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IAT Journal Animal Technology and Welfare INCLUDES G G G G G IAT Congress 2013 posters Mouse endotracheal intubation simplified Discussion on improving lighting for non human primates Congress 2013 Severe suffering workshop report Xenopus tropicalis assessment of a stand alone system Official Journal of the Institute of Animal Technology and European Federation of Animal Technologists ISSN 1742 0385 Vol 12 No 2 August 2013

OFFICERS President Dr Robin Lovell Badge FRS Animal Technology and Welfare Immediate Past President Professor Sir Richard Gardner MA PhD FSB HonFIAT FRS Vice Presidents David Anderson MRCVS Stephen Barnett BA MSc CBiol FSB RAnTech John Bleby TD JP DVetMed DLAS CBiol FSB MRCVS Brian Cass CBE Miles Carroll PhD Gerald Clough BSc PhD EurBiol CBiol MSB SFZSL Paul Flecknell MA Vet MB PhD DLAS DipLECVA MRCVS Barbara Mortimer BVetMed DLAS MRCVS Judy MacArthur Clark CBE BVMS DLAS CBiol FSB MRCVS Fiona McEwen BSc BVM S MSc MRCVS Tim Morris BVetMed PhD DipACLAM DipECLAM CBiol FSB CertLAS MRCVS Jos Orellana BVSc MSc Clive Page PhD BSc Sophie Petit Zeman PhD Gail Thompson RLATG Robert Weichbrod PhD RLATG Sheila Whitehead BVMS MSc CertLAS MRCVS Lord Robert Winston FMedSci DSc FRCOG FRCP FRCS Ed FSB Vol 12 No 2 August 2013 CONTENTS Editorial Jas Barley Chair of the Editorial Board ix Honorary Members John Frogley FIAT RAnTech Andrew Jackson MIAT John Lesley FIAT RAnTech Brian Lowe MSc FIAT RAnTech Ronald Raymond FIAT RAnTech Peter Russell FIAT RAnTech David Spillane FIAT Ray Thatcher FIAT RAnTech Peter Willan DMS FInstLM MIAT Members of Council Ken Applebee Jas Barley Kate Burton Charlie Chambers Steven Cubitt Andy Cunningham Glyn Fisher Carol Fox Cathy Godfrey Alan Graham John Gregory Patrick Hayes Linda Horan Andy Jackson Elaine Kirkum Adele Kitching Sarah Lane Norman Mortell Steve Owen Wendy Steel Allan Thornhill Lynda Westall Debbi Young Members of Executive Committee 79 Consistency of ventilation in IVCs and open cages their normoxic atmosphere and its impact on haematological parameters of mice Gianpaolo Milite and Lucia Buccarello 83 Discussion paper reducing severe suffering Penny Hawkins 87 PAPER SUMMARY TRANSLATIONS Life Members Roger Francis MSC FIAT RAnTech Pete Gerson MSc FIAT RAnTech John Gregory BSc Hons FIAT CBiol FSB RAnTech Patrick Hayes FIAT DipBA RAnTech John Kelly FIAT Robert Kemp FIAT Hon RAnTech Keith Millican FIAT CBiol MSB Phil Ruddock MIAT RAnTech Ted Wills HonFIAT RAnTech Dorothy Woodnott FIAT A simple method of endotracheal intubation in mice Anastasia Papastefanou Evangelos Balafas and Nikolaos Kostomitsopoulos 93 TECH 2 TECH GA passports Workshop Report Nikki Osborne Caroline Chadwick Mary Ann Haskings Jenny Salisbury and Amanda Pickard 105 The problem with the dark bottom row cage for non human primates A discussion by the Laboratory Animal Refinement Enrichment Forum Vera Baumans Kaile Bennett Marcie Donnelly Genevieve Andrews Kelly Stefanie Nelsen Kimberly Rappaport Allison Reiffer and Viktor Reinhardt Moderator 111 AFOS vet minimises stress and pain in Rodent Euthanasia Desiree Betts 115 POSTER PRESENTATIONS Improving animal welfare by providing extra environmental enrichment with play time Natalie Edwards 119 Welfare for wobbly mice Helen Cooper 123 Webcams as a tool to aid communications diagnosis and treatment Darren Egan 127 Single handed jugular blood sampling in conscious rats Kate Heath 130 Determination of recovery period after intra abdominal transmitter and EEG electrode placement in common marmosets Callithrix jacchus Leo van Geest Milene Rijnbeek Herbert Brok Jaco Bakker Rianne Klomp Ingrid Philpens Saskia Arndt and Jan Langermans 133 Maximising operational and research activities through the use of disposable caging Rebecca Varrall David Smith and Stephen Baker 136 Creation of a complex environment for NZW rabbits within the confines of an animal unit Ashley Grimmer Natalie Edwards and Kate Tansley 138 The chicken as a research animal Lejla Bektic 141 143 Chair Board of Educational Policy Ken Applebee FIAT CBiol FSB RAnTech A study to assess the suitability of a stand alone aquatic system for housing Xenopus tropicalis frogs Nick Harman Peter Thompson David McDonald Nicola Goodwin Diane Hazlehurst and Lynda Westall Chair Board of Moderators Cathy Godfrey FIAT RAnTech Challenging taboos in Animal Breeding and Welfare Jackie Harrison Carol Copley and Nichola Chrobot 146 The changing face of flexible film isolators at the Mary Lyon Centre Quarantine Suite Mark Harrison and Alison Gallop 150 Chair Registration Accreditation Board Charlie Chambers MIAT RAnTech Our experience as GSK apprentices Jack Armes and Ben Jaggs 152 Instructions to Authors 155 Council Officers Chair Steve Owen FIAT RAnTech Vice Chair Ken Applebee FIAT CBiol FSB RAnTech Honorary Secretary Wendy Steel BSc Hons FIAT RAnTech Honorary Treasurer Glyn Fisher FIAT RAnTech Assistant Treasurer Charlie Chambers MIAT RAnTech Chair ATW Editorial Board Jas Barley MSc FIAT RAnTech continued on page ii i

BRANCH SECRETARIES 2013 Officers continued from page i Bulletin Editor Sarah Lane MSc FIAT RAnTech Aberdeen Ms Donna Wallace Rowett Research Institute Greenburb Road Bucksburn Aberdeen AB21 9BJ Assistant Bulletin Editor Elaine Kirkum MIAT RAnTech MIScT Branch Liaison Officer Lynda Westall BSc Hons FIAT DMS RAnTech EFAT Representatives Charlie Chambers MIAT RAnTech Kate Burton MSc FIAT RAnTech Cambridge Ms Fran Flack Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton Cambridgeshire CB10 1SA Cheshire Ms Julie Humphreys Block 53 Mereside Astrazeneca Alderley Edge Nr Macclesfield Cheshire SK10 4TG Council Website Coordinator Allan Thornhill FIAT RAnTech IAT INFORMATION Animal Welfare Officers LABA Representatives Sarah Lane Debbi Young ATW Bulletin Editorial Board Jas Barley Chair Patrick Hayes Editorial Assistant Elaine Kirkum Sarah Lane Lynda Westall Board of Educational Policy Ken Applebee Chair Steven Cubitt Secretary Carol Fox CPD Officer Sarah Lane Board of Moderators Cathy Godfrey Chair Glyn Fisher Secretary Moderators Gary Childs Joanna Cruden Nicky Gent Linda Horan Sue McHugh Communications Group Norman Mortell Chair Kate Burton Haley Daniels Linda Horan Elaine Kirkum Allan Thornhill Lynda Westall Registration and Accreditation Board Ken Applebee Charlie Chambers Chair Gerald Clough Carol Fox John Gregory Cathy Godfrey Sarah Lane Ron Raymond Wendy Steel Secretary Steve Owen Stuart Stevenson Carol Williams Observers Charles Gentry Certificate Holders Forum Adrian Deeny LASA Kathy Ryder Home Office Lucy Whitfield LAVA Advertisement Managers PRC Associates Ltd Email mail prcassoc co uk IAT OFFICERS MAY BE CONTACTED VIA Edinburgh Ms Janice Young Evans Building Western General Hospital Crewe Road Edinburgh EH4 2XU Hertfordshire Essex Ms Hazel Sleight GSK Safety Assessment Toxicology Support Park Road Ware Hertfordshire SG12 0DP Huntingdon Suffolk Norfolk Ms Jo Martin Huntingdon Life Sciences Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS Ireland Mr Colin Travis Trinity Biomedical Sciences Institute Trinity College 157 160 Pearse Street Dublin 2 Ireland London Ms Karen Robinson BSU Rayne 4th Floor Lambeth Wing St Thomas Hospital London SE1 1UL Midlands Mr Ian Fielding Tecniplast UK BCM Box 3058 London WC1N 3XX North East England Ms Nicky Windows Datesand Ltd PO Box 45 Manchester M11 1XD Oxford Mr Adrian Woodhouse Prosidion Windrush Court Transport Way Watlington Road Cowley Oxford OX4 6LT IAT Administrator iat101 btconnect com OR VIA THE IAT WEBSITE AT www iat org uk OR VIA THE REGISTERED OFFICE 5 South Parade Summertown Oxford OX2 7JL 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 2013 Institute of Animal Technology All rights reserved No part of this publication may be reproduced without permission from the publisher ii Surrey Hampshire Sussex Ms Lesley Hughes c o Building 200 ASU Veterinary Laboratories Agency Woodham Lane New Haw Addlestone Surrey KT15 3NB West Middlesex Mrs Wendy Steel Faculty of Medicine Imperial College London Central Biomedical Services South Kensington Campus London SW7 2AZ West of Scotland Ms Linda Horan Biological Procedures Unit University of Strathclyde SIBS Building c o Phys and Pharm 27 Taylor Street Glasgow G4 ONR

August 2013 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 AND TO RECOGNISE THAT GOOD LABORATORY ANIMAL WELFARE IS AN ESSENTIAL COMPONENT OF GOOD LABORATORY ANIMAL TECHNOLOGY AND SCIENCE Editorial Jas Barley Chair of the Editorial Board Welcome to our Congress 2013 review issue Congress this year was a joint venture with the Laboratory Animal Veterinary Association LAVA and it was encouraging to see so many of our Veterinary colleagues attend and participate in the event We hope to bring as many of the presentations including platform speakers contributions to you in the coming months albeit in possibly different formats to our usual style This is also a truly international issue with contributions from Greece Italy The Netherlands Sweden and the United States of America The Biomedical Industry is subject to increasing globalisation and I am pleased to say that despite the difficult financial times which many of our colleagues are experiencing interest in Animal Technology and Welfare ATW remains high with it being distributed to readers in over 28 countries worldwide The importance of sharing information between animal technologists and our veterinary colleagues both within the UK and overseas increases with this globalisation as it enables us wherever possible to work to similar standards and principles Obviously the unique opportunity to discuss and share our practices face to face at meetings such as Congress is never going to be entirely replaced by Journals or on line discussions but by using the best of all technologies available we hope to be able to make much of the material at Congress accessible to those of you who were unable to attend Of course education is the key and through sharing information we both learn and teach others The discussion document by Penny Hawkins on reducing severe suffering is a result from the workshop held at Congress and the resulting exchange of ideas and protocols Similarly the contribution from the Laboratory Animal Refinement and Enrichment Forum LAREF is just one of many discussions that members of the Forum have benefited from over recent years and which they have been generous enough to share with our readers thanks to the efforts of the moderator Viktor Reinhardt Nikki Osborne s report on GA passports again is a collaboration between workshop participants Many of the papers and posters presented in this issue demonstrate that solutions to animal welfare problems need not necessarily be expensive or complicated and that the overwhelming factor is the desire to give the animals in our care the best life possible The age old cry we can t afford it is not an excuse even in these straitened times and the paper on endotracheal intubation of mice by the team at the Centre for Experimental Surgery in Athens demonstrates this Natalie Edwards s poster on playtime for rats provides a simple solution to a perpetual problem of adult single housed rats Similarly the team from the Phenomics laboratory at the University of Cambridge has approached improving the environment of rabbits in a low cost manner For those of you that do not know what Phenomics are either I m reliably informed that it s an area of biology concerned with the measurement of phenomes the physical and biochemical traits of organisms as they change in response to genetic mutation and environmental influences see you ve learnt something already ix

