combined Measurement of noise was also performed inside the changing station type ARIA CS48 The measurements were carried out inside the complete IVC cage covered with the lid and without the lid with changing station on or off trying to replicate the conditions during cage changing The IVC cage used for noise measurement had both clips holding the lid in place removed to avoid the clicking noise interfering with measurement when the lid was being closed and the same cage was used throughout the noise measurement The cage was positioned on the IVC rack closest to the door and directly opposite to the changing station in column 4 and row C Lignocel bedding one fun tunnel one chew stick and one disc of Bed r nest were present in the cage No food or water was added to the IVC cage during the noise measurement All sound recording was performed for 3 min Female Male Figure 3 Percentage comparison of mice sex having a spontaneous seizure during the study The majority of reported seizures during the study were black mice although we do not have comparable numbers of white light colour mice within our unit there is around one hundred white NSG mice stock and around fifty white BALB c mice as sentinels within the unit Breeding Breeding in our unit is run independently by several research groups All groups manage their own breeding colonies according to their experimental requirements To support the breeding performance a 12 hours light dark cycle is maintained within the unit The majority of animals identified as having seizures during the study were kept on breeding and maintenance protocols Following the advice from our NVS any mice experiencing a seizure were not used for future breeding stock As a consideration of animal welfare mice which were notified with three or more seizure attacks were humanely killed Statistical analysis A Kruskal Wallis test was carried out to determine the relationship between seizure severity and coat state score P

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 163 Increased incidence of spontaneous seizures in laboratory mice in an IVC environment trigger a seizure attack Every spontaneous seizure was recorded for the study consequently a range of different mice strains were observed without any preselection Some animals had only one or two notified attacks and lived for many months Certain animals that showed one or two seizures were already under an experiment and lived to the end of the study The highest coat score in our study was 4 the lowest 0 with the score of 0 5 being most common Some mice with coat score 0 were observed with severe seizure and some with score 4 were noticed with only moderate seizure There were also animals notified with mild seizure when their coat score was 1 or 1 5 However a Kruskal Wallis statistical analysis showed that overall there was a significant relationship between coat condition score and seizure severity p 0 027 However the coat score was recorded after the seizure was observed so there is a possibility that the attack could have contributed to the animal coat state as opposed to mice in poor health having seizures Figure 1 generation when at least one parent was affected could indicate that spontaneous seizure is in their genetic background Figure 4 This is also consistent with a rapid decrease in the number of seizures observed from September 2018 after eliminating all affected animals from future breeding stock However we still observe single cases of spontaneous seizure in mice in colonies where breeding could not be stopped on time for various reasons data not published This also suggests genetic implication to spontaneous seizure when dealing with subsequent generations Reduction of seizures by excluding animals with those exhibiting attacks from future breeding stock is one of the factors that instantly decreased spontaneous seizures in mice within our unit Noise The base line of noise measurement was within the complete IVC cage with lid on The noise increased about 3 during measurement performed inside the cage with 1 a cage changing station switch on or 2 hoovering in the room one activity at the time During both activities 1 and 2 simultaneously noise was greater at approximately 6 When measurement was carried out outside the IVC cage noise elevated about 12 with just the cage change station on and approximately 3 purely by hoovering Both activities 1 and 2 simultaneously increased the noise in animal room roughly by 12 when measurement was performed outside of the cage Noise measurement inside the cage increased by 15 when the IVC cage was placed onto the working cage changing station and about 20 when the lid was removed from the IVC cage All sound frequencies measured during our study were too low for mice to hear Independent noise measurement was also undertaken within the Wohl BSU in March 2019 which also confirmed that the frequencies are too low for mice to be able to hear on the IVC rack or in the cage changing station Breeding Breeding was stopped if one of the parents exhibited any seizures Offspring with at least one parent identified with epilepsy were not used for future breeding stock Observing seizures in first and second Figure 4 Breeding out of spontaneous seizures in mice Discussion This study was carried out to address concerns in relation to spontaneous seizures in laboratory mice after transfer from traditional open top cages into IVCs which has been obser ved across the industr y regardless of the IVC supplier The primary concern is the welfare of animals especially when multiple seizures were obser ved in individuals Fur ther concerns relate to the impact on experimental results particularly for long term studies where cohort numbers could be compromised necessitating repeat experiments When the incidence of seizures in laboratory mice at King s College London was first identified a policy was agreed in discussion with the Named Veterinar y Surgeon NVS and consultation with the Personal Licence Holder PIL concerned to humanely kill any mouse that had experienced 3 seizures or had welfare issues that may have arisen from a seizure Records of the incidence of seizures in all mice held in IVCs in the Biological Ser vices Unit in the Maurice Wohl Neuroscience Institute at King s College London were 163

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 165 Increased incidence of spontaneous seizures in laboratory mice in an IVC environment loss The same strains also show tendency to noiseinduced seizures The author found that even strains resistant to seizures may develop tendency to those attacks if exposed to a few seconds of loud noise between 24 42 days of age It is important to note that sound provides environmental information for nocturnal animals like mice By stimulating central and peripheral systems sound controls autonomic motor responses learning and memory planning and executing and much more According to Turner DBA 2 lose their hearing when they are around 2 weeks old and are deaf when they reach 3 4 months of age and their exposure to loud noise between 3 4 weeks of age may results in fatal seizures Popular background strains as C57BL6 and BALB c also show genetically progressive hearing lost which starts early in their lives but progresses slowly 5 Summary and conclusion Mice have different hearing range to that of humans which is supported by published finding 6 8 10 Unpleasant and noisy sounds for people may not be a disturbance to mice as the sound frequency is simply too low for the animals to hear 8 10 Two separate noise measurement exercises confirmed that mice could not be able to hear the sound of technical equipment in the animal rooms Our study has compared noise measurement for different activities in the animal room and in IVC caging however further study should be performed to find out if other factors other than noise may be a stimuli for spontaneous seizures in mice Animal Technologists to understand the strains they are working with and to differentiate unwanted side effects of genetic mutation in order to spot genetic drift as soon as possible and take proper action to improve animal welfare Ideally tissue samples should be obtained from any mouse that has a spontaneous seizure for polymerase chain reaction PCR analysis to detect a possible genetic cause of seizures Acknowledgements Our thanks go to Ken Applebee Glyn Fisher and Martin Lawton for their support We are very grateful to all of the Wohl BSU staff for their commitment to record data for over eight month period and for their support throughout this study Thank you to Jolene Hammonds Liza Nabi and Nicholas Steinmetz for all their help References 1 2 3 4 By selecting only future breeding stock that had not exhibited seizures in any consecutive generations spontaneous seizures almost completely 99 decreased in number of observed attacks throughout the unit At that time no environmental changes or adjustments were undertaken The authors consider that the findings of our study of the incidence of spontaneous seizure in laboratory mice could impact on animal welfare It is vital to identify spontaneous seizure activity in mice as early as possible to ensure that they are eliminated from future breeding stock This requires animal care staff and researchers to be trained to identify all forms of seizures The elimination of the cause of seizures will not only improve animal welfare but will also reduce the number of animals required to complete studies as seizures would result in the loss of an experimental cohort thus leading to a refinement in welfare and a reduction in the numbers of animals used 5 6 7 8 Future work Importance of genetic drift needs to be addressed as a main point of breeding and maintaining any animals colonies Sufficient training should be given to all those responsible for breeding including researchers and 9 Blood D C Studdert V P and Gay C C 2007 Saunders Comprehensive Veterinar y Dictionar y 3rd edition Saunders Frankel W Ph D 2014 What is Epilepsy Research Highlight h t t p s w w w j a x o r g n e w s a n d i n s i g h t s 2 0 1 4 december what is epilepsy Mirsky I A Elgart S and Aring C D 1943 Sonogenic convulsions in rats and mice I Control studies Journal of Comparative Psychology 35 3 249 253 Steinmetz N A 2017 Aberrant Cortical Activity in Multiple GCaMP6 Expressing Transgenic Mouse Lines eNeuro Vol 4 Issue 5 Research Article Methods New Tools https www eneuro org content 4 5 ENEURO 020717 2017 Low Marchelli J 2017 Strategies to Minimize Genetic Drift and Maximize Experimental Reproducibility in Mouse Research Charles River Laboratories International Inc Research Models http animalab eu sites all pliki white_paper_eng pdf Turner J G 2015 Hearing in Laboratory Animals Strain Differences and Nonauditory Effect of Noise Comparative Medicine https www ncbi nlm nih gov pmc ar ticles PMC 3725606 Committee for Update of the Guide for Care and Use of Laboratory Animals Guide for Care and Use of Laboratory Animals 2011 Eighth Edition The National Academies Press Washington D C https www ncbi nlm nih gov books NBK54050 Carman R A Quimby F W and Glickman G M 2008 Vibration Effects on Laboratory Mice during Building Construction The Journal of Acoustical Society of America https asa scitation org doi 10 1121 1 2935010 Reynolds R P Kinard W L Degraff J J Leverage N and Norton J N 2010 Noise in a Laboratory Animal Facility from Human and Mouse Perspectives J Am Assoc Lab Anim Sci 165

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 December 2019 Page 167 Animal Technology and Welfare Non invasive extraction of Cnidarian venom through the use of autotomised tentacles PHILLIP ROBINSON 1 3 STEVEN TRIM2 and CAROL TRIM3 1 2 3 The Deep Aquarium Kingston upon Hull HU1 4DP Venomtech Ltd Discovery Park Sandwich CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury CT1 1QU Correspondence Dr Carol M Trim carol trim canterbury ac uk Based on a Congress 2019 Platform Presentation Summary The animals contained within the phylum Cnidaria Sea Anemones Corals Sea Pens Jellyfish Boxjellies and Hydra have origins that can be dated back to around 750 million years ago mya and as such they represent what is potentially the oldest known venomous lineage that is recognised today 1 2 The phylum Cnidaria which includes Sea Anemones Corals and Jellyfish are also one of the most understudied as far as toxins go likely a result of the constraints involved in obtaining samples Over the last two decades there have been increased efforts to further our ability to obtain samples however the sampling techniques developed were invasive and generally required the dissection of tissues from the organism Within recent years there have been some developments in the chemical extraction of Cnidarian venom using ethanol to trigger nematocyst firing These developments have led to the formation of this research which uses ethanol to elicit stimulation of nematocysts on naturally autotomised tentacles whilst being observed under light microscopy before having protein content measured using microspectrophotometry This paper focusses on a unique observation of Cnidaria that is unknown in any other animal taxa passive autotomy of envenomation apparatus the tentacles Introduction Cnidaria is an exclusively aquatic phylum of invertebrate animals which have been shown to have diverged between 686 819 mya which implicates the entire taxon as one of the oldest venomous lineages that are still present to the current day 1 2 3 Although the taxa are well known as being venomous with some species being renowned as medically significant to humans they have been relatively underutilised as research subjects despite being a promising and almost untapped source of novel proteins 4 5 6 One of the largest attributes to the underutilisation of Cnidarians in the field of venom research stems from the difficulties encountered when attempting to obtain samples of venom 4 6 Since the 1960s the ability to obtain samples of venom and develop our comprehension of Cnidarian toxins has gradually increased 7 However most of the sampling techniques that have been utilised have commonly necessitated dissection of the organism s tissue to acquire toxins There have been diverse methods of extraction employed over time using procedures such as triggering the release of nematocyst with amnion obtained from human embryos chemical or osmotic discharge using mechanical means to achieve the destruction of tissues sonication of tissues to rupture cnidocytes maceration and using bead mills to homogenise tissues however it has been stated that many of these methods are either of unknown reliability or can lead to proteins contaminating the samples 6 7 8 9 10 11 Over the last 5 years there has been progress made in the use of chemical induced firing in order to sample Cnidarian venom A significant conclusion from this research highlights that ethanol is an effective means to chemically initiate the firing of nematocysts with a secondar y advantage being that this method significantly mitigates the contamination of the samples with lysed tissues and non target proteins 6 7 8 Cnidaria like many other invertebrate taxa are capable of autotomy of tissue these mechanisms are presumed to have evolved to protect the rest of the organism 12 Long trailing tentacles evolved for prey capture can inadvertently entangle other jellyfish or excessively large prey and predators in which it may be advantageous to autotomise the entangled tentacle to 167

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 168 Non invasive extraction of Cnidarian venom through the use of autotomised tentacles escape As in other autotomising organisms Cnidaria have co evolved a regenerative ability to maintain function Casual observation of medusa stages in captivity has highlighted that autotomy frequently happens potentially through contact or entanglement with tank mates Authors have also anecdotally observed and identified autotomised tentacles in the coral sea of Australia highlighting that there may potentially be some slight in situ implications for aspects of this study As our current understanding of Cnidarian toxins is comparatively lacking when considering the research outputs focussing on terrestrial organisms within the field of toxinology 6 13 a focus on developing further knowledge of the toxins that are produced within this taxa will not only increase our comprehension for their medical significance but will also generate further research of the potential for Cnidarian venom based novel drug leads As the advancements of new extraction techniques have provided a multitude of opportunities the work presented within this paper aims to provide a novel means of obtaining Cnidarian toxins whilst simultaneously improving their welfare As a result of this there may be potential to increase our current knowledge base of Cnidarian venoms whilst promoting a non invasive means of sample acquisition Ethical considerations The Cnidarian used within this research are not protected under the Animals Scientific Procedures Act 1986 However prior to undertaking any research all aspects of the Cnidarian work detailed within this paper were subjected to an ethical application and subsequent ethics review by the ethics committee at The Deep Aquarium The Deep Hull Furthermore the research has been performed with the improvement of ethics in mind and as such has been carried out in a manner that promotes zero live animal contact through the use of tissues obtained as a result of natural autotomy Rat er ythrocytes were har vested in accordance with the 3Rs and Animals Scientific Procedures Act 1986 from Schedule I culled animals used for other studies Figure 1 A 274L kreisel system housing Chrysaora pacifica This image taken from the back of the system with the backdrop removed illustrates the general set up and plumbing of a kreisel system It is worth noting that this specific enclosure is technically a pseudokreisel as a true kreisel is fully rounded rather than possessing a flattened wall as in this image Methods Husbandry The Chrysaora medusae adult life stage are kept and displayed in a kreisel system Figures 1 and 2 a specially designed aquarium specifically for housing pelagic jellyfish and Ctenophores The jellyfish display system consists of a large kreisel with a volume of 824L a smaller kreisel with a volume of 297L a sump containing around 1 000L and altogether including pipework and life support the system Figure 3 holds a total volume of 2 222L of artificial saltwater 168 Figure 2 The same kreisel system depicted in Figure 1 this time from the exhibition side with the backdrop in place Note that the jellyfish have just been fed and small Aurelia can be seen as well as a cloud of Artemia nauplii

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 170 Non invasive extraction of Cnidarian venom through the use of autotomised tentacles Medic Titan 2000 to ensure temperatures do not unintentionally fall outside of these parameters A 90 100 water change is performed on the tubs every two weeks on average however the frequency in which this may be required can often depend on the number of polyps present and factors such as algae build up To induce strobilation metamorphosis from the polyp stage into the ephyra stage the temperature of the water bath is very gradually increased by 1 2 C at the rate of around 1 C per week The water is then held at this temperature for around a week before being slowly reduced by 3 4 C over the space of at least a week The strobilation is induced by the stress of enduring the changing temperature and once the cue to strobilate has been provided it may take a minimum of 2 4 weeks before any ephyrae are produced depending on the species in question Before strobilation is initiated the polyps must be in good condition which can be ascertained through having a plump appearance with an almost pink colouration to them As strobilation occurs the polyps will be seen to elongate and the colour will change to brown At this point the ephyra will begin to detach from the strobilae and become free swimming individuals Ephyrae can be carefully transferred from the tub into a 5L glass fishbowl that is gently aerated through a 6mm airline attached to a diaphragm air pump Hiblow HP40 the aeration should be gentle enough to ensure ephyrae remain suspended in the water column but not so severe that they are unable to freely swim through pulsating of the bell Transfers of ephyrae should be performed using a plastic Pasteur pipette which will need the tip cutting so the hole is larger than the size of the ephyra If the ephyra is too large to fit in a pipette the transfer should be performed using a plastic or glass beaker As each ephyra grows and their lappets begin to fuse into a bell they require carefully transferring from the glass fishbowl into a small 30L kreisel Initial flow within the kreisel should be much lower than is required in a kreisel housing fully developed medusae but this will necessitate gradual increases as the ephyrae develops in size and becomes a medusa When increasing the flow of the kreisel it is imperative to ensure that the increase is done over several days or more as to minimise stress potential tissue damage and the possibility of tentacles becoming tangled once they have developed Unlike some species of jellyfish Chrysaora do not have a symbiotic relationship with zooxanthellae and as such are not photosynthetic which means that each life stage requires active feeding The type of food and frequency of feeding changes as polyps grow and metamorphose with polyps requiring feeds at least 5 times a week using between 1 3mL of either live rotifers L strain Brachionus plicatilis ZM Systems UK or live nauplii When using live rotifers the 170 cultures which are kept in a lower salinity of 20 22ppt should gradually be brought up to the same salinity of the system that is being fed Live rotifers are the best staple to provide when feeding the ephyra life stage although supplementary power feeding several times a week is often the most ideal method to ensure that enough food is acquired This is undertaken using around 150mL of system water in a beaker and adding 1 2mL of live rotifers or live nauplii and allowing the ephyrae to feed for around 10 minutes keeping a close eye on water quality during this period As the ephyrae grow and start to form a bell additional power feeds of mucous taken from Aurelia species are provided several times a week for a duration of no longer than 10 minutes This gradually increases into being target fed small chunks of chopped Aurelia as the tentacles develop Adult medusae are fed 3 times a day with the first being a targeted feed of roughly chopped Aurelia chunks which are squirted into the tentacles and mesenteric arms of each individual using a turkey baster this is supplemented with 800 1200mL of live nauplii dependent on the size of the kreisel stocking density and the volume of Artemia in the culture The second feed that is offered is a free feed of around 800mL live nauplii suspended in the water column and the final feed of the day is either defrosted Calanus species or occasionally defrosted Mysis species along with another 800 1200mL of nauplii Calanus species is the preferred food as it remains suspended in the water column for a greater length of time being a smaller organism The nauplii that are used are cultured on site and are always decapsulated using sodium hydrochloride before culturing takes place Decapsulation protocol The Artemia cysts were rehydrated in fresh water that was aerated using an air pump and air stone for approximately 2 hours All the water from the rehydrated Artemia cysts was strained away using a fine filter sock 70g of sodium hydroxide was added to a bucket containing 2 5L of fresh water and allow to dissolve In a separate container 1 65L of sodium hypochlorite was added to 3 35L of fresh water and then mixed This was then added to the sodium hydroxide solution The filter sock containing the Artemia cysts was then added into the mixed solution ensuring that the top of the sock remained out of the water so cysts were retained within the filter As the reaction took place over several minutes and caused the decapsulation to occur the cysts changed colour from their original brown into an orange colour Whilst the decapsulation was taking place a solution of sodium thiosulphate was made in a separate bucket by dissolving 70g of sodium thiosulphate in 6L of fresh water Once around 90 of the cysts appeared to have changed the reaction was inhibited by removing the filter sock and rinsing with fresh water thoroughly before the filter sock was placed into the sodium thiosulphate

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 171 Non invasive extraction of Cnidarian venom through the use of autotomised tentacles solution and soaked for around 2 3 minutes After the filter sock was removed from the sodium thiosulphate solution it was rinsed under fresh water for several minutes before having all the excess water strained away Decapsulated cysts were stored in a fridge inside a container with just enough artificial saltwater to cover the cysts To hatch the Artemia the desired volume of cysts was placed into a vessel containing artificial seawater that was heated to 26 28 C and aerated for around 24 hours Ensuring that the aeration was strong enough to ensure the cysts remained suspended within the water column throughout the pre set BSA calibration This process was repeated at 1 minute long intervals in order to calculate the ideal immersion time required to obtain the largest possible extract volume within the sample Figure 5 Once a guideline for timings had been estimated a new sample was produced this time using two sections of tentacle one measuring 110mm and the other 72mm The tentacles were immersed in ethanol for around 8 minutes before being removed to prevent contamination of the sample through tissue lysis Sample preparation Venom extraction procedure In order to conduct the primary trials for this method a small section of naturally autotomised tentacle was removed from the kreisel using a 30mL Universal specimen bottle and was stored in system water at 4oC until it was processed later the same day This was to initially assess whether the nematocyst firing would be elicited using this method The small section of tentacle 2mm was placed onto a clear glass microscope slide and as much of the remaining seawater as possible was blotted dry before the slide was placed onto the stage of the microscope Olympus BX40 Once the view had been focussed at a magnification of x20 a small amount of ethanol was added to the slide using a Pasteur pipette to initiate the firing of the nematocysts The secondary stage of development was to assess the extract for the presence of protein and in turn ascertain if there was the potential presence of a usable amount of venom within the sample This was achieved through placing a section of tentacle 10mm into a 0 5mL centrifuge tube containing ethanol and analysing the quantity of protein found within the solution using a DS 11 Spectrophotometer Denovix USA After blanking the microvolume reader a 1 L sample of the ethanol tentacle extract mix was pipetted onto the reader and the protein concentration was measured using absorbance at 260 280nm using Samples were concentrated using 0 5mL 10 000 Da centrifugal filters Amicon Ultrafree MC and each 10K filter was centrifuged Hermle Z160M at 5000g for 10 minutes The retentate from within each filter tube was then pooled into a separate 0 5mL centrifuge tube Owing to the potential instability of Cnidarian toxins 14 15 16 the extracts were lyophilised to improve stability during transport of samples from the sampling site to the laboratory The lyophilisation was achieved using a desiccation method attained by placing the centrifuge tube containing the sample into a 530mL airtight food grade lockable container Sistema To Go Sistema Plastics UK Limited Surrey UK containing 50g of silica gel SiO2 with the lid of the centrifuge tube left open The container was then sealed shut with Parafilm wrapped around the lid to ensure the seal was as air tight as possible The sample was desiccated at ambient temperature 26 C until it had been fully lyophilised which took around two days for an entire 0 5mL tube The lyophilised samples were then stored at 20 C as an additional measure to preserve the integrity of the extract Protein analysis The lyophilised venom was dissolved in 40ul of HPLC grade water The DS 11 spectrophotometer was blanked with 1 L HPLC grade water wiped and the three successive 1 L aliquots of extract were measured for protein concentration Erythrocyte lysis Figure 5 Rate of protein release into extraction media from autotomised tentacle Rat er ythrocytes were collected passively into heparinised phosphate buffered saline PBS Scientific Lab Supplies post cer vical dislocation and decapitation Cells were chilled on ice until counted with a haemocytometer In a conical well microamp 96well plate Applied Biosystems 100 L of 10mg ml venom along with separate rows of 100 Dimethylsulphoxide DMSO Fisher Scientific and 100uL sterile water was serially diluted 1 in 10 into PBS 100 L of 1x107 cells ml was added to each well and the plate covered with sealing film and incubated at 37 C for six hours Supernatants were removed from each well into a polystyrene 384 well plate and read on a Fluostar plate reader BMG Labtech at 414nm and 620nm as per Eschbach et al 17 171

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 172 Non invasive extraction of Cnidarian venom through the use of autotomised tentacles Results Light microscopy of nematocysts firing Nematocysts were observed to fire from the tentacle in response to ethanol stimulation Most nematocysts were fired within the first 12 seconds with a relatively small number being slower and a complete halt in firing being observed from around 20 seconds Figure 6 shows tentacle section before stimulation Figure 7 shows tentacle post firing of nematocysts bright white lines protruding from tissue contained 89 57 3 6 g L protein thus 3 583mg of protein was obtained The high ratio of 260 280nm 0 94 0 01 indicates nucleic acid contamination presumably from cellular lysis from the broken ends of the tentacle but which may also be a component of the venom itself Identification of active venom At a final concentration of 5 g L Chrysaora venom from autotomised tentacles caused a modest amount of lysis in rat erythrocytes Figure 8 This caused release of 13 3 of haemoglobin into the supernatant when compared to the positive control of 50 DMSO Although small the effect is significantly different p 0 013 from the negative control PBS i e null hypothesis of no effect using a non paired student T Test in Excel Microsoft Figure 6 A small section of autotomised tentacle obtained from Chrysaora pacifica observed Pre firing at x20 magnification Figure 8 Erythrocyte lysis of Chrysaora colorata venom compared with 50 DMSO control and 50 sterile water The venom collected from autotomised C colorata tentacle causes 13 3 5 lysis of isolated rat erythrocytes Discussion Figure 7 A post firing image of the tentacles seen in Figure 5 The fired nematocysts can be clearly observed as white lines extending from the segments of tentacle Protein analysis The investigation of rate of protein venom release into stimulation media Figure 5 demonstrates that although the nematocysts have finished firing after 20 seconds the protein continues to increase before reaching a plateau at eight minutes This could be because the venom is either being released for longer or it is an artefact of diffusion of the released venom into the stimulation media The 40 L sample stimulated in microcentrifuge tube 172 This study demonstrates that protein concentration increases in the media surrounding fired nematocysts from autotomised tentacles This protein has venom like properties of causing erythrocyte lysis and thus is expected to be venom that would have been injected by the animal on envenomation Further work is planned to characterise the venom such as identifying proteins contained within and their functional effects Thus further development could lead to a novel means of obtaining Cnidarian venom samples without the need to undertake any animal contact Despite the potential beneficial implications linked to this study there are also some areas in which these techniques are less efficient than the currently accepted methods Some of the foreseen downfalls associated to the use of the autotomy method are the unreliability of sample availability the inconsistent size and weight of tentacles that can be obtained and the potential loss

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 173 Non invasive extraction of Cnidarian venom through the use of autotomised tentacles of tissue integrity encountered from autotomised tentacles going unnoticed for a length of time Further work is expected to include investigation of autotomised tentacle longevity in order to examine how long they retain viability post autotomy refining the processing and storage of samples and developing a standard for sample quality which allows for the most efficient assay results 9 10 11 The results indicate nucleic acids are present in the venom this could either be from the broken ends of the tentacles or the venom itself Nuclei acids such as RNA and DNA have been reported from vertebrate venoms so may be a part of the normal composition of Cnidarian venoms 19 This study highlights an unusual aspect of Cnidarian biology that presents a unique opportunity to collect and study the venom of these animals without trauma or euthanasia and thus we feel is a major improvement in their welfare 12 13 14 Acknowledgements Thanks to The Deep Aquarium for the provision of autotomised tentacles and Aquarist Shoshana Levine for providing information on husbandry regimens 15 16 References 1 2 3 4 5 6 7 8 Park E Hwang D Lee J Song J Seo T and Won Y 2012 Estimation of divergence times in cnidarian evolution based on mitochondrial protein coding genes and the fossil record Molecular Phylogenetics and Evolution Vol 62 329 345 Jouiaei M Yanagihara A A Madio B Nevalainen T J Alewood P F and Fry B G 2015 Ancient Venom Systems A Review on Cnidaria Toxins Toxins Vol 7 2251 2271 Gacesa R Chung R Dunn S R Weston A J JaimesBecerra A Marques A C et al 2015 Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera Cnidaria Anthozoa Scleractinia BMC Genomics Vol 16 No 774 Fraz o B Vasconcelos V and Antunes A 2012 Sea Anemone Cnidaria Anthozoa Actiniaria Toxins An Overview Marine Drugs Vol 10 1812 1851 Mariottini G L and Pane L 2014 Cytotoxic and Cytolytic Cnidarian Venoms A Review on Health Implications and Possible Therapeutic Applications Toxins Vol 6 108 151 Jouiaei M Casewell N R Yanagihara A A Nouwens A Cribb B W Whitehead D et al 2015 Firing the Sting Chemically Induced Discharge of Cnidae Reveals Novel Proteins and Peptides from Box Jellyfish Chironex fleckeri Venom Toxins Vol 7 936 950 Barnes J 1967 Extraction of Cnidarian Venom from Living Tentacle In Animal Toxins Russell F E and Saunders P R Pergamon Press Oxford UK 115 129 Carrette T and Seymour J 2004 A rapid and repeatable method for venom extraction from Cubozoan nematocysts Toxicon Vol 44 135 139 17 18 19 Berking S and Herrmann K 2005 Formation and discharge of nematocysts is controlled by a proton gradient across the cyst membrane Helgol Mar Res Vol 60 180 188 Helmolz H Ruhnau C Sch tt C and Prange A 2007 Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata L and Cyanea lamarckii P ron L slieur Toxicon Vol 50 53 64 Ponce D Brinkman D L Luna Ram rez K Wright C E and Dorantes Aranda J J 2015 Comparative study of the toxic effects of Chrysaora quinquecirrha Cnidaria Scyphozoa and Chironex fleckeri Cnidaria Cubozoa venoms using cell based assays Toxicon Vol 106 5767 Bickell Page L R and Mackie G O 1991 Tentacle Autotomy in the Hydromedusa Aglantha digitale Cnidaria An Ultrastructural and Neurophysiological Analysis Phil Trans R Soc Lond B Vol 331 155 170 Turk T and Kem W R 2009 The phylum Cnidaria and investigations of its toxins and venoms until 1990 Toxicon Vol 54 1031 1037 Brinkman D L and Burnell J N 2009 Biochemical and molecular characterisation of cubozoan protein toxins Toxicon Vol 54 1162 1173 Doi 10 1016 j toxicon 2009 02 006 Nagai H 2012 Marine Protein Toxins In Handbook of Marine Natural Products Fattorusso E Gerwick W and Taglialatela Scafati O Springer Dordrecht 1388 1419 Ponce D Brinkman D L Potriquet J and Mulvenna J 2016 Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle Chrysaora fuscescens Cnidaria Scyphozoa Toxins Vol 8 4 102 Doi 10 3390 toxins 8040102 Eschbach E Scharsack J P John U and Medlin L K 2001 Improved erythrocyte lysis assay in microtitre plates for sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae Journal of Applied Toxicology Vol 21 6 513 519 https doi org 10 1002 jat 797 Bernheimer A W and Avigad L S 1976 Properties of a toxin from the sea anemone Stoichactis helianthus including specific binding to sphingomyelin Proceedings of the National Academy of Sciences of the United States of America Vol 73 2 467 471 https doi org 10 1073 pnas 73 2 467 Currier R B Calvete J J Sanz L Harrison R A Rowley P D and Wagstaff S C 2012 Unusual stability of messenger RNA in snake venom reveals gene expression dynamics of venom replenishment PLoS ONE Vol 7 8 1 10 173