August 2013 Animal Technology and Welfare A simple method of endotracheal intubation in mice ANASTASIA PAPASTEFANOU EVANGELOS BALAFAS and NIKOLAOS KOSTMITSOPOULOS1 Centre for Experimental Surgery Biomedical Research Foundation of the Academy of Athens 4 Soranou Ephesius Street 11527 Athens Greece 1 Correspondence to nkostom bioacademy gr Summary Endotracheal intubation in mice is commonly used when airway management is necessary The procedure is difficult to do because of the small size of mice and their oropharynx Various methods for endotracheally intubating mice have been described in the scientific literature and most of these methods require the use of expensive devices and or special training In this report we describe our experience of an easy reliable and inexpensive method for endotracheally intubating mice through the mouth developed in our Centre using low cost materials which can be found in almost all surgical laboratories Introduction Endotracheal intubation is done for a air way management and mechanical ventilation when animals are anaesthetised with an inhalation anaesthetic agent b the intratracheal instillation of a test compound or c assessment of pulmonary function Depending on the specific needs of the research the endotracheal tube can be either inserted into the trachea through the mouth orotracheal intubation or by performing a tracheostomy1 2 3 4 Of the two methods orotracheal intubation is less traumatic and can be repeated in the same animal1 developed in our Centre and used in authorised experimental protocols Description of the method In order to surmount the problem of smallness and circumvent the problems of equipment we have developed a non traumatic method for rapid and repeated endotracheal intubation of mice through the mouth orotracheal intubation In this method the following materials are required a 22G Teflon intravenous cannula one disposable insulin syringe one pair of anatomic forceps a powerful flashlight 4 0 coated braided silk non absorbable surgical suture Silkam B BRAUN Aesculap AG CO Tuttligen Germany 10 lidocaine Xylocaine Astra Zeneca Sodertalje Sweden surgical tape a plastic compact disc CD case which is used as the working table normal saline solution gauze and one pair of scissors The 22G Teflon intravenous cannula is used as the endotracheal tube The tip approximately 5mm of the metal needle of the intravenous cannula is cut in order to prevent the needle damaging the tissues while intubating the mouse Figure 1 The rest of the needle is used as a guide wire in the catheter in order to keep The endotracheal intubation of mice is well described in the scientific literature and these published methods vary in technique and the required equipment1 5 6 Endotracheal intubation of mice is difficult to do because of the small body size of a mouse and its narrow mouth cavity and glottic opening This diminutiveness necessitates the use of specific and often expensive equipment such as a surgical microscope a flexible or rigid fibre optic laryngoscope an otoscope or other custom made instruments to endotracheally intubate a mouse1 7 8 9 The aim of this paper is to present our experience from the implementation of an intubation method which was Figure 1 The tip approximately 5mm of the metal needle of the Teflon intravenous cannula is cut before passing it into the mouse s trachea 79

A simple method of endotracheal intubation in mice the tracheal tube straight for intubating the mouse Before passing the catheter into the trachea its tip is sprayed with 10 lidocaine in order to facilitate its passage by preventing laryngospasm and numbing the throat during its passage The mouse is usually anaesthetised using a combination of 85 mg kg ketamine Imalgene 1000 Merial Lyon France and 10 mg kg xylazine Rompun Bayer Animal Health GmbH Leverkusen Germany The depth of anaesthesia is determined by the absence of swallowing and righting reflexes The presence or absence of the righting reflex can be determined by pinching the rear foot or the tail of the mouse When the righting reflex is abolished the anaesthetised mouse is placed on the CD case in a supine position Its forelimbs are immobilised with surgical tape and its upper incisors are secured with the 4 0 silk suture Figure 2 The anaesthetised mouse is then suspended at an angle of 60 to 70 on its back in a head up position on the CD case Figure 3 The mouse s tongue is gently pulled out of the mouth using the anatomical forceps and is held aside using the thumb and index fingers In order to avoid the tongue slipping from the fingers a small piece of cotton gauze can be used to improve the grip of the fingers The area of the vocal cords is transillumated in order to permit a clear view of the tracheal opening by focussing the light beam of flashlight which is held by an assistant on the ventral neck of the mouse Figure 4 Figure 2 Immobilisation of the mouse s forelimbs using surgical tape and the upper incisors using a 4 0 silk suture Figure 4 The tongue is held with a small piece of gauze and the intravenous cannula is inserted into the mouth Figure 3 The anaesthetised mouse is secured to a plastic compact disc case which is then placed in a head up position at an angle of approximately 60 70 80 Figure 5 The intubated anaesthetised mouse

A simple method of endotracheal intubation in mice The intravenous catheter is inserted into the mouth and the epiglottis is opened by circular movements of the catheter s tip The catheter is then advanced through the larynx and across the vocal cords into the trachea Resistance and lar yngospasm may be encountered if the tip of the catheter touches the vocal cords In such circumstances the catheter should be retracted and the procedure repeated When the mouse is intubated the needle of the catheter is removed carefully and the CD is restored to the horizontal position Figure 5 Finally the tube is secured with surgical tape and the intubated mouse is ready to be connected to the ventilator A small dental mirror can be used to con rm correct placement of the catheter in the trachea For this purpose the mirror should be chilled in a 20 C freezer and kept cold until use It is then positioned over the catheter s visible inlet after placing the catheter If the catheter has been successfully placed in the trachea a visible condensate will form on the mirror s surface due to the moist exhaled air Although this method of confirming the correct placement of the catheter has been described as unreliable because the formed mist is occasionally vague10 we found this method reliable for confirming the correct placement of the endotracheal tube Other published methods for confirming proper placement of the endotracheal tube in mice include closing the end of the tube with one s fingertips or placing a plastic pipette onto the end of the tube or an extension tube of intravenous infusion that contains one drop of water10 11 In our opinion all these methods for confirming proper placement of the endotracheal tube can potentially compromise respiratory function of the mice Discussion conclusion The endotracheal intubation of mice requires special skills Based on our experience the described technique is easy to be learned and adopted even by moderate experienced personnel Although the method requires an assistant in order to per form the intubation we never faced any coordination problems in the everyday practice Main advantage of the method is that it does not necessitate the use of expensive instrumentation like a surgical microscope special tables extra guide wires or instruments such as a laryngoscope or an otoscope It is used routinely in our centre in different research protocols that require monitoring the airway management and ventilation of anaesthetised mice To date we have used this method of orotracheal intubation in more than 200 mice and all animals have recovered without any problems or residual adverse effects be found in almost all surgical laboratories Investigators and laboratory animal technicians with relatively little experience can be trained to use this method of orotracheal intubation when they need to intubate mice in their research References 1 2 3 4 5 6 7 8 9 10 11 Spoelstra E N Ince C Koeman A Emons V M Brouwer L A Van Luyn M J A Westerink B H C and Remie R 2007 A novel and simple method for endotracheal intubation of mice Laboratory Animals 41 1 128 135 Miller D L and Van Winkle D M 1999 Ischemic preconditioning limits infarct size following regional ischemic reperfusion in in situ mouse hearts Cardiovasc Res 42 3 680 684 Hoetzel A Schmidt R Vallbracht S Goebel U Dolinay T Kim H P Ifedigbo E Ryter S W and Choi A M K 2009 Carbon monoxide prevents ventilator induced lung injury via caveolin 1 Crit Care Med 37 5 1708 1715 Huang C S Kawamura T Lee S Tochigi N Shigemura N Buchholz B M Kloke J D Billiar T R Toyoda Y and Nakao A 2010 Hydrogen inhalation ameliorates ventilator induced lung injury Critical Care 14 6 R234 Cambron H Latulippe J F Nguyen T and Cartier R 1995 Orotracheal intubation of rats by transillumination Lab Anim Sci 45 3 303 304 Brown R H Walters D M Greenberg R S and Mitzner W 1999 A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice J App Physiol 87 6 2362 2365 Costa D L Lehmann J R Harold W M and Drew R T 1986 Transoral tracheal intubation of rodents using a fiberoptic laryngoscope Lab Anim Sci 36 3 256 261 Hastings R H and Summers Torres D 1999 Direct laryngoscopy in mice Contemp Top Lab Anim Sci 38 6 33 35 Hamacher J Arras M Bootz F Weiss M Schramm R and Moehrlen U 2008 Microscopic wire guidebased orotracheal intubation description evaluation and comparison with transilllumination Laboratory Animals 42 2 222 230 Watanabe A Hashimoto Y Ochiai E Sato A and Kamei K 2009 A simple method for confirming correct intubation in mice Laboratory Animals 43 4 399 401 Rivera B Miller S R Brown E M and Price R E 2005 A novel method for endotracheal intubation of mice and rats used in imaging studies Contemp Top Lab Anim Sci 44 2 52 55 In conclusion we describe an easy reliable and inexpensive method for endotracheally intubating mice through the mouth using low cost materials that can 81

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August 2013 Animal Technology and Welfare Consistency of ventilation in IVCs and open cages their normoxic atmospheres and its impact on haematological parameters of mice GIANPAOLO MILITE1 and LUCIA BUCCARELLO2 1 2 Corresponding author Scientific Consultant Udine Italy Dipartimento di Scienze e Tecnologie Veterinarie Universit di Milano Italy Correspondence to gianpaolo milite fastwebnet it Abstract Oxygen concentration in an Individually Ventilated Caging system IVCs was recently found to be slightly lower than optimal for an ideal microenvironment to house laboratory mice Given the substantial technical differences between IVCs of different designs which may yield dissimilar intra cage environmental conditions it was decided to monitor the microenvironment of an Individually Ventilated Cage IVC system and an open cage system and compare the impact on Red Blood Cell RBC count and related physiological parameters in C57Bl 6J mice housed in open cages A small decrease in the intra cage oxygen level was detected in both cage systems when approaching the cage change procedure performed every two weeks No differences in the haematological parameters were detectable after 6 weeks conditioning of mice in either of the two systems The IVC microenvironment was shown to be free from any impact on the haematological parameters taken into consideration in this trial when compared to the open cage set up Introduction There are a number of parameters used to assess the welfare of animals used in research each contributing to the description of the physical and psychological health status From a simple evaluation of appearance coat condition body weight gait posture feeding and drinking behaviour urination and defecation to faecal or blood cortisol the list of welfare indicators can be very long The choice of the most appropriate indicator is mainly down to the potential effect caused by the factor under investigation and haematological parameters are certainly very good indicators of state of animal health Red Blood Cell count total haemoglobin Hgb and haematocrit HCT are potential indicators when studying the environmental fresh air availability in terms of gas composition in Individually Ventilated Caging systems The impact on the above mentioned blood parameters may be due to a reduced quality of the air because a decreased percentage of oxygen lower than 20 9 or an increased CO2 concentration If alterations of intracage atmosphere are accepted when an IVC is not ventilated static it is not acceptable to detect a constant modification of its microenvironment in terms of oxygen reduction during standard operations Removal of pollutants like ammonia NH3 and CO2 from a ventilated environment stability of desired temperature and relative humidity is achieved through an efficient ventilation an acceptable concentration of oxygen O2 is also maintained provided that at room level the same parameters are controlled and within regulatory acceptable ranges Intra cage microenvironment has been widely investigated especially in Individually Ventilated Cages due to their confined environmental condition Consistent differences between IVC systems of different designs and operating at different air changes per hour and mode of air distribution have been shown to create diversity in the microenvironment ammonia CO2 and oxygen with possible impact on breeding performances1 Some authors failed to find a relation between different concentrations of NH3 in the cage and respirator y tract lesions2 whereas more recently others have described the connection between cumulative pollutants NH3 and nasal histological identifiable lesions3 nevertheless intra cage oxygen concentration was not reported by these investigators 83

Consistency of ventilation in IVCs and opencages their normoxic atmosphere and its impact on haematological parameters of mice Oxygen concentration was taken into consideration when a disposable cage was tested under static conditions up to six hours showing consistent reduction of O2 in relation with cage occupancy and time4 A recently published scientific paper5 describes the impact due to reduced oxygen concentration on mouse Red Blood Cells count haemoglobin and haematocrit all of which increased when mice are maintained in a ventilated caging system operated at 60 Air Changes per Hour ACH and the air is injected at animal level vs the open cage control group Individually ventilated caging systems var y considerably in terms of solutions found to move the air inside the cages and the flow rate suggested by the manufacturer The main consequence of the two mentioned substantial differences is the efficiency of ventilation responsible for the quality of the microenvironment in the cage6 Owing to these differences in design and performance it was decided to test an IVC system with technical and operating solutions very different from that tested by York et al5 to understand if the problem identified by the authors can be generalised to IVCs or is specific to that equipment Materials and methods Animals and housing A total of 24 Male C57Bl 6J Harlan Laboratories Italy Specified Pathogen Free mice 4 weeks old at arrival weaned and maintained in open cages were randomly distributed 4 to a cage three cages x 2 groups open and IVC In addition four mice were distributed two to a cage one cage x group two mice each Prior to distribution to the assigned caging group all mice were maintained at the same density in open cages for one week Oxygen Carbon dioxide Ammonia Humidity and Temperature Intra cage air oxygen CO2 and ammonia were measured using a Drager X Am 7000 inserting a flexible probe alternatively through the flap used for the bottle nipple in the IVC configuration or through the bar lids in the open one to a distance from the bedding of approximately 2 5 cm Temperature and Relative Humidity RH were monitored by means of Data Loggers USB 502 Measurement Computing Norton MA USA positioned on top of the wire bar lid of the cages and set to record the two parameters every 30 minutes One Data Logger was also positioned on top of the rack to monitor the environment Treatments and testing IVC mice were 11 weeks old at testing and had spent almost 6 weeks in IVC or open cages Open cage mice were 11 weeks old either at testing and had spent all their life in open cages Haematology All mice were euthanised by carbon dioxide at ten weeks of age and blood was drawn by cardiac puncture The approximate volume of blood was 0 4 to 0 6 ml A maximum volume of 0 5 ml was placed in EDTA coated microtainer tubes Becton Dickinson Franklin Lakes NJ USA Analysis for Red Blood Cells count RBC total haemoglobin content Hgb haematocrit HCT Mean Corpuscular volume MCV Mean Cell Haemoglobin MCH Mean Cell Haemoglobin Concentration MCHC Red Blood Cell Distribution width RBCw and Platelets PLT were performed on a Abacus Junior Vet Diatron Messtechnik GmbH Austria set for mice profile Results Each cage was filled with 150g of autoclaved corn cob bedding Autoclaved diet was distributed and weighed on a weekly basis Autoclaved filtered water was provided by bottles 270 ml capacity bottles were singly weighed when filled and after one week during change All mice were weighed after the random distribution to each cage Cages were GM500 Tecniplast S p a VA Italy 500 cm2 each of usable surface with wire top cage lids that were either open to the environment or connected to a positive pressure mode Air handling Unit set at 75 Air Changes Hour ACH in positive pressure according to the manufacturer s specification The Air handling unit is connected to the rack by flexible hoses and HEPA filtered air enters and exits the cages from the rear of the plastic cage cover 84 Air oxygen and other gases Oxygen was maintained at a concentration of 20 9 throughout the trial in both systems with minor fluctuations down to 20 6 two days before cage change day 14 in both open cages and the IVCs Ammonia concentration was not detected in either systems during the first 12 days before change but increased up to 3 p p m in the IVC system and 5 p p m in the open cages during the last few days before cage cleaning Carbon dioxide CO2 on day 14 day of cage change was 0 067 and 0 037 respectively in both IVCs and open cages Body Weight Feed and Water intake Mean body weight of mice in the two groups were