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 174 Animal Technology and Welfare December 2019 RSPCA workshop Congress 2019 everyday 3Rs for Animal Technologists PENNY HAWKINS and JULIET DUKES RSPCA Research Animals Department Wilberforce Way Southwater West Sussex RH13 9RS Correspondence research animals rspca org uk Background This workshop aimed to encourage Animal Technologists to actively promote and help to implement the 3Rs within their daily work in the animal facility The focus was on refinement as this R benefits most from the expertise and perspectives of Animal Technologists and care staff Animal Technologists can encourage and implement refinement by Bringing new information e g from journals such as Animal Technology and Welfare meetings and websites to the attention of colleagues the Named Information Officer NIO and the Animal Welfare and Ethical Review Body AWERB Actively suggesting refinements and offering to help evaluate these Communicating with scientists about animal behaviour biology and welfare Helping to define and implement welfare assessment protocols and participating in actual severity assessment and retrospective review Raising any concerns they may have Participating in the AWERB and supporting its initiatives Sources of information on the 3Rs The workshop began with a presentation setting out some background information on the 3Rs with some routes to finding out more about each R Replacement Replacement includes both full replacement e g computer or mathematical models established cell lines that do not use animal sera human volunteers human cell or tissue cultures and partial replacement e g use of invertebrates that are not believed to suffer such as Drosophila cell lines that do require animal sera such as Fetal Bovine Serum out of scope developmental stages use of cells or tissues from animals killed for the purpose Avoiding animal use can also be regarded as replacement for example 174 through a change in research direction or identifying a different approach to addressing a human or animal health problem Animal Technologists can make a big difference with respect to replacement in education and training by monitoring developments in training materials developing training resources and replacing animals during early stages of training in animal handling so that people are more confident when they come to handle living animals Some training materials e g model animals are high tech and relatively expensive but there are also some very good low tech resources that can be purchased or crafted see norecopa no and search for homemade Sources of information about replacement include the NC3Rs nc3rs org uk Norecopa norecopa no and an RSPCA slide set please email for a copy For more in depth information see the Altweb search engine altweb jhsph edu Reduction Reduction is often considered in terms of optimising animal numbers through better experimental design and statistical analysis There are other ways of using flexible thinking within experimental design to reduce numbers for example it is sometimes possible to use historic control groups from other experiments Animal Technologists can directly contribute to reducing animal numbers by helping to ensure that tissues are shared effectively within and between establishments and by implementing good colony management minimising wastage in breeding programmes and sharing and archiving lines of genetically altered GA animals For information about reduction see the NC3Rs and Norecopa as above and for information about experimental design reproducibility and research integrity see ResponsibleAR on Twitter Information on sharing and archiving GA mice can be found at nc3rs org uk minimising use ga mice Animal Technologists can and do contribute to

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 175 RSPCA workshop Congress 2019 everyday 3Rs for Animal Technologists implementing replacement and reduction with respect to training and minimising wastage When it comes to encouraging the two R s more widely it is up to you how much you want to research maintain and use your knowledge The web links mentioned above are all helpful and useful newsletters are produced by the RSPCA Research Animals Depar tment email for details NC3Rs including Tech3Rs aimed at Animal Technologists and care staff and Norecopa You could also ask the researchers you work with to explain how they search for and monitor progress with Replacement and how they implement Reduction Hopefully you are working in a culture where these kinds of conversations are accepted and welcomed if they are not you could ask the AWERB to review how effectively it is per forming the task of promoting a Culture of Care Refinement The workshop then moved on to the topic of refinement thinking about the whole life experience of the animal This R used to be considered solely in terms of reducing suffering caused by experimental procedures but now it is understood that there are many other potential sources of discomfort pain and distress and more thought is given to promoting positive welfare For example events and procedures that can have a negative effect on welfare include early maternal separation including in rodents who are removed from the dam before they would disperse naturally transpor t marking for identification genotyping housing in inappropriate social groups or individually for social animals under stimulating and or uncomfortable housing husbandry procedures e g cage change necessary for good health but still a stressor scientific procedures and their after effects and the humane killing process Challenging and testing This was followed by a second presentation on effectively challenging and testing yourselves potential refinements and also your colleagues With respect to challenging yourselves the Ratlife video youtu be giu5WjUt2GA was used to highlight the fact that wild type behaviours are innate in domesticated and laboratory animals if they are given oppor tunities to express them The laborator y environment can become normalised so it is important to remember the contrast between large complex natural habitats and the laboratory no matter how much is done to refine the latter Potential refinements may also need to be evaluated to assess their impact on i animal welfare ii the science and iii animal house resources This needs an assessment process that has been set out in liaison with all those concerned including relevant researchers managers and budget holders so that everyone has bought into the process and will be prepared to implement the outcome You may also need to challenge others constructively of course You can keep a running conversation going about refinement day to day and outside of more formal interactions such as project licence review and AWERB discussions The paper Communicating the Culture of Care lists a 10 point plan for good communications including picking the right moment s emphasising the positives and always ending with an agreement on something even if it is that you need to talk about the topic some more 1 There are also some good tips for influencing people on the Human Behaviour Change for Animals website hbcforanimals com and the Culture of Care network has produced a sheet with some ideas for improving communication between scientists and animal technologists and named persons see norecopa no coc The key point to remember is that you will always be able to find some common ground with a researcher even if your starting positions appear to be different You can assume that everyone wants better science and welfare and if you want to gain a better understanding of the other pressures that scientists are under the Nuffield Council on Bioethics project on research culture is ver y helpful nuffieldbioethics org project research culture All of these can combine to have a significant effect on animal welfare but the good news is that much more is known about the behaviour and welfare needs of animals and how to implement refinement to reduce the lifetime impact on animals We discussed the concepts of a life not worth living a life worth living and a good life a good life should be the ideal state for any animal kept by humans for any purpose Refinement can obviously move an animal s welfare status towards the good life and can drive the other two Rs seeing animals in more natural surroundings can lead to greater empathy for them encouraging people to seek humane alternatives optimise numbers and minimise wastage Case studies We had an interactive discussion on ways in which participants accessed information on refinement and how well supported they were in accessing information including attending external meetings training courses and passing information on within their establishments We then went on to discuss some hypothetical case studies on a number of different topics in which Animal Technologists had to consider how they would inform and persuade colleagues to implement refinement or improve their practice Three examples are set out below if you would like the full list please email research animals rspca org uk 175

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 176 RSPCA workshop Congress 2019 everyday 3Rs for Animal Technologists 1 Capturing mice Your establishment was quick to pick up on the issue of capturing mice using a tunnel or cupped hands rather than picking them up by the tail Staff attended workshops run by the NC3Rs which described positive outcomes for both animal welfare and science and a programme was initiated to achieve a change to standard practice for catching mice in which staff were trained to use cupped hands However this did not last long and staff reverted to catching by the tail There was a perception that it was taking too long to catch the animals and that this was affecting husbandry practices such as cage change Some people were also reluctant to change their behaviour based on animal welfare science feeling that their own practical experience gave them a better understanding of what was in the animals best interests What should you do about this Several participants had direct experience of this issue and the following suggestions were made Set up a properly controlled pilot study using just a few racks to i measure the time it takes to train the mice and staff and ii compare cage change times using capture by the tail vs in cupped hands or a tunnel Use a stopwatch and ensure the data analysis in ii is done by someone blinded to the experimental conditions Promote open communication between people having issues Make sure enough time is allowed for training including videos If tunnel capture seems to cause aggression do not give up seek further advice in case technique could be improved Simply make it a policy to use a tunnel or hands Supply information in each animal room put up posters Persistence Use soft skills to get technical staff and users on board Emphasise that the outcome is better welfare for mice less stress It may take a while to achieve change but it will be beneficial for the future 2 Assessing welfare in a new protocol A researcher at your establishment is keen to add a new protocol to their project studying brain and behaviour in mice using a virtual reality system in which a head fixed mouse runs on an air cushioned spherical treadmill The objective is to investigate decision making and navigation in mice obtaining electrophysiology recordings via a head cap while the mouse responds to computer generated patterns The researcher says that the protocol is well tolerated and will not cause significant distress You are not so sure as you know that mice are thigmotactic and seek to make contact with the walls of their enclosure which 176 they can never do in the virtual reality set up You are also concerned that their ability to display a range of behaviours including signs of distress will be physically limited How would you go about getting evidence to support your concerns what could you do to help ensure the protocol was adequately refined to take account of the mouse s experience including appropriate welfare assessment Participants made the following points The applicant can be asked for discussion regarding what is the benefit of using this particular technique what are the alternatives e g telemetry how will animals be housed between recording sessions could they habituate animals more effectively further refine the surgery The applicant can also be asked for fur ther evidence e g scientific literature to show how the technique has been used in the UK and elsewhere All of these can be consulted and asked for support NACWO NIO NVS HO AWERB Expertise can be sought on mouse behaviour including in the wild to understand how distress behaviours may be compromised and to identify potential behavioural indicators of discomfort pain or distress This is a very technical set up with the mouse in an unfamiliar situation so it would be appropriate to suggest a pilot study 3 Enrichment for Zebrafish You have come across a series of papers by fish biologists and animal welfare scientists which describe how they evaluated environmental enrichment for Zebrafish In one study they used preference tests to evaluate whether groups of male and female Zebrafish would choose to spend more time in areas of the tank with enrichments such as gravel pictures of gravel on the bottom of the tank plants or air stones They found that pictures of gravel were preferred over a barren tank floor Another study discovered that artificial grass significantly improved fecundity the number of viable eggs and further papers reported that substrate plus grass reduced anxiety and improved brain development You want to tr y providing similar enrichment for the Zebrafish in your care but there are a lot of tanks with a great many fish How would you prepare to make your case Suggestions included Seek approval from the NACWO and the AWERB Acquire some data to back up the case for enrichment either from the literature or by doing a study in house Make it relevant to helping the researchers making it clear that healthy fish with good welfare means good science

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 177 RSPCA workshop Congress 2019 everyday 3Rs for Animal Technologists Ask for a discussion on enrichment for Zebrafish for example via the AWERB or a user group Draw up a future plan and protocol for change including not just what to change but how Resources to help keep up with the Three Rs Participants were asked to write a take home message on a Post it note some examples are listed below In the materials we made available to the participants after the workshop we included a PDF document with live links to the journals and websites listed below All URLs accessed 1 July 2019 If you would like this resource list please email research animals rspca org uk General principles Journals Refinement is the key If science is worth doing it is worth doing well There are many options relating to the 3Rs Refinement can be achieved in several ways and a technician can make a difference Replacement is always the goal but we can still have a massive impact until then A life for an animal used in research must be worth living Highlighting animal welfare issues is everyone s responsibility and to challenge bad practice These journals often publish papers on laboratory animal behaviour and environmental enrichment PLOS ONE and Animals are free to access but most of the others require subscriptions However if you cannot access these journals an alternative option is to subscribe to updates of the content pages then request papers of interest directly from the authors You could ask your NIO to do this on behalf of the team Feedback from participants Communicating and keeping an open mind Even when researchers or technical staff are resistant to change understanding their reasons and supporting their views is really important when explaining why the new husbandr y can be advantageous to them given time to adjust The impor tance of the role of effective communication Various routes to approach users researchers about possible refinement opportunities in their work Far more awareness than we thought beforehand I feel more encouraged and motivated to push forward with the refinement I am currently doing with more info on how to go about it It is everyone s responsibility to make a change Look for common ground between a scientist and an Animal Technologist Do not be afraid to speak up about refinement procedures Researching and using resources Ensure my 3Rs knowledge is continually up to date and ask the researchers about their refinement techniques and research into refinement There are many resources to find information that people might not have been aware of Have website information available for all to access at work Make time for training Impor tant to keep up to date with current refinements Individual responsibility do not rely on others to distribute information PLOS ONE open access journal which publishes in a wide range of disciplines Very easy to search and you can set up alerts such as zoology or research integrity to go to your mailbox journals plos org plosone Animals open access animal science and animal welfare journal mdpi com journal animals see the special issue on 60 years of the Three Rs in October 2019 Animal Technology and Welfare of course Lab Animal a Nature Research journal covering in vivo research and technology nature com laban Laboratory Animals not to be confused with the journal above this is the official journal of a number of bodies including the Federation of European Laboratory Animal Science Associations journals sagepub com home lan ILAR Journal journal of the US Institute for Laboratory Animal Research often publishes useful themed issues academic oup com ilarjournal Animal Welfare the journal of the Universities Federation for Animal Welfare this covers all areas of human animal interaction including laboratory animals ufaw org uk the ufaw journal animal welfare Applied Animal Behaviour Science the official journal of the International Society for Applied Ethology ISAE This reports mainly on farmed animal behaviour but includes some papers on animals used in the laborator y or their wild equivalents appliedethology org Applied_Animal_Behaviour_Science html Journal of Applied Animal Welfare Science JAAWS 177

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 178 RSPCA workshop Congress 2019 everyday 3Rs for Animal Technologists covers companion farm wild and laboratory animals animalsandsociety org human animal studies jaaws the UFAW series an excellent and accessible background to animal welfare science with a chapter on enrichment Online G G G G G G G The Animal Welfare Institute AWI Enrichment and Refinement Database awionline org content enrichment and refinement databases NC3Rs nc3rs org uk including the Tech3Rs newsletter nc3rs org uk tech3rs Norecopa the Nor wegian national Three Rs platform which has produced a ver y helpful database and guidance documents all in English norecopa no Altweb the global clearinghouse for alternatives altweb jhsph edu RSPCA repor ts and resources science rspca org uk sciencegroup researchanimals reportsand resources for our newsletter email research animals rspca org uk Human Behaviour Change for Animals aims to help people improve animal welfare by understanding the key principles of human behaviour change and how to apply them hbcforanimals com Social media e g Zebrafish Information Network ZFINmod Other organisations with an interest in the Three Rs G G FRAME Fund for the Replacement of Animals in Medical Experiments emphasis on replacement and experimental design but supports all Three Rs frame org uk UFAW Universities Federation for Animal Welfare ufaw org uk Meetings Keep an eye on the NC3Rs events calendar nc3rs org uk events and IAT calendar iat org uk calendar Books Hubrecht R C 2014 The Welfare of Animals Used in Research Wiley Blackwell Oxford ISBN 978 1 11996707 1 This is one of a series of books produced by UFAW Young R J 2003 Environmental Enrichment for Captive Animals Blackwell Science Oxford ISBN 0632 06407 2 Also in the UFAW series this covers the reasons for enrichment and principles behind designing and evaluating it very well including a chapter on designing and analysing enrichment studies Largely zoo based but very useful Mellor D J Patterson Kane E and Stafford K J 2009 The Sciences of Animal Welfare WileyBlackwell Oxford ISBN 978 1 4051 3495 8 Also in 178 Acknowledgements Thank you to all the workshop participants and to the IAT for their support for the workshop Thanks also to Chloe Stevens for helping to facilitate Reference 1 Boden T and Hawkins P 2016 Communicating the Culture of Care how to win friends and influence people Animal Technology and Welfare 15 3 151 156

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 December 2019 Page 179 Animal Technology and Welfare PAPER SUMMARY TRANSLATIONS INHALTVERZEICHNIS RSPCA Workshop Kongress 2019 3R f r den Tiertechniker Alltag PENNY HAWKINS und JULIET DUKES Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Korrespondenz research animals rspca org uk Abstract Zweck dieses Workshops war es Tiertechniker anzuregen die Umsetzung der 3R aktiv bei ihrer t glichen Arbeit in der Tiereinrichtung zu f rdern und zu unterst tzen Der Schwerpunkt lag auf Refinement Verbesserung da Expertise und Verst ndnis von Tiertechnikern und Pflegepersonal diesem R am meisten zugute kommen Tiertechniker k nnen Refinement wie folgt f rdern und umsetzen Kollegen den benannten Informationsbeauftragten NIO und den Animal Welfare and Ethical Review Body AWERB auf neue Informationen aufmerksam machen Aktiv Vorschl ge f r Verbesserungen vorbringen und Hilfe bei deren Bewertung anbieten Der Austausch mit Wissenschaftlern ber Tierverhalten biologie und schutz sowie die Unterst tzung bei der Festlegung und Umsetzung von Protokollen zur Tierschutzbeurteilung und die Beteiligung an konkreter Schweregradbewertung und nachfolgender berpr fung sind ebenfalls wichtig Tiertechniker m ssen eventuell bestehende Bedenken u ern und nat rlich sollten sie im AWERB mitwirken und seine Initiativen unterst tzen Schlagw rter Tiertechniker 3R Verbesserung Kommunikation 179

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 180 Paper Summary Translations Erh hte H ufigkeit spontaner Anf lle bei Laborm usen in einer IVC Umgebung ANNA MORGUNOWICZ CHELSEA CAVANAGH SOFYA NIKITOCHKINA und JULIA KEEBLE Biological Services King s College London Maurice Wohl Clinical Neuroscience Institute 5 Cutcombe Road Denmark Hill London SE5 9RX Korrespondenz anna morgunowicz kcl ac uk Basierend auf einer Pr sentation anl sslich eines IAT Kongresses Abstract In den letzten zehn Jahren wurden oben offene K fige f r Nagetiere zunehmend durch einzelbel ftete K figsysteme IVC verdr ngt Dies hat viele Vorteile mit sich gebracht so zum Beispiel eine bessere Kontrolle des Gesundheitszustands und die Minimierung der Kontamination immunsupprimierter Tiere Am King s College London KCL ging der Transfer von M usen aus offenen K figen in IVC mit einer scheinbar erh hten H ufigkeit spontaner Krampfanf lle bei M usen einher Die meisten Anf lle wurden beim ffnen von K figen in Wechselstationen beobachtet sodass geschlussfolgert wurde dass eine signifikante Ver nderung von Umweltreizen das Auftreten von Anf llen beg nstigen kann Ziel der vorliegenden Studie war es die H ufigkeit von Anf llen bei M usen nach ihrer Verlegung in IVC zu erfassen und die unterschiedlichen Umweltbedingungen zwischen dem Inneren und dem u eren von IVC zu untersuchen insbesondere den Ger uschpegel Die Studie beleuchtet die Zunahme des Auftretens spontaner Anf lle bei M usen in IVC und die Notwendigkeit die Auswirkungen der Umwelt auf Anf lle bei Versuchstieren zu untersuchen um k nftig ein H chstma an Tierschutz zu gew hrleisten Schlagw rter M use Anf lle einzelbel ftete K fige IVC 180

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 181 Paper Summary Translations Nicht invasive Extraktion von Nesselgiften durch Verwendung autotomisierter Tentakel PHILLIP ROBINSON 1 3 STEVEN TRIM2 und CAROL TRIM3 1 2 3 The Deep Aquarium Kingston upon Hull HU1 4DP Gro britannien Venomtech Ltd Discovery Park Sandwich CT13 9ND Gro britannien School of Human and Life Sciences Canterbury Christ Church University Canterbury CT1 1QU Gro britannien Korrespondenz Carol Trim Canterbury ac uk Basierend auf einer Pr sentation anl sslich des IAT Kongresses 2019 Abstract Der Ursprung der zum Stamm der Nesseltiere Cnidaria geh renden Tiere kann rund 750 Millionen Jahre1 2 zur ckverfolgt werden sodass sie heute als potenziell ltester bekannter giftiger Stamm gelten Nesseltiere zu denen auch Seeanemonen Korallen und Quallen geh ren sind in Bezug auf Toxine eine der am wenigsten untersuchten Arten was vermutlich auf die eingeschr nkten Bedingungen f r Probennahmen zur ckzuf hren ist In den letzten zwei Jahrzehnten wurden verst rkte Anstrengungen unternommen um unsere F higkeiten zur Probennahme zu verbessern die entwickelten Methoden waren jedoch invasiv und erforderten generell die Gewebedissektion der Organismen In den letzten Jahren gab es einige Entwicklungen in der chemischen Extraktion von Nesselgift mit Ethanol um das Abfeuern von Nesselkapseln Nematocysten auszul sen Diese Entwicklungen begr ndeten die Aufnahme dieser Forschungsarbeit bei der Nesselkapseln an nat rlich autotomierten Tentakeln mit Ethanol stimulier t und unter Lichtmikroskopie beobachtet werden bevor der Proteingehalt mithilfe von Mikrospektralphotometrie gemessen wird Dieser Artikel konzentriert sich auf eine einzigartige Beobachtung bei Nesseltieren die bei anderen Tierst mmen unbekannt ist die passive Autotomie von Giftapparaten den Tentakeln Schlagw rter Cnidaria Giftextraktion Nematocysten Mikrospektralphotometrie 181

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Animal Technology and Welfare Page 182 December 2019 CONTENU DE LA REVUE Atelier de la RSPCA Congr s 2019 les 3R au quotidien pour les technologues animaliers PENNY HAWKINS et JULIET DUKES RSPCA Research Animals Department Wilberforce Way Southwater West Sussex RH13 9RS Correspondance research animals rspca org uk R sum Cet atelier avait pour objectif d encourager les technologues animaliers promouvoir activement et aider la mise en uvre des 3R dans leur travail quotidien au sein des installations animali res L accent tait mis sur le raffinement ce R tirant au mieux parti de l expertise et des perspectives des technologues et du personnel charg de s occuper des animaux Les technologues animaliers peuvent encourager et mettre en uvre le raffinement en Apportant de nouveaux renseignements l attention de leurs coll gues du responsable des informations d sign NIO et des autorit s charg es du bien tre des animaux et de l examen thique AWERB et en sugg rant activement des am liorations afin d aider valuer ces aspects Communicant avec les scientifiques sur le comportement animal la biologie et le bien tre social tant galement importants pour aider d finir et mettre en uvre des protocoles d valuation du bien tre social et participer l valuation de la gravit r elle ainsi qu l examen r trospectif Les technologues animaliers doivent exprimer leurs ventuelles pr occupations et doivent bien s r participer l AWERB et soutenir ses initiatives Mots cl s Technologue animalier 3R raffinement communication 182

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 183 Paper Summary Translations Augmentation de l incidence des convulsions spontan es chez la souris de laboratoire dans un environnement de CIV ANNA MORGUNOWICZ CHELSEA CAVANAGH SOFYA NIKITOCHKINA et JULIA KEEBLE Biological Services King s College London Maurice Wohl Clinical Neuroscience Institute 5 Cutcombe Road Denmark Hill London SE5 9RX Correspondance anna morgunowicz kcl ac uk Bas sur une pr sentation de la plateforme du congr s IAT R sum Le passage de cages sans couvercle des cages ventil es individuellement IVC a consid rablement augment ces 10 derni res ann es pour les rongeurs Cela s accompagne de nombreux avantages notamment un meilleur contr le de l tat de sant et la r duction de la contamination des animaux immunod prim s Au King s College London KCL le transfert de souris de cages sans couvercle des CIV a co ncid avec une apparente augmentation de l incidence des convulsions spontan es chez la souris La majorit des convulsions sont observ es lorsque la cage est ouverte dans une station de changement aussi pense t on qu un changement significatif dans les stimuli environnementaux peut contribuer l incidence des convulsions Cette tude avait pour but d enregistrer l incidence des convulsions chez les souris apr s leur transfert dans des CIV et d examiner les diff rences environnementales entre l int rieur et l ext rieur des CIV en particulier les niveaux de bruit Cette tude met en vidence l augmentation de l incidence des convulsions spontan es chez la souris log e en CIV et la n cessit d tudier l impact de l environnement sur les convulsions des animaux de laboratoire afin d assurer dor navant le plus haut niveau de bien tre animal Mots cl s Souris convulsions CIV 183

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 184 Paper Summary Translations Extraction non invasive du venin des cnidaires par l utilisation de tentacules autonomis es PHILLIP ROBINSON 1 3 STEVEN TRIM2 et CAROL TRIM3 1 2 3 The Deep Aquarium Kingston upon Hull HU1 4DP Venomtech Ltd Discovery Park Sandwich CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury CT1 1QU Correspondance Carol Trim Canterbury ac uk Bas sur une pr sentation de la plateforme du congr s IAT 2019 R sum Les animaux contenus dans l embranchement des cnidaires ont des origines qui remontent environ 750 millions d ann es1 2 et en tant que tels ils repr sentent ce qui est potentiellement la plus ancienne lign e d animaux venimeux connue aujourd hui L embranchement des cnidaires qui comprend les an mones de mer les coraux et les m duses est aussi l un des plus sous tudi s en ce qui concerne les toxines probablement du fait des contraintes li es l obtention d chantillons Ces vingt derni res ann es des efforts accrus ont t mis en uvre pour am liorer notre capacit obtenir des chantillons mais les techniques d chantillonnage qui ont t d velopp es sont envahissantes et exigent g n ralement la dissection des tissus de l organisme Ces derni res ann es des d veloppements d extraction chimique du venin des cnidaires ont permis de d clencher la r action des n matocystes et de recueillir le venin des cnidaires en utilisant de l thanol Ces d veloppements ont conduit la formation de cette recherche qui utilise l thanol pour stimuler les n matocystes des tentacules naturellement autonomis es tout en les observant en microscopie optique avant d en mesurer la teneur en prot ines par microspectrophotom trie Ce document porte sur une observation unique de cnidaires inconnue dans les autres taxons animaux de l autotomie passive de l appareil venimeux et des tentacules Mots cl s Cnidaires extraction de venin n matocystes microspectrophotom trie 184

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 185 December 2019 Animal Technology and Welfare INDICE DE LA REVISTA Taller de la RSPCA Congreso de 2019 Las 3R diarias para los tecn logos de animales PENNY HAWKINS y JULIET DUKES RSPCA Research Animals Department Wilberforce Way Southwater West Sussex RH13 9RS Correspondencia research animals rspca org uk Resumen Este taller trat de fomentar entre los tecn logos de animales el uso activo y la implementaci n de las 3R en su trabajo diario en instalaciones de animales El taller se centr en el refinamiento ya que esta R se beneficia principalmente de la experiencia y la perspectiva de los tecn logos de animales y del personal de cuidado Los tecn logos de animales pueden fomentar e implementar el refinamiento aportando nueva informaci n a sus compa eros al Named Information Officer NIO y al Animal Welfare and Ethical Review Body AWERB as como sugiriendo activamente refinamientos y ofreciendo ayuda para su evaluaci n La comunicaci n con los cient ficos sobre el comportamiento la biolog a y el bienestar de los animales tambi n es importante as como ayudar a definir e implementar protocolos de evaluaci n de bienestar y participar en evaluaciones de la gravedad real as como en revisiones retrospectivas Los tecn logos de animales deben informar sobre cualquier preocupaci n que tengan y por supuesto deben participar en el AWERB y respaldar sus iniciativas Palabras clave Tecn logo de animales 3R Refinamiento comunicaci n 185

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 186 Paper Summary Translations Mayor incidencia de convulsiones espont neas en ratones de laboratorio dentro de un entorno IVC ANNA MORGUNOWICZ CHELSEA CAVANAGH SOFYA NIKITOCHKINA y JULIA KEEBLE Biological Services King s College London Maurice Wohl Clinical Neuroscience Institute 5 Cutcombe Road Denmark Hill London SE5 9RX Correspondencia anna morgunowicz kcl ac uk Basado en una Presentaci n de Plataforma en un Congreso de IAT Resumen Durante la ltima d cada se ha producido un cambio enorme pasando de utilizar jaulas abiertas para ratones a utilizar jaulas ventiladas individualmente IVC por sus siglas en ingl s Esto ha tra do consigo muchos beneficios como un mejor control del estado de salud y una minimizaci n de la contaminaci n de animales inmunocomprometidos En King s College London el traslado de ratones de jaulas abiertas a IVC ha coincidido con un aparente aumento de los casos de convulsiones espont neas de algunos ratones La mayor a de las convulsiones se observan cuando se abre la jaula en una estaci n de cambio y por lo tanto se ha propuesto que un cambio significativo en los est mulos ambientales puede contribuir a la incidencia de las convulsiones El objetivo de este estudio era registrar la incidencia de convulsiones en ratones despu s de su transferencia a las IVC e investigar las diferencias ambientales entre el interior y el exterior de las IVC en concreto los niveles de ruido Este estudio destaca el aumento de las incidencias de convulsiones espont neas en ratones dentro de la IVC y la necesidad de investigar el impacto del ambiente respecto a las convulsiones de animales de laboratorio para poder garantizar el mayor nivel posible de bienestar animal en el futuro Palabras clave Ratones convulsiones IVC 186

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 187 Paper Summary Translations Extracci n no invasiva de veneno cnidario mediante el uso de tent culos automatizados PHILLIP ROBINSON 1 3 STEVEN TRIM2 y CAROL TRIM3 1 2 3 The Deep Aquarium Kingston upon Hull HU1 4DP Venomtech Ltd Discovery Park Sandwich CT13 9ND School of Human and Life Sciences Canterbury Christ Church University Canterbury CT1 1QU Correspondencia Carol Trim Canterbury ac uk Basado en una Presentaci n de Plataforma en un Congreso de IAT Resumen Los animales contenidos en el filo Cnidaria tienen or genes que se remontan a unos 750 millones de a os1 2 y como tales representan lo que es probablemente el linaje venenoso m s antiguo que se conoce a d a de hoy El filo Cnidaria que incluye an monas de mar corales y medusas tambi n es uno de los mbitos menos estudiados en lo que se refiere a las toxinas probablemente debido a las limitaciones que hay a la hora de obtener muestras En las ltimas dos d cadas se han incrementado los esfuerzos para aumentar nuestra capacidad de conseguir m s muestras sin embargo las t cnicas de muestreo desarrolladas eran invasivas y por lo general requer an la disecci n de tejidos del organismo En los ltimos a os ha habido algunos avances en la extracci n qu mica del veneno cnidario utilizando etanol para activar el disparo de nematocistos Estos avances han llevado a la formaci n de este estudio que utiliza etanol para estimular los nematocistos en tent culos naturalmente automatizados mientras se observan bajo el microscopio de luz antes de medir el contenido de prote na mediante la microespectrofotometr a Este estudio se centra en una nica observaci n de Cnidaria que se desconoce en cualquier otro tipo de taxonom a de animales la autonom a pasiva del aparato de envenenamiento los tent culos Palabras clave Cnidario extracci n de veneno nematocistos microespectrofotometr a 187

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 188 Animal Technology and Welfare December 2019 INDICE DELLA REVISTA Seminario della RSPCA Congresso 2019 le 3R per stabularisti nel lavoro quotidiano PENNY HAWKINS e JULIET DUKES RSPCA Research Animals Department Wilberforce Way Southwater West Sussex RH13 9RS Corrispondenza research animals rspca org uk Studio basato sulla presentazione data in occasione del Congresso IAT 2019 Abstract Questo seminario si proponeva di incoraggiare gli stabularisti a promuovere attivamente e a favorire l implementazione delle 3R nell ambito del loro lavoro quotidiano all interno della struttura per animali L enfasi era sul perfezionamento refinement dal momento che questa R trae maggior beneficio dalle competenze e dai punti di vista degli stabularisti e del personale addetto alla cura degli animali Gli stabularisti possono incoraggiare e implementare il perfezionamento Portando nuove informazioni all attenzione di colleghi del Named Information Officer NIO e dell Animal Welfare and Ethical Review Body AWERB Suggerendo attivamente perfezionamenti e rendendosi disponibili a valutarli oni e naturalmente dovrebbero far parte dell AWERB e sostenere le sue iniziative Parole chiave Stabularista 3R perfezionamento refinement comunicazione 188

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 189 Paper Summary Translations Maggiore incidenza di crisi epilettiche spontanee nei topi da laboratorio stabulati in gabbie ventilate individualmente IVC ANNA MORGUNOWICZ CHELSEA CAVANAGH SOFYA NIKITOCHKINA e JULIA KEEBLE Biological Services King s College London Maurice Wohl Clinical Neuroscience Institute 5 Cutcombe Road Denmark Hill London SE5 9RX Corrispondenza anna morgunowicz kcl ac uk Studio basato sulla presentazione data in occasione di un Congresso IAT Abstract Nel corso dell ultimo decennio si assistiti a un notevole allontanamento dall uso di gabbie per roditori con tetto apribile verso quelle ventilate individualmente IVC I vantaggi sono stati molteplici e includono un controllo pi accurato dello stato di salute unitamente alla riduzione al minimo della contaminazione di animali immunocompromessi Nell ambito di King s College London KCL il trasferimento da gabbie con tetto apribile a gabbie IVC ha sembrato comportare una maggiore incidenza di crisi epilettiche spontanee nei topi La maggioranza di esse si osservata al momento dell apertura della gabbia in una stazione di cambio e pertanto si pensa che un notevole cambiamento negli stimoli ambientali possa contribuire all insorgenza degli attacchi epilettici Lo studio ivi proposto ha mirato a registrare l incidenza degli attacchi nei topi a seguito del trasferimento alle gabbie IVC valutando le differenze ambientali tra l interno e l esterno di tali gabbie con particolare enfasi sui livelli di rumorosit Lo studio evidenzia l aumento nell incidenza di crisi epilettiche spontanee nei topi stabulati in gabbie IVC sottolineando il bisogno di indagare l impatto dell ambiente sugli attacchi epilettici verificatisi tra gli animali da laboratorio al fine di garantire loro il massimo livello di benessere in futuro Parole chiave Topi crisi epilettiche gabbie IVC 189