Consistency of ventilation in IVCs and opencages their normoxic atmosphere and its impact on haematological parameters of mice comparable and respectively in IVC 26 77 0 68 in open 27 01 0 45 group 95 C I of difference 1 154 to 1 639 two tailed Student s t test t 0 357 df 26 p 0 723 The mean food intake during the last two weeks of the trial was 3 68 g 0 21 and 3 54 g 0 18 respectively in the IVCs and in open cages Daily water intake was 4 21 g 0 1 and 4 43 g 0 18 respectively in the IVC and the open cages Temperature Mean Sd Relative humidity Mean Sd Room Cage 1 Cage 2 Cage 3 Cage 4 Cage 5 Cage 6 IVC Open IVC Open IVC Open 21 0 22 9 22 9 23 6 22 7 22 9 21 5 0 1 0 7 0 7 0 6 0 7 0 6 0 4 64 4 68 3 64 7 70 5 65 4 75 4 67 0 2 9 3 3 2 9 4 3 2 9 4 4 3 3 Temperature and Relative humidity All cages with 4 mice were equipped with a Data Logger An additional Data Logger was positioned on top of the rack to record temperature and Relative Humidity in the room One IVC cage had a mean value of RH outside the acceptable field of variation 40 to 70 Table 1 IVC GM500 Open GM500 p values total n 14 14 Red Blood Cells 109 L 9 08 0 25 9 08 0 11 p 0 991 Haemoglobin g dl 15 22 0 29 15 22 0 16 p 0 982 Haematocrit 40 82 0 82 40 26 0 42 p 0 551 Mean Cell Volume fL 44 71 0 24 44 5 0 20 p 0 505 Mean Cell Haemoglobin pg 16 67 0 14 16 82 0 14 p 0 445 Mean Cell Haemoglobin Concentration g dl 37 15 0 25 37 73 0 31 p 0 469 RBC distribution width 18 15 0 22 18 28 0 14 p 0 628 t Student test two tailed probability Table 1 Red Blood Cell RBC count and related RBC parameters from group housed 4 and 2 group Haematology A preliminary statistical evaluation for differences between the means of RBC count and related parameters in mice housed at a density of 2 and 4 to a cage in their respective groups IVC and open was performed Table 2 Intra cage and Room Temperature and Rel Humidity were sampled by means of Data Loggers during the last 4 weeks of the trial every 30 minutes Discussion The trial was carried out in the area of Milano Italy located at a mean altitude of 122 m above sea level It is important to note that IVCs pressurised environment is not capable of modifying the normobaric condition 760 mm Hg The conditioning period of approximately 6 weeks is sufficient to determine the complete turnover of RBC in mice7 8 9 which allows possible modifications due to altered environmental conditions to be expressed In our trial the differences were found to be limited to an expected slightly higher relative humidity and temperature in IVCs vs Open cages and the room Both RH and temperature can alter the reading of O2 concentration in air never theless recording instruments are able to maintain a linear reading in a range of temperature that includes values close to 0 C and up to 50 C On the other hand as the humidity in air increases water vapour molecules displace O2 molecules causing the output of the sensor to decrease Of course the effect is larger at higher temperatures because there is more water vapour in the air Relative humidity in the test room was unfortunately constantly close to the upper acceptable limit stated by international guidelines regulation field of variation 45 to 65 and was consequently unavoidable to find mean RH above 65 in the cages Since no statistical significant difference between the means was detected it was decided to pool the results 14 mice from IVC vs 14 mice from open cages During the last two days before the cage change procedural interval was 14 days RH in some IVCs was higher in comparison with both the room and open cages This might have played a minor role in the minor fluctuations of O2 concentration detected in that very short phase Table 2 shows that no differences were detected between the mean values of the haematological parameters tested for the two housing systems However no significant differences in mean Red Blood Cell RBC count and related RBC parameters body weight feed and water consumption were seen 85

Consistency of ventilation in IVCs and opencages their normoxic atmosphere and its impact on haematological parameters of mice between mice from the two systems ventilated and open confirming that the concentration of O2 in the cages of both groups was comparable and optimal for the physiological process of hematopoiesis Differences in the design and performance of IVCs such as the number of Air Changes per Hour 75 and air delivery point is probably the key point for the interpretation of the differences between our results and those described by York et al5 as an efficient turnover of air in the cage makes its microenvironment similar to the outside macroenvironment minimising differences in the atmosphere and any impact on mice physiology References 1 2 3 4 5 6 7 8 9 86 Grignaschi G Corsi L Zennaro E and Martino P A 2010 Breeding Performances as Welfare Indicator A Comparative Study on C57Bl 6J Mice in Three Different Individually Ventilated Caging Systems Lab Animal Europe Vol 10 6 40 41 Reeb Whitaker C K Paigen B Beamer W G Bronson R T Churchill G A Schweitzer B and Myers D D 2001 The impact of reduced frequency of cage changes on the health of mice housed in ventilated cages Laboratory Animals 35 58 73 Vogelweid C M Zapien K A Honigford M J Li L Li H and Marshall H 2011 Effects of a 28 Day CageChange Interval on Intracage Ammonia Levels Nasal histology and Perceived Welfare of CD1 Mice JAALAS Vol 50 6 868 878 Nagamine C M Long C T McKeon G P and Felt S 2012 Carbon Dioxide and Oxygen Levels in Disposable Individually Ventilated Cages after Removal from Mechanical Ventilation JAALAS Vol 51 2 155 161 York J M McDaniel A W Blevins N A Guillet R R Allison S O Cengel K A and Freund G G 2012 Individually ventilated cages cause chronic low grade hypoxia impacting mice hematologically and behaviorally Brain Behaviour and Immunity 26 951 958 Rosenbaum M D VandeWoude S Volckens J and Johnson T E 2010 Disparities in Ammonia Temperature Humidity and Airborne Particulate Matter between the Micro and Macroenvironments of Mice in individually Ventilated Caging JAALAS Vol 49 2 177183 Dod K S Bierman H R and Shimkin M B 1951 The disappearance of sulfhemoglobin in the blood as measure of erythrocyte in normal rabbits and mice and in tumor bearing mice J Nat Cancer Inst 2 1093 Burwell E L Buckley B A and Finch C A 1953 Erytrocyte life span in small animals comparison of two methods employing radio iron Am J Physiol 172 718 Van Putten L M and Croon F The Life Span of Red Cells in the Rat and the Mouse as Determined by Labeling with DFP32 in Vivo Blood 13 789 794

August 2013 Animal Technology and Welfare Discussion paper reducing severe suffering PENNY HAWKINS Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Report of a workshop held at the Institute of Animal Technology Laboratory Animal Veterinary Association Congress 2013 Correspondence to penny hawkins rspca org uk Abstract Structure of the workshop A workshop on reducing severe substantial suffering was held at a UK meeting for animal technologists and laborator y animal veterinarians in March 2013 Delegates discussed the roles that they were able to play in refining severe procedures including identifying any scientific or practical obstacles to reducing suffering and how these might be addressed Participants also suggested new resources or initiatives that could help to further reduce suffering and improve welfare The report of the meeting includes recommendations for ethical or animal care and use committees such as Animal Welfare Bodies trainers researchers and animal technologists and their professional bodies A total of 32 people attended of whom the majority were animal technologists and Named Animal Care and Welfare Officers NACWOs with several laboratory animal veterinarians The session began with some brief examples of how input from animal technologists has already successfully refined or avoided severe suffering within research projects The rest of the workshop was devoted to a discussion session in which delegates could exchange ideas and experiences with respect to refining severe procedures explore whether there were scientific or practical obstacles to avoiding severe suffering and discuss the roles that animal technologists can play in reducing severity in general People were also asked to think of any resources or initiatives that could help them to achieve further reductions in severe suffering Introduction Animal technologists and care staff hereafter referred to as animal technologists may be required to monitor and care for animals during procedures that cause severe substantial suffering They also play essential roles in ensuring that procedures are refined and that animals are effectively monitored and assessed so that suffering can be alleviated as far as possible The new European Directive 2010 63 EU1 which includes explicit requirements to refine procedures and reduce suffering was implemented in the UK as the revised Animals Scientific Procedures Act 1986 ASPA from 1 January 20132 With this in mind a workshop on Reducing Severe Suffering was held at the IAT LAVA Congress in March 2013 The workshop aimed to consider the role of the animal technologist in implementing both legal and ethical requirements to reduce suffering including through membership of the new local Animal Welfare and Ethical Review Body AWERB which will have similar roles to the local Ethical Review Process under the previous ASPA This paper aims to report on the workshop generate further discussion within establishments and make recommendations for further initiatives to help reduce severe suffering on the basis of the discussion and delegates conclusions on the day Workshop participants provided many examples of their opportunities to influence how protocols are conducted and refined within their own establishments three specific examples are summarised in Boxes 1 to 3 as well as some practical difficulties that they sometimes need to overcome The majority of the discussion and conclusions related to alleviating and avoiding suffering in general but they were nevertheless highly relevant to the primary focus of tackling severe suffering The recommended actions on training and communication within the text were suggested by participants on the day as new initiatives that would further help to reduce suffering General good practice within establishments Participants agreed that a good culture of care with high levels of trust and interaction between animal technologists and scientists is essential for the effective reduction of suffering This works best if technologists feel empowered with good support from Named Persons NACWOs and Named Veterinary Surgeons and facility management and there is also an effective and well suppor ted AWERB Animal 87

Discussion paper reducing severe suffering technologists should be among the AWERB members and all technologists should have good access to the AWERB regardless of whether they are members With a good culture of care there is increased opportunity for animal technologists to have input into protocols procedures and study design especially including refinements at the project design stage This may be through AWERB membership or involvement in project teams Several delegates explained how project licence applications were circulated to animal technologists and Named Persons at an early stage within their establishments This kind of positive open culture can be supported and informed by Home Office Inspectors More experienced participants felt that Inspectors have become much more approachable than they used to be with the result that the views and concerns of animal technologists were respected and supported by Inspectors and the relationship is more of a partnership Opportunities to attend and participate in animal welfare or 3Rs meetings such as those held by the RSPCA UFAW the NC3Rs IAT and LASA3 were highly valued Meeting with colleagues to discuss welfare and refinement issues is especially important at these events and the IAT was mentioned as a good means of liaising with other animal technologists Delegates also shared a variety of examples of good practice within their establishments relating to project review welfare assessment and refinement as listed below Mice were used in the safety assessment of an anticancer drug that caused endotoxic shock at some doses Animal technologists liaised with external colleagues caring for animals undergoing similar adverse effects where chips were used to transmit body temperature and enable better definition of humane endpoints Temperature chips were used in the study and it was discovered that an endpoint of a 2 C drop in temperature could be used to prevent avoidable suffering G Assessing welfare recording and communicating observations G G G G Reviewing projects and monitoring outcomes G 88 If licence applications are circulated to the AWERB in good time any significant welfare issues and potential refinements can be identified and addressed well before the project begins In some instances AWERBs have suggested pilot studies of protocols involving severe procedures so that the actual impact on the animals can be better assessed and understood and refinements identified and trialled Some establishments completed a form for each Effective day to day welfare assessment is critical e g using scoring or traffic light systems tailored to each project Spending quality time with the animals including extra handling and socialisation also helps to understand their normal behaviour and thus to better identify when there could be a welfare problem Internal passports help to alert staff to potential welfare issues for genetically altered GA animals including heterozygous crosses These documents are similar to the passpor ts that have been developed for use when transporting GA animals3 but with an emphasis and focus on local information requirements For example internal passpor ts define in detail what is normal for each line or cross making it easier to detect animals with additional welfare problems Technology can be used to communicate about animals within large or multi site facilities For example some participants explained how they can take photographs of animals and send the images to Named Persons via secure email Others used streaming video linkups to enable Named Persons or researchers to assess disease progression in animals Refinement Box 1 Refining an endpoint G protocol setting out the species strain age sex housing and care procedures and humane endpoints These are located near the animals housing so that they are accessible to all In addition a clearly defined plan of action for all animals in the event of welfare problems including who to contact when out of hours is displayed in all areas Regular or ad hoc retrospective reviews can be conducted in which actual severity is assessed and the potential for refinement is reconsidered rather than just one review at the end of the study G Housing husbandry and care refinements such as ensuring appropriate groupings of social animals and providing environmental enrichment can help to reduce suffering due to procedures as well as improving the quality of life for animals in general Providing good quality housing has been shown to reduce both behaviours associated with pain and analgesic requirements5 6 Some establishments regularly focus on refining humane endpoints taking advantage of new technology where possible This requires monitoring of relevant literature on both endpoints and potentially useful technological developments For This is within the remit of the Named Information Officer whose role is discussed later but others can also contribute towards accessing new knowledge