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 190 Paper Summary Translations Estrazione non invasiva del veleno dei Celenterati tramite tentacoli autotomizzati PHILLIP J ROBINSON 1 3 STEVEN TRIM2 e CAROL TRIM3 1 2 3 The Deep Aquarium Kingston upon Hull HU1 4DP Regno Unito Venomtech Ltd Discovery Park Sandwich CT13 9ND Regno Unito School of Human and Life Sciences Canterbury Christ Church University Canterbury CT1 1QU Regno Unito Corrispondenza Carol Trim Canterbury ac uk Studio basato sulla presentazione data in occasione del Congresso IAT 2019 Abstract Gli animali che costituiscono il phylum dei Celenterati hanno origini che possono essere fatte risalire a circa 750 milioni di anni fa1 2 e pertanto rappresentano quella che potenzialmente la stirpe velenosa pi vecchia nota oggi Il phylum dei Celenterati che include anemoni di mare coralli e meduse anche uno dei meno studiati in termini di tossine probabilmente a causa delle difficolt nell ottenere campioni Negli ultimi due decenni ci si impegnati fortemente ad ottenere un numero pi elevato di campioni tuttavia le tecniche di campionamento sviluppate sono risultate invasive e generalmente richiedevano la dissezione dei tessuti dall organismo Pi di recente si assistiti ad alcuni sviluppi nell estrazione chimica del veleno dei Celenterati che fa ricorso all etanolo per attivare il firing di nematocisti Tali sviluppi hanno dato il via a questa ricerca che utilizza l etanolo per provocare la stimolazione delle nematocisti su tentacoli autotomizzati in modo naturale durante l osservazione al microscopio ottico prima di misurare il contenuto proteico tramite microspettrofotometria Questa relazione incentrata su un esclusiva osservazione dei Celenterati sconosciuta in altri ordini animali ovvero l autotomia passiva dell apparato di avvelenamento vale a dire i tentacoli Parole chiave Celenterati estrazione del veleno nematocisti microspettrofotometria 190

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 191 December 2019 Animal Technology and Welfare TECH 2 TECH Haven t the time to write a paper but want to get something published Then read on This section offers readers the opportunity to submit informal contributions about any aspects of animal technology Comments observations descriptions of new or refined techniques new products or equipment old products or equipment adapted to new use any subject that may be useful to technicians in other institutions Submissions can be presented as technical notes and do not need to be structured and can be as short or as long as is necessary Accompanying illustrations and or photos should be high resolution NB Descriptions of new products or equipment submitted by manufacturers are welcome but should be a factual account of the product However the Editorial Board gives no warranty as to the accuracy or fitness for purpose of the product Refining handling for small laboratory fishes CHLOE STEVENS RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Correspondence chloe stevens rspca org uk Based on an IAT Congress 2019 Platform Presentation Introduction and removal of the fish from the water causing potentially damaging severe or repetitive stress Refining the way that fish are handled may therefore help to reduce the stress that the fish experiences which in turn can help to improve fish health and welfare Ensuring that study animals are not stressed is also key to achieving reliable scientific data Fishes in captivity such as those used in laboratory research may experience handling on a regular basis Laboratory fish may need to be handled for a variety of reasons for example when being moved between tanks for use in experiments or for breeding for veterinar y treatment or when being packed for transportation Unfortunately handling is known to be stressful to fish 1 4 Refining handling Stress which is a general term for the suite of physiological psychological and behavioural changes in response to a challenge to homeostasis may not cause problems for an organism if the stressor is short lived However when stress is severe longlasting or repetitive it can start to have damaging effects including a drop in growth and reproductive rate suppression of the immune system and even death 5 In the laboratory handling is usually done with dip nets and involves pursuit confinement and capture So how can handling be refined and handling stress reduced Being removed from the water is often considered to be one of the more stressful elements of being handled so avoiding this or at least minimising the amount of time fish spend out of water is a good starting point 6 This approach is already being used in the aquaculture industry where some have started using fish pumps or transfer piping to move fish avoiding removing the fish from the water and limiting the amount of time the fish spends in a confined space 7 191

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 192 Tech 2 Tech Some studies have started to examine the effects of handling fish using standard dip nets or using methods which keep fish submerged in water when handled with somewhat mixed results In one study using a scoop to catch fish was found to cause lower ventilation rates in sticklebacks and Panamanian bishops suggesting they were less stressed but this was not the case for rainbow trout 8 However that study also found that Panamanian bishop fish Brachyrhaphis episcopi showed more anxious behaviour after scoop handling they were slower to leave a shelter and more reluctant to approach a novel object than net handled fish Another study found similar behaviours in three spined sticklebacks Gasterosteus aculeatus caught in a box rather than a net less willingness to leave a dark sheltered area or to approach a novel object 9 The results of these studies may seem contradictory at first but there are some possible explanations For example some research has suggested that physiological stress can lead to higher levels of risktaking behaviour Being willing to leave the shelter and approach novel objects would be considered risk taking behaviour so this might suggest that the net handled fish were more stressed than the scoop or box handled fish Another possible explanation is that fish which are experiencing more physiological stress might become worse at making decisions and so pay less attention to staying in shelters or avoiding novel objects However even though interpreting the results is not easy they do show that the way fish are handled can make a difference to fish stress levels and behaviour so this area is worth exploring further One aspect of handling which has not yet been considered is the behaviour and technique of the handler The training given to those handling fish in laboratories or other captive settings like the ornamental fish industry can be varied so the skill level of handlers is likely to also be varied This raises the question does the skill level of the handler affect stress levels in handled fish To answer this question we asked people who had never handled fish before to watch either a training video explaining good handling technique or a control video then asked them to catch fish from a tank Stevens et al unpublished data We saw definite differences in the technique used by trained and untrained handlers untrained handlers were much more likely to splash chase the fish and use sudden movements But this did not translate into higher stress levels in the handled fish However we also examined the behaviour of the fish left behind in the tank after the handling event and found that fish who had been exposed to but not handled by trained handlers showed less behavioural signs of stress than those exposed to untrained handlers The possibility that handling technique can affect the fish left in the tank is very important as a single fish could be exposed to hundreds of stressful 192 handling events in their lifetime The effects of a single handling event might even go further recent research has shown that zebrafish can experience emotional contagion that is zebrafish who could see stressful events happening in adjoining tanks can also become stressed and the effect is worse is the observer fish are familiar with the stressed fish 10 Anyone aiming to develop interventions to reduce handling stress in laboratory fish should therefore also consider the effects of their intervention on non handled fish Although there are currently few studies on refining fish handling the results of these studies suggest that improving handling methods can be beneficial for captive fish To help those looking to try and refine handling methods in their own facility here are some action points to consider Although more evidence is needed minimising the amount of time fish spend out of water when being handled is likely to benefit fish welfare Using slow calm movements and avoiding chasing fish is likely to be beneficial for the welfare of fish who are not themselves handled but are exposed to a handling event Removing fish from the water may increase the severity of procedures the fish experiences this should be discussed with the scientists working with the fish NTCOs may want to consider reviewing how people at their facility are trained to handle fish Remember that one size does not fit all different fish species may require different types of interventions to help reduce stress Consider the effect that stress from handling may be having on the science for example this may mean allowing fish longer recovery times after being handled for an experimental procedure When studying the effects of a handling intervention on fish stress and welfare consider measuring a range of parameters using only one indicator of stress can give an incomplete picture of the welfare of the animal References 1 2 3 4 Sneddon L U Wolfenden D C C and Thomson J S 2016 Stress Management and Welfare In Biology of Stress in Fish Fish Physiology pp 463 539 Academic Press Ramsay J M Feist G W Varga Z M Westerfield M Kent M L and Schreck C B 2009 Whole body cortisol response of zebrafish to acute net handling stress Aquaculture 297 157 162 Biswas A K Seoka M Takii K Maita M and Kumai H 2006 Stress response of red sea bream Pagrus major to acute handling and chronic photoperiod manipulation Aquaculture 252 566 572 Barton B A 2000 Salmonid fishes differ in their cortisol and glucose responses to handling and transport stress N Am J Aquac 62 12 18

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 194 Animal Technology and Welfare December 2019 Sperm cryopreservation and in vitro fertilisation in Zebrafish facilities at King s College London DIMITRA MANTZOROU THOM BERRIMAN WILL HAVELANGE JACQUELINE GLOVER SAM BERRY and BRUNO CORREIA DA SILVA Zebrafish Facilities Biological Services Unit Hodgkin Building King s College London Guy s Campus London SE1 1UL Correspondence dimitra mantzorou kcl ac uk Winner of the young presenters prize at the 2019 West Middlesex Branch Technicians Symposium Introduction King s College London KCL Zebrafish Facilities have the capacity to house 50 000 zebrafish Danio rerio There are currently 491 distinct lines of Zebrafish on the racks however not all lines are actively and constantly in use This created the need to establish an in house Sperm Cr yopreser vation and In Vitro Fertilisation programme At the time of writing KCL has successfully frozen 94 lines Sperm Cryopreservation is one of the most effective methods of large scale and long term storage of genetic material and offers multiple advantages such as extension of the reproductive period of an individual male Zebrafish the Reduction of the number of live Zebrafish in a facility a significant drop in cost and space required for maintenance of live populations prevention of loss of important and irreplaceable lines and finally allows the re introduction of any lost alleles back into the population 1 4 Sperm cryopreservation Sperm cryopreservation is scheduled to take place every two weeks on Wednesday mornings between 9 00 and 11 00 The first step to a successful Sperm Cryopreservation session is the selection of the male Zebrafish Ideally only male Zebrafish younger than 1 5 years old will be used for Sperm Cryopreservation because older Zebrafish tend to produce sperm of impaired quantity and quality Two weeks prior to a scheduled Sperm Cr yopreser vation session the selected males are set up in breeding pairs with wildtype females to breed The male Zebrafish that successfully fertilise eggs are considered to have good quality of sperm These male Zebrafish are separated from the females fed extra diet and are used in the next scheduled Sperm Cr yopreser vation session The evening prior to Sperm Cryopreservation gated breeding tanks are set up with 3 male Zebrafish which have 194 previously been selected on one side and 2 female wildtype Zebrafish on the other side as seen in Figure 1 and food is withheld for 12 hours to avoid regurgitation Figure 1 Gated Zebrafish breeding tanks with 3 male Zebrafish on one side and 2 female Zebrafish on the other side Materials The materials Figure 2 needed for collection of the sperm are G a microscope Figure 2 Set up for sperm collection

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 195 Tech 2 Tech G G G G G G G G G G a sponge with a slit a small tank anaesthetic solution analgesic solution a plastic spoon paper towels cotton swabs a post anaesthesia recovery tank microcapillary tubes microcapillary tube pipette controller The materials Figure 3 needed for sperm freezing are G G G G G G G G G G G a 200 l Gilson pipette 2ml orange lid cryovials a Styrofoam box with ice 0 5ml microcentrifuge tubes freezing medium a box with Dry Ice Ethanol Bath 15ml blue lid falcon tubes a Timer a cryovial freezing box a box with liquid Nitrogen a Liquid Nitrogen storage tank Figure 3 Materials used in sperm freezing Tip Only certified cryopreservation cryovials should be used to freeze the sperm at the correct rate Sperm cryopreservation technique The Cryopreservation technique is time sensitive and requires two experienced technicians to run efficiently The technicians carry out the following steps 1 Technician 1 prepares the anaesthetic and the analgesic solution Analgesia is added to the breeding tanks and to the recovery tank 30 minutes prior to the procedure Stock solutions and dosage rates can be found in Table 1 2 Technician 2 prepares a box with Dry Ice Ethanol bath the tubes with 145 l freezing medium solution details on preparation can be found on Table 1 the box with liquid Nitrogen containing a cryovial freezing box labels the cryovials and prepares the Record books Tip It is important that the freezing medium is prepared fresh before each Sperm Cryopreservation session 3 4 5 Technician 1 prepares the microscope set up as seen in Figure 2 Once prepared Technician 1 immerses 2 3 male Zebrafish to be anaesthetised into the anaesthetic solution Once the fish lose balance and become immobile the Technician checks that they are fully anaesthetised by gently touching with the spoon monitoring for a reflex response Technician 1 removes one anaesthetised fish from the anaesthetic solution with the plastic spoon head facing towards the handle of the spoon and tilts the spoon slightly so that excess water is removed The fish is dried carefully with paper 195

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 196 Tech 2 Tech 6 towel especially around the urogenital pore and placed in the slit of the sponge Technician 1 gently spreads the anal fins with a cotton swab to expose the urogenital pore A microcapillary tube attached to the microcapillary tube pipette controller is placed at the urogenital pore and gentle abdominal pressure is applied to obtain sperm Figure 4 These steps are repeated for the next male Zebrafish 8 Usually sperm is pooled together from 2 3 male Zebrafish with a target sperm volume of 3 3 l and minimum accepted volume of 1 5 l 5 9 Technician 2 hands Technician 1 a 0 5ml microcentrifuge tube containing 145 l of freezing medium into which the sperm is expelled 10 Technician 2 pipettes up and down once to mix and then aliquots 35 l of sample into four cryovials then places each cryovial into a pre chilled falcon tube in dry ice and sets a timer for 30 minutes Tip It should only take 30 seconds from the moment the sperm touches the freezing medium to the moment the cryovial is placed in the falcon tube in the dry ice 11 When the time elapses Technician 2 quickly transfers the cryovials into the cryovial freezing box with liquid nitrogen In vitro fertilisation IVF At King s College London Zebrafish In Vitro Fertilisation IVF is used to assess if the Sperm Cryopreservation has been successful It can also be used to recover a frozen line or to get a new generation when the parents do not spawn IVF is scheduled to take place in the main facility every two weeks alternating with Sperm Cryopreservation on Wednesday mornings between 9 00 and 11 00 The first step to a successful IVF is the selection of the female Zebrafish Three stocks of female wildtype Zebrafish are used in rotation for the IVF procedures Two weeks prior to a scheduled IVF session the selected females are set up in breeding pairs with wildtype males to breed The female Zebrafish that successfully lay healthy fertilised eggs are selected These female Zebrafish are separated from the males fed extra diet and will be used in the next scheduled IVF The evening prior to IVF gated breeding tanks are set up with 3 female Zebrafish on one side and 2 male Zebrafish on the other side and food is withheld for 12 hours to avoid regurgitation The materials Figure 5 needed for In Vitro Fertilisation are G G G G G G Figure 4 Collection of sperm from male Zebrafish G G 7 196 Technician 1 transfers the fish into the recovery tank containing fresh system water with analgesia monitoring the fish to ensure that it recovers fully G G G a microscope a small tank anaesthetic solution analgesic solution a plastic spoon a post anaesthesia recovery tank 90mm petri dishes 200 l AquaBoost OvaCoat solution per petri dish a fine brush paper towels a box with liquid Nitrogen

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 197 Tech 2 Tech G G G G a heat block set at 37 C parafilm cut into squares E3 Medium solution fructose solution 8 solution without activation for 30 minutes This gives enough time to perform IVF with frozen sperm Technician 2 removes the excess OvaCoat solution from the eggs with some paper towel carefully Figure 5 In vitro fertilisation set up IVF technique IVF is carried out by two experienced Technicians to run efficiently The Technicians carry out the following steps 1 2 3 4 5 6 Figure 6 Female Zebrafish immersed in a tank with anaesthetic solution Technician 1 prepares the anaesthetic and the analgesic solution Analgesia is added to the breeding tanks and to the recovery tank 30 minutes prior to the procedure Stock solutions and dosage rates can be found in Table 1 Technician 2 prepares the E3 Medium solution fructose solution adds 200 l of OvaCoat solution to each petri dish and sets the heat block at 37 C Technician 1 prepares the microscope set up as seen in Figure 5 Once prepared Technician 1 immerses 2 3 Zebrafish to be anaesthetised into the anaesthetic solution Figure 6 Once the fish lose balance and become immobile the Technician checks that they are fully anaesthetised by gently touching with the spoon monitoring for a reflex response Technician 1 removes one anaesthetised fish from the anaesthetic solution with the plastic spoon head facing towards the handle of the spoon and tilts the spoon slightly so that excess water is removed The fish is dried carefully with some paper towel especially around the urogenital pore and placed into a piece of parafilm Figure 7 Technician 1 then applies gentle abdominal pressure to obtain the eggs Good egg clutches are transferred into the OvaCoat solution in the petri dish with a fine brush Figure 7 Tip Big egg clutches improve fertilisation rates 7 The eggs can stay hydrated in the OvaCoat Figure 7 Collection of eggs from an anaesthetised female fish 197