Discussion paper reducing severe suffering example infrared thermometers and implantable temperature transponders have been successfully used to refine endpoints and significantly reduce suffering in vaccine studies and some disease models7 8 Training Good training in assessing and reducing suffering was regarded as essential Those present at the workshop were satisfied with the level of training that they had received through a combination of modular licensee training IAT qualifications and National Vocational Qualifications NVQs The consensus was that all animal technologists should do Home Office training modules 1 to 3 which include ethics the biology and husbandry of the species and recognising pain and distress even if they did not conduct regulated procedures This was because the modules were recognised to instil the correct mind set in trainees which then has to be consolidated and built on through good in house training and Continuing Professional Development CPD G G severe procedures should at least go and see the animals undergoing these and observe the effects More training in animal behaviour and biology should be provided for researchers to enable better understanding of the impact of research on animals the benefits of refinement and techniques for recognising and alleviating suffering Work should be done to facilitate better recognition of more subtle indicators of poor welfare or suffering as this can help to refine endpoints and ideally avoid severe suffering This could include producing training resources including pictures and video clips of animals at different levels of suffering not just severe Figure 1 Some material like this is already available at the Assessing the Health and Welfare of Laboratory Animals website http www ahwla org uk C57 BL6 mice were bought in at six to eight weeks old and housed in groups of ten This led to fighting so that groups had to be split up and animals disrupted Animal technologists suggested altering husbandry protocols to permit some used nesting material to be transferred when cage cleaning and males and females were group housed in separate rooms Problems with aggression ended and the mice are now successfully group housed Box 2 C57 BL6 management It was felt that training should include up to date information on new approaches to assessing animal behaviour and welfare such as grimace scales 9 Although it is not always feasible to implement techniques such as these locally delegates took the view that keeping up with developments in these fields encourages people to think more about ways of assessing animals and to keep open minds about what causes animals to suffer and how animals may show subtle signs of suffering A good level of ongoing access to literature e g the IAT and UFAW Handbooks10 11 and other resources is essential Recommended actions on training G Ideally all scientists should have to be animal technologists for a while This would help researchers to gain better understanding of animals and their behaviour and to understand how technologists feel so that they might perhaps consider more fully what they are asking technologists to do when severe procedures are involved All researchers whose projects include Figure 1 This mouse is beginning to experience adverse effects associated with a tumour Body shape and posture are slightly altered and the coat is slightly ruffled in appearance Appropriate action at this point and or increased monitoring of the animal could help to prevent more severe suffering Photo credit AHWLA Communication All delegates emphasised that good communication was the most important factor with respect to reducing severe suffering and promoting consistency in how this is done The US based Laboratory Animal Welfare Training Exchange www lawte org was mentioned as a good example of facilitating communication on training issues including the exchange of training materials In the UK communication about all 3Rs is one of the roles of the Named Information Officer NIO This post is one of the specific requirements for personnel in Directive 2010 63 EU1 as set out in Article 24 1b Member States shall ensure that each breeder supplier and user has one or several persons on site who shall ensure that the staff dealing with animals have access to information specific to the species housed in the establishment 89

Discussion paper reducing severe suffering The NIO may well be a senior animal technologist although this is not a foregone conclusion and it is up to the local establishment to appoint the most suitable person Whatever the background of the NIO others can also take on the task of retrieving new information For example animal technologists can gather information from courses meetings and colleagues With respect to reducing severe suffering technologists can encourage the NIO to specifically seek out information on refinement that is relevant to procedures at their establishment or pass their own findings on to the NIO for further dissemination The AWERB was also regarded as a valuable means of internal communication about refinement The importance of sharing information between animal technologists was a recurring theme and the ability to discuss the issue of severe suffering and exchange ideas and information about refinement and the culture of care were both rated highly in par ticipants feedback forms at the end of the session Delegates also mentioned actively using the IAT EFAT journal Animal Technology and Welfare and meetings to communicate and exchange ideas on a range of topics including refinement A neuroscience researcher wanted to house guinea pigs individually post surgery due to concerns that they would gnaw one another s wound dressings Animal technologists discussed further options with the researcher and pointed out that going from group housing to individual housing in an unfamiliar environment post surgery could well affect results It was agreed to pair house the guinea pigs following surgery and there were no problems Box 3 Guinea pig housing post surgery Approachable animal technologists who cultivate good relations with scientific staff were viewed as essential for building a culture of cooperation and good communication with respect to reducing and avoiding severe suffering and to refinement in general This requires confidence and support as outlined earlier one group described how positive it was to be able to ask colleagues for a second opinion about an animal and be sure that they would be suppor ted if necessary Relationships can be greatly improved by initiatives that bring people with different roles together such as internal seminars and discussions where researchers present an overview of their work to animal technologists to provide a better understanding of the objectives animal models and potential benefits of their projects This can help staff to suggest appropriate refinements and very importantly gives animal technologists more confidence to raise welfare concerns with researchers and ask for fur ther discussion or for refinements to be implemented 90 Recommended actions on communication G G G Research and testing facilities should set up exchange programmes in which animal technologists and NACWOs could work at or visit other facilities to learn about refinement techniques and how these are researched implemented and supported Establishments should ensure that research staff include details of the level of suffering experienced by animals in their publications with the refinements that were put in place to alleviate any pain or distress so that other researchers can implement these The ARRIVE guidelines provide a good basis for this approach12 The IAT should set up an online chat room for animal technologists As an example the American Association for Laboratory Animal Science AALAS hosts the Techlink mailing list http www aalas org online_resources listser ves aspx but a UK equivalent is needed Some difficult issues Some delegates had found researchers reluctant to implement refinements to husbandry or procedures that would reduce or avoid severe suffering This was usually due to concerns about introducing new variables or generating data that would not be compatible with previous research or sometimes for economic reasons or simply the fear of change and a desire to maintain traditional practice Par ticipants had also experienced problems with respect to assessing animal suffering and therefore being able to reduce it which can drift towards suboptimal practice which then becomes established as the norm Under implementation of refinement and ineffective assessment of severity can clearly both impact on the ability to reduce severe suffering and animal technologists need support if they are to challenge the status quo and improve practice As minimising suffering is a requirement of the ASPA support should always be forthcoming However some delegates reported that they were increasingly able to negotiate with researchers about changing and refining experimental and husbandry protocols and about improving welfare assessment due to the improved status of animal technologists and better trust between people with different responsibilities This was also expressed as less of an us and them situation A further problem identified by participants was the lack of time that there can be to implement some refinements such as making proper observations of animals and undertaking habituation programmes although it was noted that breeders could initiate the latter The time factor was also an issue in pre study

Discussion paper reducing severe suffering briefings where implementation was patchy and animal technologists less likely to be at these meetings than Named Persons even though input from technologists is recognised as being highly valuable In practice most of the problems identified within the workshop were fundamentally financial ones For example allocating sufficient staff time to monitor animals effectively conducting pilot studies funding CPD implementing refinements such as heat pads or additional nesting material and purchasing technology such as webcams for monitoring or temperature chips all cost money The consensus was that all of these measures are money well spent for both animal and human welfare as well as the potential scientific benefits Acknowledgements Thank you to the IAT for accepting and promoting the workshop to Elliot Lilley and Maggy Jennings for their input into its structure to Kate Heath of GSK for her presentation to all the participants for their open discussion and ideas and finally again to Maggy for her helpful comments on the layout of this paper References 1 2 3 Conclusion The workshop participants were strongly motivated to reduce suffering at all levels especially severe suffering and in general felt well supported in their attempts to do so This is of course a relatively small sample of animal technologists and results may well be biased given that the delegates were IAT members seeking Continuous Professional Development and supported in their wish to attend Congress so were arguably more likely to receive positive encouragement than others On that basis it would be a useful exercise for all AWERBs to review and consider both the examples of good practice and the difficult issues to see whether any of the positive statements apply or could be implemented locally and whether any of the negative factors also apply and ought to be addressed In particular AWERBs could ensure that full use is made of experienced and empathetic animal technologists when designing running and reviewing research projects The recommendations within this report relate to training bodies providers of training material and the IAT as well as research and testing establishment staff and AWERBs They will be submitted to relevant people and bodies and there are also some closing recommendations for animal technologists that can be drawn from this report 4 5 6 7 8 9 10 11 G G G If you do not currently interact with the AWERB at your establishment see whether you can join or attend meetings or ensure that the AWERB knows of your interest and provides you with feedback Pass this paper to your NIO and AWERB for consideration and discussion are there any useful ideas or does it highlight issues that ought to be dealt with Go through this paper again and see whether it inspires you to play a more active role in refinement within your establishment or to communicate more both internally and externally about your efforts so far 12 European Commission 2010 Directive 2010 63 EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes Official Journal of the European Union L 276 33 79 A definitive version of the revised Act will be made available at www legislation gov uk Universities Federation for Animal Welfare National Centre for the Three Rs Laboratory Animal Science Association RSPCA GA Passport Working Group 2010 GA Passports The Key to Consistent Animal Care RSPCA Southwater Free download at http tinyurl com 6fvcctn Gabriel A F Marcus M A E Honig W M M and Joosten E A J 2010 Preoperative housing in an enriched environment significantly reduces the duration of post operative pain in a rat model of knee inflammation Neurosci Lett 469 219 223 Pham T M Hagman B Codita A Van Loo P L Str mmer L and Baumans V 2010 Housing environment reduces the need for pain relief during postoperative recovery in mice Physiol Behav 99 663 668 Warn P A Brampton M W Sharp A Morrissey G Steel N Denning D W and Priest T 2003 Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections Laboratory Animals 37 126 131 Kort W J Hekking Weijma J M TenKate M T Sorm V and VanStrik R 1998 A microchip implant system as a method to determine body temperature of terminally ill mice Laboratory Animals 32 260 269 Leach M C Klaus K Miller A L Scotto di Perrotolo M Sotocinal S G and Flecknell P A 2012 The assessment of post vasectomy pain in mice using behaviour and the Mouse Grimace Scale PLoS ONE 7 4 e35656 doi 10 1371 journal pone 0035656 Barnett S ed 2007 Manual of Animal Technology Blackwell Publishing Oxford Hubrecht R and Kirkwood J eds 2010 The UFAW Handbook on the Care and Management of Laboratory and Other Research Animals 8th edition WileyBlackwell Oxford Kilkenny C Browne W J Cuthill I C Emerson M and Altman D G 2010 Improving bioscience research reporting the ARRIVE guidelines for reporting animal research 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August 2013 Animal Technology and Welfare PAPER SUMMARY TRANSLATIONS INHALTVERZEICHNIS Eine einfache methode zur endotrachealen intubation bei m usen ANASTASIA PAPASTEFANOU EVANGELOS BALAFAS und NIKOLAOS KOSTOMITSOPOULOS1 Centre for Experimental Surgery Biomedical Research Foundation of the Academy of Athens 4 Soranou Ephesius Street 11527 Athen Griechenland 1 Korrespondierender autor nkostom bioacademy gr Abstract Endotracheale Intubation wird bei M usen gew hnlich im Zusammenhang mit einer erforderlich werdenden Atemwegssicherung angewandt Es handelt sich aufgrund der geringen Gr e von M usen und ihres Mundrachenraums um ein schwieriges Verfahren In der wissenschaftlichen Literatur werden verschiedene Methoden endotrachealer Intubation f r M use beschrieben wof r meist kostspielige Ger te bzw spezielle Schulung erforderlich sind In diesem Bericht beschreiben wir unsere Erfahrungen mit einer einfachen verl sslichen und kosteng nstigen Methode zur endotrachealen Intubation bei M usen die durch den Mund erfolgt und in unserem Zentrum entwickelt wurde Sie basiert auf dem Einsatz kosteng nstiger Materialien ber die nahezu alle chirurgischen Labors verf gen 93

Paper Summary Translations Konsistenz der bel ftung in IVCs individually ventilated cages einzelbel fteten k figen und offenen k figen ihre normoxische atmosph re und ihr einfluss auf h matologische parameter von m usen GIANPAOLO MILITE1 und LUCIA BUCCARELLO2 1 2 Wissenschaftlicher Berater Udine Italien Dipartimento di Scienze e Tecnologie Veterinarie Universit di Milano Mailand Italien Korrespondierender autor gianpaolo milite fastwebnet it Abstract In einer einzelbel fteten K figanlage IVCs wurde k rzlich eine Sauerstoffkonzentration festgestellt die leicht unter dem f r eine ideale Mikro Umgebung f r Versuchs Hausm use optimalen Wert lag Angesichts der betr chtlichen technischen Unterschiede zwischen IVC Bauarten die zu unterschiedlichen Umgebungsbedingungen in den einzelnen K figen f hren k nnen wurde beschlossen die Mikro Umgebungen einer IVC Anlage und einer offenen K figanlage zu berwachen und die entsprechenden Auswirkungen auf die Anzahl roter Blutk rperchen und damit verbundener physiologischer Parameter von in offenen K figen gehaltenen C57Bl 6J M usen zu vergleichen Dabei wurde kurz vor dem K figwechsel der alle zwei Wochen stattfindet in beiden K figanlagen ein geringer R ckgang des Sauerstoffgehalts im K fig festgestellt Unterschiede zwischen den h matologischen Parametern wurden nach 6 w chiger Konditionierung der M use in keiner der beiden Anlagen festgestellt In der IVC Mikro Umgebung wurden beim Vergleich mit der Versuchsanlage des offenen K figs keine Auswirkungen auf die bei diesem Versuch betrachteten h matologischen Parameter festgestellt 94

Paper Summary Translations Diskussionspapier verringerung von starken belastungen f r versuchstiere PENNY HAWKINS Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Bericht ber einen Workshop im Rahmen des Kongresses der Laboratory Animal Veterinary Association 2013 am Institute of Animal Technology Korrespondierender autor penny hawkins rspca org uk Abstract Im M rz 2013 fand im Rahmen der britischen Tagung f r Tiertechniker und Versuchstier rzte ein Workshop ber Methoden zur Verringerung von starken wesentlichen Belastungen f r Versuchstiere statt Die Teilnehmer diskutierten die Aufgaben die sie bei der Verfeinerung diesbez glicher Methoden bernehmen k nnen wie z B die Identifizierung wissenschaftlicher oder praktischer Hindernisse f r eine Verringerung der Belastungen f r Tiere und wie diese berwunden werden k nnen Die Teilnehmer schlugen zudem neue Mittel und Initiativen zur weiteren Senkung der Belastungen f r Tiere und zur Verbesserung des Tierschutzes vor Der Tagungsbericht enth lt Empfehlungen zu Tierethik und pflege und f r Gremien wie Tierschutzorganisationen Trainer Forscher und Tiertechniker und deren Berufsverb nde 95

Animal Technology and Welfare August 2013 CONTENU DE LA REVUE M thode simple d intubation endotrach ale des souris ANASTASIA PAPASTEFANOU EVANGELOS BALAFAS et NIKOLAOS KOSTOMITSOPOULOS1 Centre de chirurgie exp rimentale Fondation pour la recherche biom dicale de l acad mie d Ath nes 4 rue Soranou Ephesius 11527 Ath nes Gr ce 1 Auteur principal nkostom bioacademy gr R sum L intubation endotrach ale des souris est couramment utilis e lorsqu une assistance respiratoire s impose Il s agit d une proc dure complexe en raison de la petite taille des souris et de leur oropharynx Diff rentes m thodes d intubation endotrach ale des souris sont d crites dans les ouvrages scientifiques et la plupart de ces m thodes n cessitent l utilisation d appareils co teux et ou une formation sp ciale Dans le pr sent rapport nous d crivons notre exp rience li e l utilisation d une m thode simple fiable et peu co teuse consistant r aliser une intubation endotrach ale des souris par la bouche m thode que nous avons d velopp e au sein de notre centre au moyen d quipements peu co teux et pr sents dans pratiquement tous les laboratoires de chirurgie 96

Paper Summary Translations Homog n it du syst me de ventilation au sein des cages ventilation individuelle CVI et des cages ouvertes atmosph re normoxique et impact sur les param tres h matologiques des souris GIANPAOLO MILITE1 et LUCIA BUCCARELLO2 1 2 Consultant scientifique Udine Italie Dipartimento di Scienze e Tecnologie Veterinarie Universit de Milan Milan Italie Auteur principal gianpaolo milite fastwebnet it R sum On a r cemment d couvert que la concentration d oxyg ne au sein d un syst me de cage ventilation individuelle CVI tait l g rement inf rieure la valeur optimale li e au microenvironnement id al pour pouvoir conserver des souris tant donn que le nombre consid rable de diff rences techniques qui existe entre les diff rents mod les de CVI peut g n rer des conditions environnementales in gales l int rieur des cages il a t d cid de surveiller le microenvironnement d un syst me de cage ventilation individuelle CVI et d un syst me de cage ouverte et de comparer l impact sur le nombre de globules rouges et sur les param tres physiologiques associ s des souris du groupe C57Bl 6J h berg es dans des cages ouvertes Une l g re diminution du niveau d oxyg ne l int rieur des cages a t d tect e pour les deux syst mes de cage l approche du processus de changement de cage effectu toutes les deux semaines Aucune diff rence n a t identifi e au niveau des param tres h matologiques apr s les 6 premi res semaines d adaptation des souris aux deux syst mes Il a t d montr qu en comparaison avec le syst me de cage ouverte mis en place les param tres h matologiques pris en consid ration lors de cet essai ne sont en aucun cas influenc s au sein du microenvironnement du syst me de cage ventilation individuelle 97

Paper Summary Translations Document de discussion r duction des souffrances aigu s PENNY HAWKINS Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Rapport d un atelier organis l institut de la technologie animale lors du congr s 2013 de l association des v t rinaires sp cialistes des animaux de laboratoire Auteir principal penny hawkins rspca org uk R sum En mars 2013 un atelier sur la r duction des souffrances aigu s substantielles a t organis au Royaume Uni lors d une r union rassemblant des technologues en sant animale et des v t rinaires sp cialistes des animaux de laboratoire Les repr sentants ont discut des r les qu ils ont pu jouer dans le cadre de l am lioration des proc dures relatives aux souffrances aigu s comme par exemple l identification d obstacles scientifiques ou pratiques la r duction des souffrances et de solutions permettant d y faire face Les participants ont galement sugg r de nouvelles ressources ou initiatives pouvant contribuer r duire davantage les souffrances et am liorer le bien tre des animaux Le rapport de cette r union inclut des recommandations destin es aux comit s d thique et de protection et d utilisation des animaux tels que les organismes de protection des animaux formateurs chercheurs technologues en sant animale ainsi qu leurs corps professionnels 98

August 2013 Animal Technology and Welfare INDICE DE LA REVISTA Un m todo simple de intubaci n endotraqueal en ratones ANASTASIA PAPASTEFANOU EVANGELOS BALAFAS y NIKOLAOS KOSTOMITSOPOULOS1 Centre for Experimental Surgery Biomedical Research Foundation of the Academy of Athens 4 Soranou Ephesius 11527 Atenas Grecia 1 Autor correspondiente nkostom bioacademy gr Resumen La intubaci n endotraqueal en ratones se suele utilizar cuando es necesario un manejo de las v as a reas El proceso es dif cil de realizar debido al peque o tama o de los ratones y de su orofaringe Varios m todos de intubaci n endotraqueal en ratones han sido descritos en documentos cient ficos y la mayor a de esos m todos requieren el uso de aparatos caros y o formaci n especial En este informe describimos nuestra experiencia con un m todo f cil fiable y asequible para realizar intubaciones endotraqueales a ratones a trav s de la boca Este m todo ha sido desarrollado en nuestro Centro utilizando materiales de bajo coste que pueden ser encontrados en la mayor a de laboratorios quir rgicos 99

Paper Summary Translations Consistencia de la ventilaci n en sistemas de jaulas ventiladas individualmente y en jaulas abiertas su atm sfera norm xica y su impacto en los par metros hematol gicos de los ratones GIANPAOLO MILITE1 y LUCIA BUCCARELLO2 1 2 Asesor cient fico Udine Italia Dipartimento di Scienze e Tecnologie Veterinarie Universit di Milano Mil n Italia Autor correspondiente gianpaolo milite fastwebnet it Resumen Recientemente se descubri que la concentraci n de ox geno en un sistema de jaula ventilada individualmente es ligeramente inferior de lo que se considerar a un nivel ptimo en un microentorno ideal para hospedar ratones de laboratorio Dadas las diferencias sustanciales t cnicas entre los sistemas de jaulas ventiladas de distintos dise os que pueden provocar unas condiciones ambientales distintas dentro de la jaula se decidi controlar el microentorno de un sistema de jaula ventilada individualmente y de un sistema de jaula abierta y comparar el impacto del recuento de gl bulos rojos y de los par metros fisiol gicos relacionados en ratones C57Bl 6J hospedados en jaulas abiertas Se detect una peque a disminuci n del nivel de ox geno dentro de la jaula de ambos sistemas al inspeccionar el proceso de cambio de jaula llevado a cabo cada dos semanas No se detectaron diferencias en los par metros hematol gicos tras 6 semanas de aclimataci n de los ratones en cualquiera de los dos sistemas El microentorno del sistema de jaula ventilada individualmente demostr que no se hab a visto afectado en cuanto a los par metros hematol gicos tomados en consideraci n en esta prueba al compararse con el sistema de jaula abierta 100

Paper Summary Translations Documento de debate reducci n del sufrimiento grave PENNY HAWKINS Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Informe sobre un taller celebrado en el Institute of Animal Technology Laboratory Animal Veterinary Association Congress 2013 Autor correspondence penny hawkins rspca org uk Resumen En marzo de 2013 se ha celebrado un taller sobre la reducci n del sufrimiento grave sustancial en el Reino Unido para tecn logos de animales y veterinarios de animales de laboratorio Los delegados debatieron sobre el papel que pod an desempe ar a la hora de refinar los procedimientos severos incluyendo la identificaci n de cualquier obst culo pr ctico o cient fico para reducir el sufrimiento y sobre c mo poder hacer frente a estos problemas Los participantes tambi n sugirieron nuevos recursos o iniciativas que pueden ayudar a reducir el sufrimiento y mejorar el bienestar El informe de la reuni n incluye recomendaciones para un cuidado animal tico y el uso de comit s como Organismos para el bienestar de los animales formadores investigadores y tecn logos de animales y sus organismos profesionales 101

Animal Technology and Welfare August 2013 INDICE DELLA REVISTA Un metodo semplice per l intubazione endotracheale dei topi ANASTASIA PAPASTEFANOU EVANGELOS BALAFAS e NIKOLAOS KOSTOMITSOPOULOS1 Centro di chirurgia sperimentale Fondazione per la ricerca biomedica dell Accademia di Atene 4 Soranou Ephesius Street Atene 11527 Grecia 1 Autore corrispondente nkostom bioacademy gr Abstract L intubazione endotracheale nei topi viene comunemente usata quando necessario gestire le vie aeree La procedura difficile da attuare a causa delle dimensioni ridotte dei topi e della loro orofaringe Nella letteratura scientifica sono stati descritti vari metodi d intubazione endotracheale dei topi e la maggior parte di questi metodi richiede l uso di dispositivi costosi e o di una formazione speciale In questa relazione descriviamo la nostra esperienza con un metodo facile sicuro ed economico per l intubazione endotracheale dei topi attraverso la bocca messo a punto nel nostro centro usando materiali a basso costo disponibili presso quasi tutti i laboratori chirurgici 102

Paper Summary Translations Coerenza della ventilazione nelle gabbie IVC e nelle gabbie aperte la loro atmosfera normossica e il relativo effetto sui parametri ematologici dei topi GIANPAOLO MILITE1 e LUCIA BUCCARELLO2 1 2 Consulente scientifico Udine Italia Dipartimento di Scienze e Tecnologie Veterinarie Universit di Milano Milano Italia Autore corrispondente gianpaolo milite fastwebnet it Abstract Recentemente stato scoperto che la concentrazione di ossigeno in un sistema di gabbie ventilate individualmente IVCs leggermente inferiore a quella ottimale per un microambiente ideale da topi di laboratorio Date le differenze tecniche sostanziali tra gabbie IVC progettate diversamente che possono quindi produrre condizioni ambientali all interno della gabbia altrettanto diverse si deciso di monitorare il microambiente di un sistema di gabbie a ventilazione individuale IVC e di un sistema a gabbie aperte e di confrontare l impatto sul conteggio dei globuli rossi e sui relativi parametri fisiologici nei topi C57BL 6J alloggiati in gabbie aperte Una piccola diminuzione del livello di ossigeno all interno della gabbia stato rilevato in entrambi i sistemi quando si avvicinava la procedura di cambio gabbia eseguita ogni due settimane Non sono state rilevate differenze nei parametri ematologici dopo 6 settimane di condizionamento dei topi in entrambi i sistemi Il microambiente IVC ha dimostrato di essere esente da qualsiasi effetto sui parametri ematologici presi in considerazione in questa prova una volta confrontato con il sistema a gabbia aperta 103