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 198 Tech 2 Tech Anaesthetic Solution The stock solution of 0 4 MS222 Tricaine methanesulfonate is made up with 1000ml RO water 4g MS222 powder 8g sodium bicarbonate NaHCO3 For anaesthesia To make a 0 02 anaesthetic solution Dilute to a 1 20 solution e g add 10ml of 0 4 MS222 to 190ml System Water Analgesic Solution 530mg Lidocaine hydrochloride powder into 1L of system water This analgesia stock solution is ready to use at a dose rate of 7ml per litre Freezing Medium 10 DMA N Ndimethylacetamide TOXIC final in BSMIS Add 200 l of DMA to 1 8ml BSMIS and vortex for 10 minutes BSMIS 4 383g NaCl 5 218g KCl 0 218g CaCl2 2H2O 0 246g MgCl2 6H2O20ml 1M Tris pH 8 0 Fructose solution 0 5 Fructose in 1XE3 medium 0 5g Fructose in 100ml 1X E3 Medium E3 Medium 16 5ml 60X E3 made up to 1L distilled water 60x E3 Medium 17 4g NaCl 0 8g KCl 2 9g CaCl2 2H2O 4 89g MgCl2 6H2O In 1L distilled water pH 7 2 OvaCoat Solution AquaBoost OvaCoat from Cryogenetics AS Table 1 Solutions used in sperm cryopreservation and in vitro fertilisation Technician 2 removes one cr yovial from the Liquid Nitrogen opens the cap tips out any liquid Nitrogen within the vial and places the cryovial into position in the heat block 10 Just before the sperm is fully defrosted Technician 2 removes the cryovial from the heat block and adds 400 l of fructose solution The activated sperm is immediately added to the eggs and a timer is set for 2 minutes 11 After 2 minutes Technician 2 floods the dish with E3 Medium solution and the eggs are transferred to the incubator at 28 5 C Later in the afternoon the Technicians check the petri dishes removing any debris and splitting the dishes in half to reduce the embryo density in each petri dish 12 The following morning Technicians 1 and 2 check the eggs under the microscope and record the fertilisation rate For sperm to be successfully preser ved we look for a minimum of 10 fer tilisation rate minimum of 100 fer tilised embryos out of 1 000 eggs At KCL in 2019 the average fertilisation rate from IVF procedures was 25 9 Summary In keeping with the principles of the 3Rs the Sperm Cryopreservation Programme at KCL has significantly reduced the number of live Zebrafish in the facilities with a subsequent decrease in cost and space needed for the maintenance of live populations Moreover Sperm Cryopreservation acts as a gene bank and a back up that prevents the loss of impor tant irreplaceable lines and allows the re introduction of any lost alleles back into the population To date KCL have successfully frozen 94 lines Cryopreservation success is evaluated through In Vitro Fertilisation which can also be used to recover a frozen line or to get new generations when parents no longer spawn Success depends on experience attention to detail and time management References 1 2 3 9 Tip The defrosted sperm should be added to the eggs as fast as possible 198 4 5 Carmichael C Westerfield M and Varga Z M 2009 Cryopreservation and in vitro fertilization at the Zebrafish International Resource Center Methods in Molecular Biology Clifton N J 546 45 65 http doi org 10 1007 978 1 60327 977 2_4 Dooley C M Scahill C F nyes F Kettleborough R N Stemple D L and Busch Nentwich E M 2013 Multi allelic phenotyping a systematic approach for the simultaneous analysis of multiple induced mutations Methods 62 pp 197 206 doi 10 1016 j ymeth 2013 04 013 Matthews J L Murphy J M Carmichael C Yang H Tiersch T Westerfield M and Varga Z M 2018 Changes to Extender Cryoprotective Medium and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation Zebrafish 15 3 279 290 doi 10 1089 zeb 2017 1521 Epub 2018 Jan 25 Yang H and Tiersch T R 2009 Current status of sperm cryopreservation in biomedical research fish models Zebrafish medaka and Xiphophorus Comp Biochem Phys C 149 224 232 doi 10 1016 j cbpc 2008 07 005 Draper B W and Moens C B A 2009 High Throughput Method for Zebrafish Sperm Cryopreservation and In Vitro Fertilization J Vis Exp 29 e1395 doi 10 3791 1395

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 04 Page 199 December 2019 Animal Technology and Welfare POSTER PRESENTATIONS Originally presented at IAT Congress 2019 Darwin s fishes keeping Malawi Cichlids JENNIFER BARTLEY University Biomedical Services sub department of Animal Behaviour High Street Madingley Cambridge CB23 8AA Correspondence jjb89 cam ac uk Introduction Cichlids in Lake Malawi have evolved different phenotypes However at the level of the genome they look very similar so research is being carried out on these fish to try and understand genome evolution African cichlids are some of the most colourful of all freshwater fish they offer a wide variety of body shapes and behaviours They are extremely active and Figure 1 Rhamphochromis chilingali can be very personable showing behaviours such as greeting technicians and begging for food This poster will provide information on the cichlids including their dietary and husbandry requirements and the breeding of these animals Information Cichlids have evolved rapidly into a large number of closely related but morphologically diverse species within large lakes There are 850 different Malawi cichlids and all of these are more similar at the genome level than humans All cichlids have some form of parental care for their eggs and fry That parental care is either guarding the eggs or mouthbrooding They have a wide range of body size from 2 5cm to 1m however the majority tend to be medium size and an oval shape Their average life span is 4 10 years but can live to 15 years Cichlids are omnivores Many cichlids are primarily herbivores feeding on algae and plants others are predatory and eat very little or no plant matter instead catching other fish eggs or young Cichlids are an aggressive species establishing territory and hierarchy through evaluating their competitors The most dominant males will have more vivid and bright colouration and subordinate males will assume a dull colour Housing requirements Figure 2 Tropheops Sp mauve Kept at 25 C 199

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 200 Poster Presentations 12 hour light dark cycle Water quality is monitored weekly Weekly water changes 20 Nets used for catching fish are kept in a 4 bleach solution Enrichment such as plants tunnels bricks and plant pots Sand substrate for tank floor Dechlorinated water Air stones and water filters Higher ratio of females to males to reduce aggression Omnivore food 5ml Novo Cichlid mix 2 5ml per cross tank Carnivore food 4 5ml Novo Cichlid mix 2 5ml black soldier fly pellets Figure 3 Tank set up Research The genetic and developmental basis of morphological variation in cichlid fish is currently being researched The focus is on understanding what makes species different at the DNA level and associating specific portions of the DNA to specific pigmentation patterns Studies are examining if it is possible to use in vitro fertilisation to create crosses between cichlid species with different colours and targeting 15 genes to determine which underlie cichlid colour differences Breeding Cichlids either mate monogamously or polygamously In the wild they will lay their eggs in caves or crevices so providing a plant pot will help to encourage these natural behaviours The courting process may vary slightly for different species but is similar for most of the mouthbrooding cichlids The male will chase the females around the tank attempting to lure one to a spawning area either a plant pot provided or a sandy area that the male will dig out by shaking his body The male and female will swim in circles in the spawning area Once the female lays her eggs she will immediately try and scoop them up into her mouth Most adult males exhibit a unique pattern of ovalshaped colour dots on their anal fin The male will gyrate his anal fin which leads the female to believe the spots are her eggs She then opens her mouth to the anal fin and he discharges sperm into her mouth and fertilises the eggs After mating the female will have a mouthful of Figure 4 Calliptera salima male with the plant pot enrichment Figure 5 Example of room set up 200 Figures 6 7 Mouthbrooders with a dropped lower jaw

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 203 December 2019 Animal Technology and Welfare Stiff as a board measuring rigor mortis in Zebrafish KAREN DUNFORD JENNA HAKKESTEEG and CAROLE WILSON University College London Zebrafish Facility Division of Biosciences Gower Street London WC1E 6BT Correspondence k dunford ucl ac uk Aim To determine the rate of rigor mortis using different anaesthetic agents Introduction Schedule one killing S1K methods require a two step process a humane method of death typically for Zebrafish an anaesthetic overdose and confirmation of death such as confirmation of rigor mortis There is widespread variation of anaesthetics used for S1K and there is debate about refining anaesthesia for Zebrafish as adverse effects are becoming a concern Anecdotal evidence suggests that different anaesthetics can inhibit or reduce the rate of the onset of rigor mortis with some taking an hour whilst others take more than three We conducted a trial in order to determine the rate of rigor mortis for four different agents Rigor mortis Rigor mortis RM is a stage of death characterised by muscle stiffness Following death lactic acid accumulates decreasing cellular pH 1 This disrupts glycolysis and in turn adenosine triphosphate ATP levels fall which breaks actin myosin cross bridges Its depletion results in prolonged muscle stiffness Figure 1 The rate of onset and duration of RM is dependent on many factors size temperature and pre mortem exhaustion Studies on anaesthetics used in Zebrafish are limited and the effects on RM are mostly unknown Four common anaesthetics are Benzocaine lidocaine 2 phenoxyethanol 2 PE and MS 222 Figure 2 Of these lidocaine benzocaine and MS 222 all reversibly bind to voltage dependent sodium channels 2 This inhibits Na uptake which stops the initiation and propagation of action potentials at the site of pain The mechanism of action for 2 PE is currently unknown in fish Figure 2 The chemical structure of each anaesthetic used Left to right Benzocaine 4 Aminobenzoic acid ethyl ester Ethyl 4 aminobenzoate chemically similar to MS222 Lidocaine hydrochloride MS222 Ethyl 3aminobenzoate methanesulfonate 2 Phenoxyethanol Attributions Benzocaine Mykhal Public domain Tricaine Edgar 181 Public domain 2Phenoxyethanol Lidocaine hydrochloride Prisonblues at the English language Wikipedia CC BY SA 3 0 http creativecommons org licenses by sa 3 0 Methods A total of 57 hybrid AB TupLF 10 month old adult fish of the same stock were used The fish were maintained in a recirculating 10L tank 28 2 28 4 C pH 7 1 7 3 in 14 10 hour light dark cycle and fed on a combination dry food diet All fish used were of similar size for each Figure 1 A Zebrafish that is in rigor mortis Table 1 The average weights of each sex The females weighed more overall 203

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 204 Poster Presentations respective sex Table 1 The fish were scheduled for euthanasia according to S1K methods as found in the Animals Scientific Procedures Act 1986 ASPA The 2PE dose followed UCL protocol whilst the other three were based on published recomendations Table 2 Table 2 Anaesthetic doses The time of no observed movement is the time of assured death which occurs after respiration ceases transferred to labelled petri dishes with the assigned post mortem media At 30 minute intervals each fish was held by the caudal peduncle and measured against a protractor to measure the angle Figure 3 Figure 3 Every 30 minutes the angle of each fish was measured using a protractor to determine onset stage of rigor mortis Most fish reached 0 Results Table 3 Lidocaine was too adverse the fish did not lose consciousness quickly enough The time points for the measured angles for each treatment were averaged and plotted to compare the rate of RM in each anaesthetic The most notable difference was 2 PE stay which reached 0 at a faster rate than Benzocaine and MS 222 2 PE reached 0 at approximately the three hour mark whereas the other two took more than five hours The maximum angle was also reached faster in the 2 PE remove media but at a slower rate Figure 4 The different post mortem media The fish in 2 PE Stay right reached the maximum angle at a faster rate than MS 222 or Benzocaine Figure 2 Fish immersed into overdose anaesthetic 10 minutes after observed death transferred into glass petri dish filled with system water remove or transferred to dish with same anaesthetic stay Lidocaine was excluded from the trial as it was deemed too adverse to use Table 3 these fish were removed from the trial and euthanised according to UCL protocol Each anaesthetic was tested into two different post mortem media removed into fresh water and staying in dosed water Figure 2 For each treatment three fish were added to the predosed water and left for ten minutes to ensure death After a knock test to ensure death individual fish were 204 Each anaesthetic was individually plotted to compare the post mortem media and its effect on the rate of RM

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 205 Poster Presentations lowered blood oxygen increased CO2 and reduced pH 3 Both high levels of cortisol and decreased pH and glucose lower the rate of glycolysis decreasing ATP which directly increases the rate of RM The potential increased stress that these animals experienced during euthanasia should be of great concern Although the data collected here is focussed on non protected animals i e dead a potential application for this information is a refined use of S1K anaesthetics for Zebrafish The exclusion of Lidocaine from the trial is a good example it is assumed that a quick death is the ideal but for more than two minutes the fish did not exhibit desirable anaesthetic depth unlike the other agents which did so into in less than two minutes Conversely 2 PE took 30 seconds yet there is now a question about cortisol stress present with this agent It is possible that we did not wait long enough in our trial to see sufficient effects from Lidocaine for S1K As a result of this we need to establish if a slower possibly less painful death would be more ethical than aiming to achieve the quickest possible induction of death Further work Figure 5 The three anaesthetics compared to the two post mortem media stay and remove The most obvious difference was 2 PE which still reached the maximum angles in the stay medium before removed Discussion It was noted during the trial that the fish euthanised with 2 PE exhibited signs of death sooner than the other anaesthetics by approximately 30 seconds Table 2 Additionally RM was induced faster with 2 PE compared to the other agents regardless of postmortem media Figure 4 It is unlikely that the small acceleration of death by 2 PE caused such an increase in the onset of RM Figure 5 Therefore we must question the possible changes in cellular physiology by 2 PE There are potential variables that were not explored or explored enough in this trial such as strain dosage and effect of sex weight This will be addressed in a repeat trial Other factors that affect stress will be controlled further such as handling and health We will also attempt to establish if the increased rate of RM with 2 PE is from the agent or the increased dosage The collected data may aid the refinement of S1K Acknowledgements Many thanks to the UCL Fish Facility staff H Calloway P Barwood E Hitchcock R Davies Green J Warmsley T Wheeler D Marks and V Moiche References 1 When comparing the post mortem media it is clear that 2 PE allows for the maximum angle to be achieved first when the fish stay in the anaesthetic Benzocaine also appeared to work faster when the fish stay rather than are removed Figure 5 Surprisingly MS 222 showed little if any difference between the media although they both work similarly at the chemical level there is the suggestion that there is even a larger difference between them than initially assumed at the beginning of the trial 2 3 Nazir D and Magar N 1962 Biochemical changes in fish muscle during rigor mortis Collymore C Tolwani A Leiggi C and Rasmussen 2 2014 Efficacy and safety of 5 anaesthetics in adult Zebrafish Danio rerio Journal of the American Association of Laboratory Animal Science 53 2 pp198203 Hedayati A A et al 2016 Effects of 2 phenoxyethanol 2 PE anaesthesia on some haematological and biochemical indices of Silver carp Hypophthalmichthys molitrix The dosages may also play a role Benzocaine and Lidocaine dosages are quantitatively small compared to 2 PE Table 2 Studies on other species suggest possible side effects of 2 PE raised cortisol and glucose levels pre mortem 205