Paper Summary Translations Documento di lavoro riduzione della sofferenza grave PENNY HAWKINS Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Resoconto del workshop svoltosi presso l Istituto di Tecnologie Animali Edizione 2013 del Congresso della Laboratory Animals Veterinary Association Autore corrispondente penny hawkins rspca org uk Abstract Nel marzo 2013 durante un meeting nel Regno Unito destinato agli stabularisti e ai veterinari specializzati in animali da laboratorio si svolto un workshop sulla riduzione della sofferenza grave sostanziale I delegati hanno discusso i ruoli che erano in grado di rivestire nell ottimizzazione delle procedure con sofferenza grave inclusa l identificazione degli eventuali ostacoli scientifici o pratici nella riduzione della sofferenza e di come questi possano essere affrontati I partecipanti inoltre hanno suggerito nuove risorse o iniziative che potrebbero contribuire ulteriormente a ridurre la sofferenza e a migliorare il benessere La relazione della riunione include le raccomandazioni per i Comitati etici o per l uso e la buona sperimentazione animale come gli Animal Welfare Bodies gli addestratori i ricercatori gli stabularisti e i loro organismi professionali 104

August 2013 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 GA Passports Workshop Report IAT LAVA Congress 2013 NIKKI OSBORNE1 CAROLINE CHADWICK2 MARY ANN HASKINGS3 JENNY SALISBURY4 and AMANDA PICKARD5 1 2 3 4 5 Research Animals Department RSPCA Wilberforce Way Southwater West Sussex RH13 9RS University of Birmingham Edgbaston Birmingham B15 2TT Cancer Research UK London Research Institute Potters Bar South Mimms Hertfordshire EN6 3LD Wellcome Trust Sanger Institute Hinxton Cambridge CB10 1SA FESA MRC Harwell Harwell Science Innovation Campus Oxfordshire OX11 0RD Corresponding author nikki osborne rspca org uk Introduction The RSPCA GA Passports Working Group GAPWG was set up in 2008 to progress and develop the idea of mouse passports 1 and welfare databases as tools for ensuring consistent standards of care throughout the lifetime of all genetically altered GA animals The idea of communicating animal care information when animals are transferred between establishments is not a new one Indeed in 2001 an APC report on Biotechnology suggested the development of databases to contain information with welfare implications for all strains of GA animals and recommended that data relating to any adverse welfare effects should be made available to any potential user of a new GA strain 2 This idea resurfaces in the 2003 JWGR report recommending that The nature of the phenotype and any specialist care required should be discussed prior to the transport of the mice and detailed in written information accompanying the GM mice 3 Three years later a GA mouse working group set up to progress many of the recommendations contained within the 2001 APC report published their 2006 report1 recommending that information on any welfare concerns should be documented and should form the basis of a welfare profile that is also used to create a mouse passport This report also provides a template mouse passport for people to use when transferring 105

Tech 2 Tech GA mice between establishments A FELASA working group report a year later4 recommends that for every transgenic strain a data sheet should be prepared at the first oppor tunity which collates all available information relating to its breeding husbandry and care This data sheet or a pointer to enable the information to be accessed via the web should be sent in advance to the receiving laboratory or central repository and a further copy should accompany the animals The FELASA paper also contains a list of the minimum level of information that FELASA working group members consider should be recorded within a data sheet By 2008 no significant progress appeared to have been made within the UK in terms of the recommendations becoming common practice so the GA Passports Working Group GAPWG was formed The group comprises representatives from a range of UK commercial academic and private organisations with an interest in the use of GA animals for research purposes and who had knowledge of the published recommendations and or experience of putting these recommendations into practice During discussions the group were unanimous that there is not one passport data sheet database entry format that works for all establishments nor is there a reason to limit the initiative to just GA mice With this in mind in 2009 the group published an easy to read quick reference booklet of recommendations with the aim of establishing a minimum standard of information to be shared between establishments when sending or receiving a GA animal of any species5 Since then group members have been raising awareness of GA passports through poster and oral presentations at meetings and conferences both within the UK and internationally The latest of these presentations was the introductory talk during the GA Passports workshop at the IAT LAVA Joint Congress 2013 Below is a summary of the talks and discussions from this workshop GA passports the story so far First to speak was Nikki Osborne convenor of the RSPCA GAPWG who gave a brief history of the background to the formation of the working group see above before introducing participants to the GA Passports the key to consistent animal care booklet5 The booklet defines a GA Passport as a record containing all the information that any staff caring for the GA animal can use to minimise the potential for pain suffering or distress and improve welfare The format of the information does not matter in terms of whether it is a paper document an electronic file an entry on a spreadsheet or welfare database so this can be varied according to resource It does however need to be intuitive to use as well as quick and easy to complete in order for it to be a practical addition to routine practice 106 There are many reasons to use GA passports to reduce the incidence of adverse events such as breeding failure disease outbreaks and health or welfare problems to minimise the need to duplicate or replicate research by sharing all known screening results or phenotypic data positive and negative to disseminate good practice around the world by sharing information on housing husbandry enrichments and procedural refinements These are just three reasons and whilst the value of specific pieces of information contained within a GA passport will vary between individuals in terms of what they consider to be of major or minor importance the crucial point is that the passport package of information serves to improve animal welfare and the quality of the scientific data There is an argument that the data contained within a GA passport should be recorded and shared for all animals regardless of species and genetic status but the passport is specifically relevant when an animal or indeed embr yos or gametes are transferred between locations where their journey ends with a new set of carers In this situation a copy of the passport should always remain with or accompany the animal or materials whilst being transported but when sending live animals it becomes vital that the information is also sent in advance so that the new set of carers can prepare in advance for the new arrival Deciding what details should be included within the GA passport proved very straightforward with ten fields of information featuring within all the passpor t or database systems that group members had developed and implemented These top ten information fields were name using formal nomenclature and local name if applicable general information such as colour diet housing enrichment behaviour phenotypic abnormalities and observable traits with welfare implications remedial actions for all adverse effects breeding strategy performance method of supply fresh frozen protocols origin who when and where created background strain or stock and the back cross inter cross generation of animals supplied if appropriate contact details person s at supplying establishment name number email postal address and other including references websites additional contact details for the originator or carer NVS extra scientific information such as PCR protocol stem cell info etc and a list of phenotypic screening undertaken to date with statement of positive negative result The final factor to consider when creating or updating passports is the language which needs to be clear easy to understand and defined such as the terms found at www mousewelfareterms org or nomenclature according to current rules to name just two examples GA Passports Building a Birmingham passport Second to present was Caroline Chadwick from

Tech 2 Tech University of Birmingham Birmingham was one of the first establishments to develop a mouse passport as early as 1999 so Caroline began by using the Turning Point system of handsets to get the participants to vote what level of importance they attribute to different fields of passport information Strain name contact details origin and background came top of the essential list with 88 of votes closely followed by phenotype and welfare information with 76 of the vote Next came breeding performance with 60 of the vote and phenotypic screening history with 53 of the vote Bedding and enrichment came top of the list for useful information with 60 of the vote followed by PCR protocols at 50 A photograph or references took joint third place in terms of useful information with 47 of the vote closely followed by diet and water with 41 of the vote The field of information considered least important of the options provided was cage type with 60 of the vote Caroline went on to discuss where passports and the information they contain should be kept The assumption is that the information arrives at the new establishment with the GA animals in this case mice and is stored on a computer somewhere or filed in the boss s office If the point of having a passport of information is to ensure consistent animal care then it does raise the question why isn t the information with the mice Each establishment will handle and disseminate information in different ways depending on the resources available but at Birmingham they have introduced cage side welfare cards These cards are coloured so they are easy for staff to identify when entering a room and contain key information For example Name Mickey This strain can experience seizures Do not disturb Close visual check only Name Minnie The homozygotes do not develop teeth Provide soft diet daily The aim of these welfare cards is to ensure relevant husbandr y welfare information is available to all staff and to highlight strains where there is an adverse phenotype This cage side approach is supported by the inclusion of the full passport record of information on a central database and means that problems getting hold of the correct nomenclature strain name and full history of the respective animals do not prevent staff access to information on the cage side welfare cards GA Passports for multiple species Next to present was Mary Ann Haskings from the London Research Institute LRI of Cancer Research UK To provide some context for her talk on developing GA Passports for multiple species Mary Ann provided some background information on mouse cages and zebrafish tanks within her establishment This highlighted the fact that the import export activity is predominantly mice and zebrafish and accounted for approximately 200 movements last year These movements were overseen by an impor t expor t co ordinator with around 80 of the GA mice sent as live mice and the remaining 20 being embryos or sperm Zebrafish are transported as embryos At the LRI the primary challenge in relation to GA passpor ts is providing gathering the complete information As mentioned by previous speakers the process and resulting information needs to be user friendly something that can be more complicated than it may first appear In the past importing and exporting has entailed two different forms because some of the details can vary but the aim was to have one format for both that can be completed for either species The chosen format also needed to be understandable by people both within CR UK and from external institutes The solution for the LRI was to ensure that all the fields recommended in the GAPWG good practice booklet were included within the internal impor t expor t database This development enabled them to incorporate drop down lists wherever possible to speed up the time taken to enter information and enable irrelevant fields to be greyed out The use of a database also facilitates the automatic generation of electronic and or paper documentation when exporting and enables the automatic upload of information on impor ted animals once the relevant fields are completed and submitted It has also enabled the inclusion of statements at relevant points within the process to remind individuals of current good practice For example the material sections states live animal transfers should be requested only when receiving tissue gametes or embryos is not practical In terms of welfare related information the LRI encompasses it all within the husbandry information section of the passport database The general section includes a description of the supplying facility in terms of whether it s a closed facility or allows introduction with quarantine or direct introduction from commercial or non commercial sources description of barrier caging tank system dietar y regime standard environmental enrichment and or special husbandry requirements The description of the mouse or zebrafish includes strain name type genotype background colour and originator details Phenotype information is expanded to include a description with phenotype age phenotype first detected preweaning early mor tality age when moribund or endpoint and breeding performance This level of information is also included when entering the details of zebrafish they hold within the Zebrafish International Resource ZIR center database www zebrafish org Like the ongoing work at other establishments the LRI passport system is still quite new and is continuing to be developed and refined over time Mar y Ann discussed the ongoing challenges in terms of 107

Tech 2 Tech perfecting how to store what will become a growing body of passport information so that it is both secure and accessible to all who need it There is also work to be done in relation to how the information will be used for example flags are being added to the data to make sure that important information is noted Another challenge for the future faced by all establishments is that each export will require a new passport to be generated with the most up to date information something which brings with it the question of who should be responsible for compiling and updating passport data and how duplicated effort can be avoided GA Passports as living records This question was progressed further by the fourth speaker Jenny Salisbury from the Wellcome Trust Sanger Institute WTSI The WTSI research support facility is one of the largest within the UK contributing to international efforts to generate and characterise targeted knock out mouse and zebrafish models through initiatives such as International Mouse Phenotyping Consortium IMPC Baylor College of Medicine Sanger Harwell BaSH consortium Knock Out Mouse Project KOMP and European Conditional Mouse Mutagenesis EUCOMM as well as generating huge amounts of phenotyping data through high throughput screening One of the key measures of success at the WTSI is the early production and sharing of mouse models with requests from the scientific community accepted as soon as one live heterozygous individual with the confirmed genotype is identified Sharing details of GA animals at such an early stage brings with it some novel challenges as well as some more common issues associated with the gathering and sharing of information with individuals and facilities on all continents and in multiple languages Both of these issues mean that the value of using passports as a communication tool when importing and or exporting GA animals was quickly recognised and the recommendations of the GAPWG adopted from the outset Like the LRI the WTSI has linked their passport to an in house database that incorporates welfare observation data The database has been quite labour intensive to set up and continues to develop to meet the facility s growing needs but led to the early realisation that to facilitate searching filtering and collation of data a controlled vocabulary would be required This marked the beginning of an ongoing collaborative effort with MRC Harwell to develop a controlled list of welfare terms to ensure accurate and consistent repor ting of phenotypes and welfare observations The whole scientific and animal care community are encouraged to contribute to this initiative through the website address www mousewelfareterms org Jenny illustrated the 108 impor tance of using controlled vocabular y and recording welfare observation data within the database to demonstrate that phenotypes and welfare concerns can and do occur throughout the life of a colony not just during the time that the line is undergoing specific phenotypic screening For example analysing data for 386 models with 28 progeny within the database enabled them to look at the percentage of offspring resulting from heterozygous inter crossing at postnatal day 14 that were lethal 0 homozygous sub viable 013 homozygous This analysis showed that 110 lines were homozygous lethal 66 lines were sub viable and 210 lines were viable Thus differentiating between these models enabled staff to improve their colony management by tailoring breeding strategies to the needs of individual lines identified as lethal or sub viable Having demonstrated the benefits of in house databases for aiding GA colony management and recording phenotypic and welfare observations Jenny went on to talk about how the WTSI share the data they record with the scientific community through their Mouse Resources Portal Using a search for Spns2 as an example Jenny showed workshop participants where to look to download a spreadsheet heat map of phenotyping information by assay which contains links to phenotyping data She also drew attention to a link that individuals can click to subscribe to a mailing list for the line of interest to be kept up to date with information as it is generated and recorded Jenny highlighted the importance of keeping a two way channel of communication open with facilities that they have sent lines to because many of their lines are sent out at a very early stage when there is very little information to accompany them so they are very happy to receive and collate information back from their collaborators GA Passports for legacy lines Last to speak was Amanda Pickard from the MRC Frozen Embryo and Sperm Archive FESA who gave us an insight into how the meaning of information changes over time and what remains useful as time passes This presentation is informed by the FESA experience of rederiving lines that were frozen 10 or more years previously FESA has been in existence since the early 1970 s and it is the UK s central archiving centre with approximately 1 500 stocks of transgenics mutants chromosome anomalies inbred strains They also have over 10 000 individual ENU mutagenised males with an associated DNA library for mutation detection and so are well established as a worldwide resource Most if not all GA passports or database entries begin with the strain name This can include a local name but should also contain the full name consistent with existing nomenclature rules It is interesting to note