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 206 Animal Technology and Welfare December 2019 Why Zebrafish RHIANNON DAVIES GREEN and CAROLE WILSON University College London Zebrafish Facility Division of Biosciences Gower Street London WC1E 6BT Correspondence rhiannondaviesgreen gmail com Aim To determine if the lack of distinct facial features in fish influences people s thoughts in species model Introduction Although fish are currently the second most used vertebrate model in research it is debatable whether the same amount of care and consideration is given to them Even the Animals Scientific Procedures Act 1986 ASPA Guidance does not consider them as sentient as other species In this preliminary work we hypothesise that this perception is in part due to the lack of facial muscles and structures that are common to all mammalian models making it difficult to empathise with them and giving them a remoteness which is not seen in and mammalian species Because Zebrafish cannot make facial expressions as mammals do it makes it extremely difficult to determine how much pain suffering and distress they are experiencing In this work we explore whether subtly altering facial expression as have an impact on people s thoughts on pain and suffering in Zebrafish and if this is related to their lack of facial expressions impacting on people s level of empathy We then go on to further examine the various reasons as to why people may or may not choose to work with Zebrafish in research Figure 1 A rodent grimace scale detailing the types of diagnostic features to look for when identifying pain suffering distress and lasting harm All pictures indicate a higher level of pain moving from left to right Top row nose and cheek flattening Middle row orbital eye tightening Bottom row whiskers tightening Grimace scale images reproduced with permission from the end NC3R s Grimace scales Grimace scales have been created for various mammalian species in the research industry in order to help those working with animals detect how much pain suffering and distress they are in Figure 1 A grimace scale is composed of various photographs of the species concerned showing dif ferent facial expressions and what they mean However a grimace scale does not exist for Zebrafish due to their inability to visually express their pain suf fering and discomfort Methods In order to find out the possible calls for people choosing to work with fish rather than mammals in research we collected and interviewed four groups of four people from different areas in the industry including researchers who had worked with fish technicians working with fish technicians that work with mammals and a group of people who do not work with animals at all We then created a false grimace scale for Zebrafish in an attempt to mimic rodent grimace scale figure 2a This was done by manipulating close up photographs of zebrafish multiple times to give the fish various distinct facial expressions 206

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 207 Poster Presentations facial expression as we well as three photographs taken from a rodent grimace scale Figure 3 Figure 2a The edited Zebrafish pictures used for the interviewees to rank in order of pain These are based on the orbital tightening portion of alien grimace scale A obvious or the highest pain B zero or no pain C moderate on some pain present in the animal Zebrafish do not have eyelids and therefore this representation is entirely fictional Figure 2b The questionnaire used to conduct interviews for those work marking the pictures Finally we compiled and analysed our data in order to compare what people from different backgrounds in the research industry thought and how they differed from one another Results We compiled the results from each group and found that 93 7 of the interviewees guess correctly when ranking the manipulated fish photographs as conveying the most pain 68 75 guess correctly when ranking the mice while 75 ranked the unedited fish as identical Figures 4 5 Figure 4 The overall number of correct and incorrect answers provided by each of the four groups laymen animal technicians fish technicians and fish researchers The animal technicians provided the most correct answers whilst the laymen provided the most incorrect Fish technicians and researchers were equal Following this we designed several questions which involved whether Zebrafish or rats had a higher percentage of use as models in research and the possible reasons behind it also whether the interviewee would personally prefer to work with fish or mammals and why Figure 2b Par t of the questionnaire also involve the individuals looking at edited photographs of Zebrafish and ranking them from 1 to 3 one being the least amount of pain and three being the most and then repeating this exercise with three photographs of fish with no fish difference in their Figure 5 The correct answers provided by each group by each category All fish technicians were able to eye up identify the unedited fish photographs whilst only some of the other groups correctly identified that the pictures were unedited Figure 3 The unedited pictures of Zebrafish The only difference between these three is the background Interviewees were asked to rank them in order of least pain to most When asked which had the higher research usage 100 of aquatic and technicians working with mammals believe that fish were more used over rats However both researchers and laymen were split 50 Finally when the interviewees were asked if they would 207

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 208 Poster Presentations prefer to work with fish or mammals the results were very mixed 100 of researchers said they would prefer to work with fish whereas for both aquatic technicians and laymen 75 preferred to work with fish as did 50 of technicians currently working with mammals Most interviewees correctly identified the unedited pictures as identical and the altered backgrounds had no effect We found most people were unable to assess pain in a fish purely based on expression as they do not have the same mammalian facial muscles Discussion We also wanted to determine if people chose zebrafish over mammals as fish inspire less empathy and therefore are easier to work with on an emotional level as they inspire less guilt When ranking the edited Zebrafish images the inter viewees tended to give anthropomorphic responses using emotional words like unhappy quote to back their choices rather than looking at physical characteristics When asked to rank levels of pain and suffering in the fish the manipulated images induce more emotive responses perhaps because the images were edited to mimic mammalian grimace scale which helps in grading the images as they were more relatable When ranking the mice images we expected the animal technicians to judge correctly as they health check mammals The majority of the other groups also guessed correctly perhaps as much as mammalian species this is intuitive as people understand features of pain and suffering in other mammals This suggests people find it easier to relate to mammals due to the similarities between facial expressions Words clouds based on the interviewees indicate that most had an emotive response to mammals as well as projecting emotions onto them Conversely fish was spoken positively in relation to ease of use and process Many interviewees gave various scientific reasons for working with fish such as an easy embryo collection and faster developmental stages But people felt less empathy and detached from fish and mostly provided negative words Figure 6 Further work In order to follow on from this concept we intend to develop a questionnaire using thematic analysis this will entail making open ended questions designed to be more probing which will improve the depth of the answers given The further topics to include would be the emotional toll on those who work with all vertebrates and why people equate pain with emotion in people but not in animals Related to this is the idea that sentience in animals only concerns the ability to feel pain yet in humans is connected to the levels of intelligence The question of an effective fish grammar grimace scale needs to be further explored Acknowledgements Many thanks to the UCL fish facility staff K Dunford H Calloway P Barwood E Hitchcock J Warmsley T Wheeler Reference 1 Figure 6 The two word clouds top and bottom The top shows the responses concerning the mammals The orange represents the emotional responses people felt about the animals and the green represents how they perceive the animals were feeling The bottom shows the responses to the fish with the red representing the responses that people feel about the fish and the brown showing technical terms used to discuss the fish No group provided an emotion provided emotional terms to represent the experience of the fish 208 Sotocinal S G Sorge R E Zaloum A Tuttle A H Martin L J Wieskopf J S et al 2011 The Rat Grimace Scales a par tially automated method for quantifying pain in the laboratory rat via facial expression Molecular Pain 7 55

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 December 2019 14 05 Page 209 Animal Technology and Welfare You are what you eat ELISE HITCHCOCK CAROLE WILSON PAUL BARWOOD and VISILA MOICHE University College London Zebrafish Facility Division of Biosciences Gower Street London WC1E 6BT Correspondence e hitchcock ucl ac uk Aim To identify any recurring differences between fish fed different diets Introduction Knowing the best techniques for healthy animals is important for all animal technicians especially when it comes to feeding Therefore UCL continued to explore a trial published in Animal Technology and Welfare which examined four commercially available feeds Figure 1 and the ways they affected University College London UCL AB Zebrafish Danio rerio 1 The previous trial used a feed which caused deformities commonly in the skull and caudal vertebrae due to this the use of the feed was terminated Specimens from fish fed this feed were fixed for bone staining for further study in order to understand the nature of the deformities Figure 2 The setup for photographing and measuring the fish Photographs were uploaded to Image J and distances measure using pixels The fish from each diet were analysed in respective sexes due to a difference in sex ratios between the previous trial and the current this affected the overall sample sizes when it came to comparative analysis Figure 1 The dry food diets used for the trial Diet F is a combination of Diet A B and C Methods Viable AB embryos were generated from various stock randomised and split into four trial groups one for each of the three feeds and one for the combination feed At five days post fertilisation dpf and subsequently in four or five day intervals until 56 dpf a random sample from each group was photographed to produce length data over time Figure 2 At day 56 the fish were sexed and evenly distributed Figure 3 At day 77 dpf a random sample of both sexes from each group was weighed and photographed to correlate with length and width data Figure 3 Comparison of sex ratios females dark bottom males light top between the previous trial light left and this trial dark right 209

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 210 Poster Presentations Bone staining Deformed fish from the previous trial were culled according to Schedule 1 methods and fixed for further study via bone staining over a five day period 2 A preliminary test of the technique with Alizarin Red was conducted on a healthy fish Figure 4 this was subsequently used as a reference and comparison to other samples caudal vertebrae Figure 6 compared to a healthy fish and fish fed other diets Chang et al 2018 found that zebrafish vertebrae become brittle thicken and calcify overtime leading to deformities such as lordosis Figure 8 3 The frequency of deformities observed with increasing age is likely because these physio chemical composition progressions Figure 4 Healthy fish bones stained with Alizarin Red Another healthy fish were gutted which caused the fish to lose it rigidity during glycerol storage and stained with Alcian Blue for car tilage staining but was overexposed and overstained This appears to have emphasised the bones further Figure 5 Figure 8 A particularly disfigured individual from Diet B exhibiting lordosis The bone staining technique greatly increases the exact deformities present Results Shapiro Wilk and Kolmogorov Smirnov tests were conducted to confirm normality all data within groups were not significantly dif ferent p 0 05 2 way ANOVAs were conducted to identify dif ferences between diets Figure 5 Healthy fish bone stained with Alcian Blue for cartilage staining In this trial there was a significant effect on growth between diet C and all other diets p

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 213 December 2019 Animal Technology and Welfare A technologist led approach to altering the culture of care regarding blood sampling NICHOLAS KAYE The Francis Crick Institute 1 Brill Place London NW1 1BF Correspondence nicholas kaye crick ac uk Abstract This poster will describe the implementation by Animal Technologists and Named Persons of a wellpublished refined bleeding technique Establishing The Francis Crick Institute from legacy institutes required a standardised approach for many techniques including blood sampling which is essential for good practice The Named Veterinar y Surgeon NVS taught the Saphenous vein method to technologists as an alternative to bleeding from the tail vein This poster will cover how we moved from previous methods the reasons why and how this was then introduced as par t of a technician led culture of care In black or pigmented mice it can be more difficult to see the tail vein through the skin This can lead to missing the vein and having to re puncture the tail The hot box was still required this increases the length of the procedure resulting in extra time for the animal outside its home cage Increased handling and pulling on the tail can potentially cause stress to the animal during the procedure Why not continue to use tail bleeds as the standard The concept of the 3Rs and especially Refinement give us a responsibility to constantly review existing techniques and assess if methods in use are still the most appropriate Using a scalpel lancet Figure 1 The tail bleed procedure Tail nicks were previously being carried out using a scalpel or lancet and we found the following concerns when we were using that method Repeated sampling can cause scarring to the tail especially if a scalpel is being used When performing the tail nick with a scalpel it can be difficult to get the correct amount of pressure needed for the initial cut in order to get enough blood without too much pressure as to result in damaging the tail and causing subsequent scarring Animals need to be warmed in a hot box prior to the procedure in order to dilate the blood vessel resulting in an extended period of time before the flow of blood stops after sampling Using a needle We tried refining the tail bleeding technique by changing to using a needle to prick the vein instead of a scalpel but we still had the following concerns Figure 2 Cross section of a mouse tail 213

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 214 Poster Presentations What alternative methods are available From performing the Saphenous bleeding technique we found that this method had several advantages Whilst bleeding from the tail vein is still a suitable option there are a variety of different alternatives although not all would be applicable as a standardised technique The Saphenous vein is easy to see in all coat colours This allows the Personal Licence PIL holder to be more confident performing the venous puncture required for sampling Either leg can be used allowing repeat sampling alternating between legs providing time for each leg to fully heal No heating of the animal is required eliminating the need for a heat chamber The bleeding stops quickly as the vein is not dilated by heating Large volumes of blood can be taken from the leg continuously with little coagulation From the list of available techniques we wanted to use one that did not require the use of anaesthetic Our NVS and a few Animal Technologists had previously used the Saphenous vein bleed so we decided to trial it for suitability as a standardised technique in our unit General anaesthesia not required General anaesthesia required General anaesthesia required non recovery G Saphenous vein G Sublingual vein G Cardiac puncture G Tail vein G Saphenous vein G Mandibular vein G Retro orbital G Tail snip G G Abdominal thoracic blood vessel G Retro orbital G Decapitation Blood vessel cannulation Figure 3 Blood collection techniques Advantages we found of using our chosen method of Saphenous vein bleeds Figure 5 Saphenous vein puncture Reduced handling of the animal from the tail may lead to a reduction the stress to the animal during the procedure Figure 6 Mouse restrained for Saphenous bleed procedure Wounds on the legs heal quickly with little to no scarring compared to what has occasionally been seen when using the tail Figure 4 Mouse leg circulatory system 214

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 215 Poster Presentations further We are looking into creating a restrainer that incorporates a thinner red plastic similar to a 50ml falcon tube and to have a variety of sizes so the most appropriate size can be used for each animal Figure 7 Wound just after procedure has finished and the bleeding has stopped Figure 9 Different sized restrainters used Technologist driven culture of care The differences and improvements of the Saphenous vein technique compared to previous methods led to it becoming the only method used in our unit where appropriate Figure 8 Healed wound 24 hours post procedure Refinements within the procedure From doing this procedure we have made further refinements This has included using higher or smaller gauge needles depending on the volume of blood required We also use different sized restraining tubes depending on the size of the mouse so that it is not too tight or too loose We found that the technician can easily and quickly restrain the animal Further refinements The restrainer we currently use can be optimised Figure 10 Blood collection presentation Scientific users were then encouraged to use this method over alternative techniques The majority of Scientific users who have been shown and taught this technique now prefer it to previous methods used this is due to the advantages of the Saphenous vein bleeding technique listed in this poster 215

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 Poster Presentations G G G G cardiac puncture abdominal thoracic blood vessel retro orbital decapitation Technologists and Scientific Users have introduced this method to visiting collaborators from partner institutes who are working on projects using blood collection highlighting the benefits to research and animal welfare Technologists have given talks internally to the whole of the Biological Research Facility on blood collection via the Saphenous vein explaining the benefits and encouraging others to take up the technique if suitable Future steps to take We will to continue to highlight the benefits of using a different procedure for blood collection to the Scientific Users and Technologists who use our facilities and standardise the technique where possible Looking forward we would like to present at symposia and inspire others to adopt this method for use in their own institutes Conclusion Although not a new technique this method demonstrates how Animal Technologists play an essential role in implementing and demonstrating good practice whilst promoting a culture of care Championing this technique at The Francis Crick Institute has highlighted the importance of Animal Technologists working closely with Scientific Users to promote methods of refinement and consistency of techniques especially when different establishments and processes are involved Acknowledgements Clare Brazill Adams Lucy Fern Yolanda Saavedra Torres Helen Bailey Luke Hitchen and Jamie Delicata References Figure 1 www nc3rs org uk mouse tail vein non surgical Figure 2 www researchgate net figure Demonstration ofmouse restraint location of blood vessels and positioning ofneedle_fig1_320737499 Figure 3 www nc3rs org uk mouse decision tree bloodsampling Figure 4 www informatics jax org cookbook figures figure 102 shtml 216 14 05 Page 216

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 December 2019 14 05 Page 219 Animal Technology and Welfare MR and CT compatible electrical heating system for mouse imaging STUART GILCHRIST VEERLE KERSEMANS PHILIP ALLEN ANA GOMES SHEENA WALLINGTON PAUL KINCHESH and SEAN SMART CRUK MRC Oxford Institute for Radiation Oncology Department of Oncology University of Oxford Old Road Campus Research Building Roosevelt Drive Oxford OX3 7DQ Correspondence sheena wallington oncology ox ac uk Introduction Active heating is required to avoid hypothermia in anaesthetised animals Small resistive MR compatible heaters are useful where space limitations prevent the use of circulating fluids but computerised tomography CT imaging is severely compromised by these due to the presence of high atomic number elements Positron emission tomography PET and Single photon emission computerised tomography SPECT imaging are unaffected by these heaters unless a corrupted CT scan is used for attenuation correction We describe a carbon fibre sheet heater element used with 100 kHz alternating current AC that is demonstrated to be MR CT SPECT and PET compatible The assembly is shown in Figure 1 A 250 resistive heater element formed from 0 75 mm thick carbonfibre sheet RS 764 8700 cut into 4 x 3 mm wide legs each 120 mm long and spaced by 1 mm which was glued to the underside of the cradles as shown in Figure 1b A PID controlled gain setting amplified a 100 kHz sine wave generated using a Pierce oscillator to maximum power of ca 2 W Figure 1a The input to the PID was derived from a fibre optic rectal temperature system Opsens Accusens Thermal stability was validated in vivo in CBA mice Figure 1 Carbon fibre sheet resistive heater embedded in a 3D printed flat base multimodal imaging cradle a Proportional Integral Derivative Controller PID b Bottom view of the cradle containing the heater element c Top view of the cradle containing an adjustable mouthpiece and physiological monitoring apparatus respiration optical thermometer and a cover sheet to contain anaesthetic gas d thermal image of the cradle surface The current wires and metal couplings to the carbon fibre are located beyond the imaging FOV so do not present image distortions Magnetic resonance MR compatibility of the heater element was tested using MR compatibility of the heater element was tested in a water gel phantom using single shot PRESS spectroscopy and 2D fast low angle shot FLASH imaging and in vivo in the mouse using respirator y gated 2D FLASH and cardiorespiratory gated 3D FLASH imaging In all cases the same acquisition was repeated for the heater turned on and off 7T Varian Inc VNMR CT compatibility MILabs Vector4CT was tested in the absence of any heater and in the presence of either copper wire or carbon fibre sheet heater elements Multimodal MR CT PET SPECT imaging of kidneys was performed using Indium Citrate In SPECT 18 F fluorodeoxyglucose FDG PET anatomical CT respiratory gated bSSFP and DCE MRI 219

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 220 Poster Presentations Results thermal stability Results The power dissipation of the carbon fibre sheet heater resulted in homoeothermic maintenance of the animals The PID based controller allowed automated temperature maintenance with a fluctuation of 0 1 C at approximately 0 5 C below the target temperature MRI compatibility The equivalence of spectra and images acquired in the presence and absence of AC heating demonstrates that the heater element delivers heat in a manner that does not corrupt the imaging process and the carbon fibre material does not lead to any marked image distortions Figure 2 A core temperature of 4 mice placed into the SPECT PET CT scanner B Core temperature of 4 mice placed into the MRI scanner The arrow indicates when respiratory gated bssfp imaging was initiated to replicate the additional heat load of high duty cycle MRI scanning Conclusion Carbon fibre sheet resistors powered with high frequency AC under PID control G G G allow homoeothermic maintenance allow automated temperature control with 0 1 C fluctuation are compatible with multimodality imaging using MR CT PET and SPECT G are small heaters G are easy to produce G are easy to integrate into cradles for multi modal imaging 220 Figure 3 Impact of current flow through the carbonfibre sheet heater element on in vivo MRI of the mouse An area close to the heater was chosen to achieve this data yellow square as image distortions originate from current flow in the MR a T1 weighted in vivo whole body respiration gated 2D FLASH MRI with the heater turned off top and on middle The bottom image displays the difference between both images

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 221 Poster Presentations CT compatibility Multimodal MR CT PET SPECT imaging The absence of attenuation derived streak artefacts when using the carbon fibre sheet heater provides good image quality that enables quantitative analysis of image intensities This was not the case where the copper wire heater was used Figure 5 Multimodal imaging of a mouse using the carbon fibre sheet resistive heater embedded in a 3Dprinted flat base multimodal imaging cradle The skeleton kidneys major vessels to the kidneys are marked up The skeleton white was imaged by CT whilst 111In citrate red 18F fluorodeoxyglucose purple and Gadodiamide green were used for SPECT PET SPECT and DCE MRI of the kidneys respectively Each panel shows an additional layer of the co registered segmented image a CT MRI b CT MRI SPECT c CT MRI PET SPECT d CT MRI PET SPECT SPECT Figure 4 Impact of the heater material on CT image quality CT imaging using a the 150 mm diameter copper wire heater or b carbon fibre sheet heater Streak artefacts due to the presence of the heater are absent when using the carbon fibre heater and image intensities remain intact 221

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 222 Animal Technology and Welfare December 2019 Food for thought the development of drug loaded diets improve both science and welfare ALISON RITCHIE PAMELA COLLIER PHIL CLARE and ANNA GRABOWSKA Division of Cancer and Stem Cells School of Medicine University of Nottingham City Hospital Campus Nottingham NG5 1PB Correspondence alison ritchie nottingham ac uk Introduction In our facility we use several hormone dependent tumour models with supplementation delivered via slow release subcutaneously s c implanted pellets implanted via trochar For example G G prostate which rely on 5 DHT breast tumours which rely on 17 Estradiol respectively females were implanted s c with 17 Estradiol 0 1 mg 21 day release pellets Pellets were implanted using a 10 gauge trochar Innovative Research of America IRA Figure 1 Males were not implanted with pellets due to unavailability 3 Supplementation via diet ssniff i males were fed 5 DHT 2 4mg kg Figure 2 ii females were fed 17 Estradiol 2mg kg Figure 3 Administration of E2 supplementation can result in side effects such as bladder calculi and urine scald Therefore to improve animal welfare and to avoid steroid supply problems we decided to develop a new way to provide hormone supplementation via the diet Following on from this we then resolved to take this methodology forward into drug therapy trials Experiment 1 LnCap and MCF 7 respectively either s c or into the mammary fat pad and were divided into the following treatment groups 1 Control no supplementation 2 Supplementation via slow release pellets IRA Figure 1 Trochar and pellet 222 Figure 2 5 DHT diet Figure 3 17 Estradiol diet

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 223 Poster Presentations Animals were weighed daily to ensure food was being consumed Diets were colour coded to aid with identification MCF 7MFP Tumour day 14 Results 1 Tumours were measured weekly using G G calipers to calculate volumes bioluminescence using the IVIS Spectrum Perkin Elmer to measure both tumour size and viability Figure 6 The imaging results and growth curves Figures 6 and 10 show the supplemented diet facilitated tumour growth in both cases In the animals which received LnCap the increase was obvious from the start but with the MCF 7 the effects were more marked from day 14 onwards The MCF 7 growth data showed an increase with diet but lower than with the pellets while both the control groups showing little or nil growth at all Figure 7 no E2 Figure 8 E2 pellet Figure 9 E2 diet LnCap SC Tumour day 35 Figure 10 MCF 7 growth curve Figure 4 no DHT Figure 5 DHT The slower growth rate of MCF 7 could allow a longer window of opportunity for treatment effects when carrying out therapy studies Equally importantly we also observed a marked reduction in side effects in mice treated with the E2 dietary supplement compared with pellets with only some slight urinary retention which was eliminated by removing the supplemented diet for a few days and is much easier and less invasive than removing a subcutaneous pellet These positive results led us to consider whether this method of delivery could also be employed in other situations For example there is a move towards therapeutic drugs being delivered orally in some cases as a food supplement so we decided to develop this methodology as an alternative to daily oral gavaging Experiment 2 Figure 6 LnCap growth curve In this experiment a potential therapy Substance 99 was formulated into diet and growth compared against control diet 223

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 224 Poster Presentations CD 1 NuNu mice were implanted subcutaneously with HCT 116 colorectal tumour cells before receiving Control or Substance 99 loaded diet with daily weights to ensure consumption Figure 16 Carrier Figure 17 Carrier Figure 18 Carrier The results of this small pilot study show a slight difference between the groups with some growth delay shown in the Substance 99 treatment group We believe this demonstrates that therapies can be delivered via diet and this study gives us the confidence to pursue this method of delivery in a full scale experiment Figure 11 Substance Sub 99 diet Results 2 Fuel3D scanning system to calculate tumour volumes and record thermal images Day 20 images Volume mm3 Bioluminescence Fuel3D vol Fuel3D thermal 500 400 0 12 3 4 300 Delivery of hormones via the diet is a major refinement in welfare terms by Reducing the need for an invasive implant and possible removal procedure Reducing deleterious side effects Promoting tumour growth Ease of delivery for the Animal Technologist Similarly our pilot therapy experiment demonstrates that delivery of drug therapies via diet rather than daily oral gavage is both 200 100 0 20 Conclusions 25 30 35 40 45 50 Days elapsed Figure 12 HCT 116 Substance 99 G G a welfare improvement and potentially a translationally more accurate model Therefore we believe the potential to deliver compounds via diet rather than by more invasive techniques should always be considered wherever experimentally appropriate Acknowledgements Thanks to ssniff Spezialdi ten GmbH and PMI TestDiet for their help and expertise in developing the diets Prof G Seymour for allowing us to incorporate this innovation into his experiment to Fuel3D in providing the technology and images in Experiment 2 and to Marian Meakin and Alison Mackie for their technical assistance Figure 13 Sub 99 Figure 14 Sub 99 Figure 15 Sub 99 224

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 225 December 2019 Animal Technology and Welfare Instructions to Authors Subjects considered for publication may include original articles technical notes and reviews pertaining to all aspects of animal science and technology management and education The Editorial Board wishes to offer par ticular encouragement to papers leading to improvements in environmental enrichment the general care and welfare of the animals used in particular those species and strains exhibiting harmful genetic defects and papers describing refinements in techniques a reduction in the number of animals that need to be used or alternatives to animal use Papers describing experimental procedures will only be accepted for publication if authors clearly state that the procedures conform to the prevailing principles and Codes of Practice of the Animals Scientific Procedures Act 1986 Papers submitted from outside the U K should state what legislation and or ethical approval the work has been carried out under In addition authors who describe surgical techniques with recovery should include details of post operative care and any analgesic therapy provided All submissions should follow the ARRIVE Animal Research Repor ting of In Vivo Experiments guidelines Kilkenny C Browne WJ Cuthill IC Emerson M Altman DG 2010 Improving Bioscience Research Repor ting The ARRIVE Guidelines for Reporting Animal Research PLOS Biol 8 6 e1000412 doi 10 1371 journal pbio 1000412 The Editorial Board reser ves the right to seek independent advice on any aspect of the content of an article but the final decision on acceptance or rejection remains with the Board to the address below together with a copy on disk CD or DVD All sheets should be typewritten on one side in double spacing and serially numbered Any photographs or graphs should be supplied as originals and conform to the format in 4 below Address for submission Journal Editorial Board Chairman 5 South Parade Summertown Oxford OX2 7JL No responsibility will be accepted for loss or damage to such articles Electronic files of submissions are required together with separate files of photographs and any graphics that appear in the manuscript Electronic submissions should be sent via email via atw iat org uk alternatively manuscript plus two copies may be sent as hard copy to the address below All sheets should be typewritten on one side in double spacing with 4 cm margins and serially numbered Additionally a copy on disk should be provided or sent by email via atw iat org uk Articles for submission should be sent to Journal Editorial Board Chairman 5 South Parade Summertown Oxford OX2 7JL No responsibility will be accepted for loss or damage to such articles Format Submission Material submitted for publication will be considered provided that it is contributed exclusively to Animal Technology and becomes the property of the Institute of Animal Technology Articles may be submitted either electronically or by hard copy as follows Electronic Articles should be submitted in Word format with double spacing to the lines and all pages serially numbered Any photographs or graphs must be submitted as separate files and conform to the format in point 4 below The relevant ar ticle must clearly indicate where photographs and or graphs are to be inserted Address for submission atw iat org uk Hard copy The original manuscript plus two copies should be sent 1 The first sheet of the article should contain the following i the full title of the paper ii the initials and last name of the author s iii the full address of the depar tment s and institution s where the work was carried out iv the address for correspondence if different to above 2 For the remainder of the paper the text should be clear and concise and where appropriate sub divided under the following headings i ii iii iv v vi vii Summary Introduction Methods Results Discussion Acknowledgements References 3 Measurements should be given in metric units see The use of S I Units 1969 British Standards Institution publication and spelling should follow that of the Oxford 225

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 226 Instructions to Authors English Dictionary Abbreviations must be defined in full at their first appearance in the text The 24 hour clock should be used for times Words to appear in italic type should be underlined Designation of inbred strains should be in accordance with the International Index of Laboratory Animals 6th edition compiled edited and published by M W Festing 1993 e g Gregory J A 1985 Principles of Animal Husbandry In Laboratory Animals An Introduction for Experimenters Second Edition Tuffrey A A John Wiley Sons Ltd Chichester 87 105 Papers accepted for publication but not yet published should be included in the list of references followed by in press Papers in preparation personal communications and unpublished observations should be referred to as such in the text only 4 Photographs should have clear and well contrasted tone values and be in colour All illustrations charts e g histograms and graphs and photographs should be submitted separately and bear on the reverse side the author s name a number corresponding to the order in which it appears in the text e g Figure 1 and an arrow pointing to the top Content Illustrations charts and photographs supplied on disk should be in JPEG TIFF or EPS formats and have a resolution of no less than 300dpi Animals The captions for illustrations charts and photographs should be typed in double spacing in numerical order on a separate sheet of paper 5 References Only essential references should be included Authors are responsible for verifying them against the original source material ATW uses the Vancouver referencing system references should be identified in the text by superscript Arabic numbers e g 12 after any punctuation and numbered and listed at the end of the paper in the order of when they are first cited in the text Automatic numbering should be avoided References should include the names and initials of up to six authors If there are more than six authors only the first three should be named followed by et al Publications for which no author is apparent may be attributed to the organisation from which they originate Simply omit the name of the author for anonymous journal articles avoid using Anonymous References should be set out as follows Journals Surname and initials of author s date title of article Name of journal in full volume number first and last page numbers e g Saigeman S 1998 Environmental enhancement of cats what why how Animal Technology Vol 49 No 3 145 154 Books Surname and initials of author s date title of book Name of publisher Town of publisher e g Flecknell P A 1987 Laborator y Anaesthesia Academic Press London Animal Chapter from a multi author book Surname and initials of chapter author s date title of chapter In title of book surname and initials of book editors Name of publisher Town of publisher first and last page numbers of chapter 226 Papers describing procedures involving the use of animals should always include full details of the animals and husbandry conditions used These would be as follows Species Breed or strain Sex Age and weight at start of procedure Genetic status inbred outbred hybrid mutant Source Microbiological status conventional specified pathogen free define which pathogens animals are free from gnotobiotic define which micro organisms are present Quarantine or acclimatisation period Husbandry during procedure Type of housing material size cage type if relevant Number of animals per cage or unit Bedding type quality any pretreatment Type of system conventional barrier ventilated rack isolator Environmental temperature C range Relative Humidity range Lighting natural artificial state hours of light and dark Ventilation number of air changes per hour Period of acclimatisation before start of procedure Feed type composition any pretreatment amount frequency Water type quality any pretreatment amount frequency Scientific procedure Number of animals and any pretreatment Time of day of procedure s Quantity and frequency of any samples Statistics Tests used should be named Reprints Free reprints are no longer provided but the ATW Editorial Board are happy to provide PDF files of articles after publication Use of these files is subject to Copyright restrictions

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Latest_2 636763158 e S Animal Technology and Welfare 3 12 19 14 05 Page 234 Institute of Animal Technology IAT Statement 18th October 2019 Undercover footage at the Laboratory of Pharmacology and Toxicology LPT near Hamburg published by Cruelty Free International and Soko Tierschutz The IAT strongly condemns the conduct of the technicians as shown in the video footage circulated by Cruelty Free International The video shows technicians routinely and inappropriately using collar and pole on juvenile macaque monkeys violently removing them from their cages and in one incident a monkey has its head smacked against a door frame Monkeys are also forced into a restraining chair with procedures performed in the presence of other distressed monkeys It is clear that these terrified monkeys have neither been habituated to interact with staff nor trained to accept the restraining chairs which is considered good practice by the IAT Some of the monkeys appear to be kept alone in undersized metal cages and exhibit stereotypic behaviour by spinning in circles indicating high levels of distress Dogs are pictured laying in what seemed to be their own blood and faeces with one beagle in a cage appearing to be bleeding The pens in which they were kept did not have any soft surfaces let alone a raised resting area or any environmental enrichment In view of the physiological and psychological distress exhibited by the monkeys and dogs it is right to question the scientific validity of any results obtained under these conditions Staff also appeared to mishandle cats with little or no empathy xviii