Tech 2 Tech that the rules and naming conventions have changed slightly over time and strain names are also updated as more information becomes known For example GENA251 became Junbo then Jbo Evi1Jbo and is now MecomJbo MGI 2158381 Knowing when a strain name was assigned gives an insight into the original meaning if protocols have changed and gives a starting point from which to determine what the current name would be General information is also a recommended part of any passport with some details that shouldn t change unless there is a problem for example colour Other details can change over time for example diets even standard ones can and do change over time housing changes as technology and species specific knowledge develops likewise environmental enrichment varies with knowledge trends All of these details can affect the phenotype behaviour or breeding so it remains important to include these details When building passports for the future an obvious challenge as highlighted by previous speakers will be how to handle the growing body of data relating to phenotypic anomalies and obser vable traits Undoubtably a lot of data is far more accessible now than it has been in the past when it was only to be found in a lab book or library somewhere However each establishment keeps their own records in their own format giving rise to questions such as what do these records tell us how meanful are they to others and how do you decide what to share or link to a passport Increased accessibility to data brings with it a responsibility to ensure it is meaningful and presented in a form such that it cannot be misinterpreted This can be greatly improved by the use of standardised vocabulary such as the mouse welfare terms already described Amanda went on to show participants an example of a record on their in house database system and how it relates to the information that gets sent out with animals from FESA She also provided a number of links to some other resources that are useful sources of information on mouse strains such as Mouse Genome Informatics www informatics jax org Mousebook www mousebook org European Mutant Mouse Welfare Terms Archive www emmanet org www mousewelfareterms org International Mouse Phenotyping Consortium www mousephenotype org and Europhenome www europhenome org This led on to a broader discussion of whether in the future it could or should be possible for the scientific and animal care community to have access to an online depository of GA passport information that is collated updated and maintained such that the entire community and animal welfare can reap the benefits of individual effor ts and minimise the potential for duplicated effort Only time will tell what the future will hold but if you have any thoughts comments or opinions on anything contained within this report the RSPCA GAPWG would like to hear from you at GA rspca org uk References 1 2 3 4 5 Wells D J Playle L C Enser W E J Flecknell P A Gardiner M A Holland J Howard B R Hubrecht R Humphreys K R Jackson I J Lane N Maconichie M Mason G Morton D B Raymond R Robinson V Smith J A and Watt N 2006 Assessing the welfare of genetically altered mice Laboratory Animals 40 2 111114 Animal Procedures Committee APC Report on Biotechnology 2001 http www apc gov uk reference biorec pdf Robinson V Morton D B Anderson D Carver J F A Francis R J Hubrecht R Jenkins E Mathers K E Raymond R Rosewell I Wallace J and Wells D J 2003 Refinement and reduction in production of genetically modified mice sixth repor t of the BVAAWF FRAME RSPCA UFAW Joint Working Group on Refinement Laboratory Animals 37 S1 Rulicke T H Montagutelli X Pintado B Thon R and Hedrich H J 2007 FELASA guidelines for the production and nomenclature of transgenic rodents Laboratory Animals 41 3 301 311 Osborne N Bussell J Carver R Chadwick C Enser B Haskings M Hubrecht R Latcham J Maconochie M Mallon A Norrington R and Wilson S 2009 GA Passports The key to consistent animal care A report of the RSPCA GA Passports Working Group RSPCA 109

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August 2013 Animal Technology and Welfare The problem with the dark bottom row cage for non human primates A discussion by the Laboratory Animal Refinement Enrichment Forum VERA BAUMANS1 KAILE BENNETT2 MARCIE DONNELLY3 GENEVIEVE ANDREWS KELLY4 STEFANIE NELSEN5 KIMBERLY RAPPAPORT6 ALLISON REIFFER7 and VIKTOR REINHARDT Moderator 1 2 3 4 5 6 7 8 Vera Baumans Utrecht University Utrecht The Netherlands Kaile J Bennett University of Michigan Ann Arbor Michigan USA Marcie Donnelly Merck Co Inc Kenilworth New Jersey USA Genevieve Andrews Kelly Huntingdon Life Sciences Somerset New Jersey USA Stefanie L Nelsen SNBL USA SRC Alice Texas USA Kimberly Rappaport University of San Francisco San Francisco California USA Allison Reiffer West Michigan Regional Laboratory Grand Rapids Michigna USA Viktor Reinhardt Moderator Animal Welfare Institute Washington DC USA Corresponding author Viktor Reinhardt viktor snowcrest net Does anyone have experience with improving the illumination in the bottom row of two tier primate caging We were playing with the idea of sticking a light on the wall behind the cage rack but are not sure if this could be risky especially when we happen to forget switching the light off at the end of the day Bennett difference It has been my experience that they last for over four years with very little maintenance required Rappaport We had the same question at our facility and came up with the following solution to improve the relatively dark living environment of the bottom row cages Yes I forgot to mention that the rooms are hosed daily Rappaport We use outdoor rope lights white and an outdoor timer We attach the rope lights to sheets of clear plastic so they can be easily removed if needed We then line the plastic sheets lights along the bottom half of the walls behind the cages rope lights towards the wall plastic towards cage This arrangement makes it impossible for curious monkey hands to grab the rope lights For a room that only occasionally has a bottom caged animal we use the same setup but attach the rope light plastic sheet to a stand that we can move around the room as needed The rope lights aren t as bright as wall mounted florescent lights but they do make a noticeable Good idea Your solution sounds waterproof as well Donnelly Thank you for sharing this practicable solution to the insufficiently illuminated bottom cage Several studies show that monkeys clearly prefer to be in the brighter and higher level of the top row versus the bottom row level of the cage rack1 4 Investigators are usually not aware whether all of their research subjects are caged at the same level of the two tier cage rack A survey of primatological articles revealed that cage location of research monkeys is usually not mentioned in spite of the fact that the environment of upper and lower row housed animals markedly differs in terms of light quality light intensity and living dimension5 Physiological and psychological parameters of the tenants are probably affected differently by the shady and relatively depressive living quarters of bottom row cages than by the bright and relatively safe living quarters of the top row cages Reinhardt 111

Tech 2 Tech ceiling mounted fixtures that can penetrate to the lower tier cages9 10 In order to get more light into the bottom row cages we replaced all solid side panels with mesh walls that were dense enough to make it impossible for the animals especially neighbors to stick their fingers through them Unlike the solid side panels these mesh walls allow the light that is reflected from the walls of the room to diffuse into the interior of the cages Replacing the light impermeable panels with lightpermeable mesh more than doubled the intensity of light in the lower row cages This not only created a healthier environment for the caged subject s thereby improving scientific methodology but it also allowed for better visual health checks and more efficient cleaning of previously dark cage corners by the attending care personnel Reinhardt We re planning on periodically rotating the bottom top row caged animals Bennett Figure 1 Macaques spend more time on their perch when they are housed in a dim bottom row versus a bright top row cage23 24 probably because it allows them to be at a greater distance from the floor and at a shorter distance to the source of the ceiling light Photo Viktor Reinhardt I wish there was more Plexiglas or at least mesh on the bottom cages to compensate for the lower light levels Bennett Illumination of the bottom row cages of our facility used to be so poor that attending personnel pushed the animals with the squeeze back to the front of the cage or used flashlights to read their tattoo IDs correctly and check the cleanliness of the cage This situation which has also been reported in the literature6 7 is in sharp contrast to Animal Welfare Regulations in the U S stipulating that lighting must emphasis added be uniformly diffused and provide sufficient illumination to aid in maintaining good housekeeping practices adequate cleaning adequate inspection of animals and for the well being of the animals 8 The prevailing two tier caging system for non human primates disregards this legal requirement in 50 of all cages as the sanitation tray beneath the upper tier is bound to cast a large shade area on all cages in the bottom tier this significantly reduces the amount of light from 112 Rotating cage position relative to the light source is also proposed by the National Research Council as a practice to reduce inappropriate light stimulation 11 Yet if we rotate the animals cage positions in the rack the fact remains that there will always be 50 of the two tier caged population that has to contend with crepuscular lighting conditions close to the ground level of the room Reinhardt Even if we had some extra lighting in the bottom cages we are still forcing arboreal animals to live close to the relatively unsafe ground at the level of our feet even though they have a biologically intrinsic need to retreat to a place above human eye level so that they can feel relatively safe I would say that providing our macaques primary enclosures with species appropriate access to the vertical dimension is almost as important as providing them with compatible social companionship Nelsen This discussion stirs up a long standing frustration that I bear with the traditional two tier caging arrangement of macaques occasionally even also of marmosets As an ethologist I would say that the two tier caging of macaques compromises the behavioral well being of animals caged in the bottom row of the rack because it does not allow them to access the vertical quasisafe dimension above human eye level of their enforced living quarters This makes them susceptible to experience fear and anxiety whenever a person a potential predator is entering the room As a veterinarian I would say that the two tier caging of macaques compromises the psychological well being of animals caged in the bottom row in the shade cast by

Tech 2 Tech the upper row Animals in the lower tier are thus relegated to a permanent state of semi gloom 12 There is data supported evidence that human primates are at a risk of suffering from depression when they are deprived of adequate illumination13 16 there is no reason to believe that this does not also apply to nonhuman primates who after all are biologically adapted to live in a naturally well illuminated outdoor environment well above ground level As a scientist I agree that the two tier caging system for macaques is outdated and not conducive to scientifically sound research unless it has been arranged that all subjects of a study are well adjusted to be caged at the same level of the rack17 If this basic condition is not met extraneous variables will affect the research data resulting in a relatively high data variability which the investigator will have to counterbalance by using excessively many research subjects This can and should be avoided If only we could house all non human primates in uppertier cages and rodents below in lower tier cages then everyone would be happy Reiffer That would be a nice solution in a dream world A more realistic solution in my opinion would be to continue pushing for the replacement of the inadequate two tier caging system with a single tier caging system for nonhuman primates in which the animals can retreat to safe locations above human eye level For pair housed animals such caging would require the installation of a privacy panel allowing subordinate partners to access the upper dimension of the cage without being hindered by their dominant companions Reinhardt References 1 2 As a psychologically healthy human being I would say that it is not ethical to confine non human primates in shady cages at ground level on a long term basis Reinhardt 3 I agree completely In addition to the compromise in well being it seems that there is a likelihood that the two tier caging of non human primates can also compromise research About a year ago we had a scientist request that we rotate our monkeys at every cage change Apparently there were some ophthalmologic findings suggesting that the difference in light levels between top row versus bottom row caged subjects influenced the data of a research study 4 5 Since there is evidence that the bottom cages can impact research this should be a good reason to eliminate the two tier system with its speciesinappropriate bottom tier cages That hasn t worked yet Andrews Kelly 6 You are so right Those cages should be banned Baumans 7 The biomedical industry has a vested interest in maintaining the traditional two tier caging system for the large number of medium sized non human primates some primatological experts have tried to defend it on quasi scientific grounds9 10 18 19 with the conclusion that the findings of research projects using two tiered housing systems are unlikely to be compromised 10 The fact remains irrefutably that the living quarters of primates in bottom row cages differ significantly from those living in top row cages It is not surprising that the International Primatological Society20 the Medical Research Council21 and the European Commission22 underline the inadequacy of the two tier caging system and hence do not recommend it Reinhardt 8 9 10 Westlund K 2002 Preference of the ver tical dimension of cyno pairs living in high cages Laboratory Animal Refinement and Enrichment Forum electronic discussion group November 28 2002 Clarence W M Scott J P Dorris M C and Par M 2006 Use of enclosures with functional vertical space by captive rhesus monkeys Macaca mulatta involved in biomedical research Journal of the American Association for Laboratory Animal Science Vol 45 No 5 31 34 MacLean E L Roberts P S Platt M L and Brannon E M 2009 Primate location preference in a double tier cage The effects of illumination and cage height Journal of Applied Animal Welfare Science Vol 12 73 81 Griffis C Martin A L Perlman J E and Bloomsmith M A 2012 Effects of play caging on the behavior and enrichment use of singly housed rhesus macaques 35th Meeting of the American Society of Primatologists Scientific Program Abstract No 33 https www asp org meetings abstractDisplay cfm abstractID 4263 confEventID 4368 Reinhardt V and Reinhardt A 2000 The lower row monkey cage An overlooked variable in biomedical research Journal of Applied Animal Welfare Science Vol 3 141 149 http www awionline org lab_animals biblio jaaws1 htm Reasinger D J and Rogers J R 2001 Ideas of improving living conditions of non human primates by improving cage design Contemporar y Topics in Laboratory Animal Science Vol 40 No 4 89 Savane S 2008 Use of flashlights in Old World nonhuman primate health monitoring American Association for Laborator y Animal Science AALAS Meeting Official Program 103 United States Department of Agriculture 2002 Animal Welfare Regulations Revised as of January 1 2002 U S Government Printing Office Washington DC http www access gpo gov nara cfr waisidx_04 9cfrv1_04 html Schapiro S J Stavisky R and Hook M 2000 The lower row cage may be dark but behavior does not appear to be affected Laboratory Primate Newsletter Vol 39 No 1 4 6 http www brown edu Resear ch Primate lpn391 html dark Schapiro S J and Bloomsmith M A 2001 Lower row caging in a two tiered housing system does not affect the 113

Tech 2 Tech 11 12 13 14 15 16 17 18 19 20 21 22 23 behaviour of young singly housed rhesus macaques Animal Welfare Vol 10 387 394 National Research Council 2010 Guide for the Care and Use of Laboratory Animals 8th Edition National Academy Press Washington DC http www nap edu catalog php record_id 12910 Mahoney C J 1992 Some thoughts on psychological enrichment Lab Animal Vol 21 No 5 27 29 32 37 Rosenthal N E Sack D A Gillin J C Lewy A J Goodwin F K Davenport Y Mueller P S Newsome D A and Wehr T A 1984 Seasonal Affective Disorder A description of the syndrome and preliminary findings with light therapy Archives of General Psychiatry Vol 41 72 80 Lewy A J Bauer V K Cutler N L Sack R L Ahmed S Thomas K H Blood M L and Latham Jackson J M 1998 Morning vs evening light treatment of patients with winter depression Archives of General Psychiatry Vol 55 890 896 Terman M Terman J S Quitkin F M McGrath P J Stewart J W and Rafferty B 1989 Light therapy for Seasonal Affective Disorder A review of efficacy Neuropsychopharmacology Vol 2 1 22 Sumaya I C Rienzi B M Deegan J F and Moss D E 2001 Bright light treatment decreases depression in institutionalized older adults A placebo controlled crossover study Journal of Gerontology Vol 56A No 6 356 360 Mulder J B 1976 Behavior patterns of laboratory animals Lab Animal Vol 5 No 5 22 28 Crockett C M Shimoji M and Bowden D M 2000 Behavior appetite and urinary cortisol responses by adult female pigtailed macaques to cage size cage level room change and ketamine sedation American Journal of Primatology Vol 52 63 80 Bentson K L Crockett C M Montgomery H B and Ha J C 2004 Cage level has little effect on behavior of macaques M fascicularis M nemestrina and M mulatta American Journal of Primatology Vol 62 Supplement 85 86 http www asp org asp2004 abstractDisplay cfm abstractID 740 confEventID 809 International Primatological Society 2007 IPS International Guidelines for the Acquisition Care and Breeding of Nonhuman Primates International Primatological Society Bronx NY http www internationalprimatologicalsociety org docs IPS_International_Guidelines_for_the_Acquisition_Care_ and_Breeding_of_Nonhuman_Primates_Second_Edition_ 2007 pdf Medical Research Council 2004 MRC Ethics Guide Best Practice in the Accomodation and Care of Primates used in Scientific Research Medical Research Council London UK http www mrc ac uk Utilities Documentrecord index htm d MRC002395 European Commission 2002 The Welfare of Non human Primates Report of the Scientific Committee on Animal Health and Animal Welfare European Commission Strasbourg France http ec europa eu food fs sc scah out83_en pdf Woodbeck T and Reinhardt V 1991 Perch use by Macaca mulatta in relation to cage location Laboratory Primate Newsletter Vol 30 No 4 11 12 114 24 http www brown edu Research Primate lpn304 html perch Shimoji M Bowers C L and Crockett C M 1993 Initial response to introduction of a PVC perch by singly caged Macaca fascicularis Laborator y Primate Newsletter Vol 32 No 4 8 11 http www brown edu Research Primate lpn32 4 html pvc

August 2013 Animal Technology and Welfare AFOS vet minimises stress and pain in rodent euthanasia DESIREE BETTS Afos Limited Unit 3 Wiltshire House Wiltshire Road Hull East Yorkshire HU4 6PA Correspondence susan higson thistlecomm com Introduction A machine that delivers anaesthesia to minimise stress and pain during the euthanasia of rodents has been developed by a UK company The AFOS ventilated euthanasia table VET was developed to address increasing concerns internationally about the physical and psychological welfare of animals during euthanasia processes that are based on slow fill Carbon Dioxide CO2 as well as the stress experienced by staff who conduct these procedures The Canadian Council on Animal Care CCAC issued guidelines for the euthanasia of animals used in science in 2010 that addressed the high aversion of rodents to CO2 exposure stating CO2 is not an ideal method for humanely killing any species and that CO2 should not be used where other methods are practical for the experiment and species 1 Subject to this instruction the guidelines advocate measures to reduce stress during CO2 euthanasia for both the animals and the staff members who carry out this procedure The CCAC guidelines categorise the use of CO2 as a conditionally acceptable method of euthanasia of rodents subject to animal welfare measures that include the animals being anaesthetised prior to the use of CO2 where practical preferably using inhalant anaesthetics the delivery of a specified CO2 flow rate the animals to be placed in the euthanasia chamber prior to the introduction of CO2 and a precise regulation of gas flow2 These guidelines quote studies on the subject by Makowska et al 20093 Niel et al 20084 Ambrose et al 20005 Britt 19876 Hornett and Haynes 1984 7 Niel and Weary 20068 20079 Hawkins et al 200610 Leach et al 201211 Liotti et al 200112 Dripps and Comro13 These guidelines also reference the International Council for Laboratory Animal Science ICLAS Working Group on Harmonization14 and recommended reference documents by the American Veterinary Medical Association AVMA 2007 15 and recommendations prepared for Directorate General of the Environment Nuclear Safety and Protection DGXI of the European Commission 1986 10 The CCAC document refers to Leach et al and Makowska and Weary s findings that While inhalant anaesthetics such as isoflurane have been shown to cause aversion in rodents11 3 during self exposure to gas anaesthetics rats appear to be closer to loss of consciousness at the onset of aversive behaviour than with CO2 and inert gases3 These conclude Because 115

Tech 2 Tech the animals are exposed to aversive concentrations of gas for a shorter duration initial induction with inhalant anaesthetics appears to be more humane than euthanasia with CO2 alone 2 Wong et al 2012 16 found that many rats willingly remain in a chamber filling with isoflurane until the point of recumbency and that this never happens when the chamber is filling with CO24 The CCAC guidelines also address the requirement for death of the animal to be ensured without it regaining consciousness through application of a second step after application of the primary method of euthanasia a gradual fill rate of less than 30 and greater than 20 of the chamber volume per minute for nonanaesthetised rodents and the need for CO2 to be delivered soon after loss of consciousness following inhalational anaesthesia to ensure that the breathing rate is high enough for death to occur rapidly5 The AFOS Vet AFOS developers have consulted with Joanna Makowska and Dan Weary at the University of British Columbia on their research into the use of anaesthesia with CO23 The conclusions derived from this research have been applied to the development of the anaesthetic euthanasia table Measures to reduce stress in the animals include the faculty to cull the rodents in the home cage The chamber can take type 3 and three type 2L cages and in a measure to reduce the possibility of alarm being 116 communicated between the rodents they are induced simultaneously each cage having an individual gas supply AFOS developers would however like to pursue further research that measures potential vocalisation The airflow is delivered via a damper to reduce noise and pressure The fan runs at 55 decibels to provide as low noise in the area as possible and low pressure of air fill on the animal The chamber is fully sealed and leak free with a clear lid and viewing panel that ensures good observation of the rodent Anaesthesia is delivered into the home cage by means of a thin tube The vapouriser is set at four percent with a thirty percent Oxygen O2 flow and the animal

Tech 2 Tech becomes unconscious within 20 to 30 seconds Trials have shown a marked reduction in indications of stress behaviour No rearing behaviour is displayed at all on the AFOS machine The animal might run once around the cage before falling under the feeding trough Whether this behaviour is due to a sense of security in this location is not established The isoflurane is carried on O2 as a diffusion through a two sandwich type lid with inlets for gas on the inside layer The lid is designed to provide a quiet process with no pressure of the flow of gas onto the animal to prevent stress resulting from the air flow As the animal is unconscious at this stage the anaesthetic is followed directly by a CO2 fill to 100 on a timed process If a CO2 fill at 20 to 30 is required the process will take longer The process from start to finish takes two and a half to three minutes and once the CO2 stage is initiated the cycle cannot be interrupted The process includes evacuation of the gas with a 15 second purge but this can be speeded up if it is necessary to draw blood or take tissues very quickly after euthanasia The lid is completely mechanical with an interlock so that it cannot be removed after the procedure starts ensuring all animals are humanely euthanised in a fast but controlled manner This control and the unit s fully contained down draught ventilation technology delivers full protection to the operator from exposure to the gases The AFOS VET can also be used for surgery induction providing a heated pad for surgery and independently controlled face mask The unit can accommodate the requirement in some facilities to euthanise immediately after surgery AFOS has applied its downdraught ventilation technology to the VET so that downdraught surgery plates and the scavenging nose cone creates a tight controlled environment in which to use anaesthetic gas Trials have indicated a 0 2 parts per million ppm exposure providing exceptional containment compared to the UK requirement at 50 ppm The unit provides the ability to transfer the animal to the pre heated surgery pad which with independently controlled face masks enables the clinician to continue with multiple inductions A touch screen enables the operator to control and visually observe the heat status of the surgery pad The machines are available as wall backed or peninsula sterility for surgery and assisting units providing a choice of two tops peripheral ventilation for necropsy and fine perforation for surgery The tops feature sump wash down and extraction of anaesthetic gas airborne pathogens allergens and aerosols as well as chemicals and odours A mobile recirculating version of the AFOS VET that excludes a surgery induction facility is currently under development A Siemens plc programme provides touch screen controls that deliver five pre set euthanasia or 117

Tech 2 Tech induction programmes that are set according to species and size controlling the level of CO2 induction with or without anaesthetic gas The screen displays a number of different selections with repeated warnings and a secure check system and dampers to protect the user The operators must observe all parameters before they can move to each stage The percentage of anaesthesia required is selected manually followed by selection for the mix of air and CO2 then the gas selection 7 8 9 10 11 12 The unit comprises a Selectatec fitting for vaporisers isoflurane seoflurane enflurane and halothane Research facilities will need to apply for a license change in order to use the anaesthesia facility on the AFOS VET However the unit is designed to be flexible to meet all facility and research needs allowing for a slow controlled CO2 fill only or a 100 CO2 fill or the use of mixed gases prior to the introduction of CO2 Two flow meters deliver medical air O2 and CO2 13 14 15 References 1 2 3 4 5 6 Charbonneau R Niel L Olfert E Von Keyserling M and Griffin G ad hoc subcommittee on euthanasia of the Canadian Council on Animal Care CCAC Guidelines Committee CCAC guidelines on Euthanasia of Animals Used in Science 2010 Page 17 18 http www ccac ca Charbonneau R Niel L Olfert E Von Keyserling M and Griffin G ad hoc subcommittee on euthanasia of the Canadian Council on Animal Care CCAC Guidelines Committee CCAC guidelines on Euthanasia of Animals Used in Science 2010 Page 5 19 http www ccac ca Makowska I J and Weary D M 2009 Rat aversion to induction with inhalant anaesthetics Applied Animal Behaviour Science 119 3 229 235 Niel et al 2008 Report of the ACLAM Task Force on Rodent Euthanasia Ambrose N Wadham J and Morton D 2000 Refinement of euthanasia Progress in the reduction refinement and replacement of animal experimentation Elsevier Science 1159 1170 Britt D P 1987 The humaneness of carbon dioxide as an agent of euthanasia for laboratory rodents Euthanasia of Unwanted Injured or Diseased Animals for Educational 118 16 or Scientific Purposes Universities Federation for Animal Welfare Hornett T D and Haynes A P 1984 Comparison of carbon dioxide air mixture and nitrogen air mixture for the euthanasia of rodents design of a system for inhalation euthanasia Animal Technology 35 93 99 Niel L and Weary D M 2006 Behavioural responses of rat to gradual fill carbon dioxide euthanasia and reduced oxygen concentrations Applied Animal Behaviour Science 100 3 4 295 308 Niel L and Weary D M 2007 Rats avoid exposure to carbon dioxide and argon Applied Animal Behaviour Science 107 1 2 100 109 Hawkins P Playle L Golledge H Leach M Banzett R Coenen A Cooper J Danneman P Flecknell P Kirkden R Niel L and Raj M 2006 Newcastle consensus meeting on carbon dioxide euthanasia of laboratory animals Meeting report London UK National Centre for the Replacement Reduction and Refinement of Animals in Research NC3Rs http www nc3rs org uk document asp id 1311 accessed on 2010 08 27 Leach M C Bowell V A Allan T F and Morton D B 2002 Aversion to gaseous euthanasia agents in rats and mice Comparative Medicine 52 3 249 257 Liotti M Brannan S Egan G Shade R Madden L Abplanalp B Robillard R Lancaster J Zamarripa F E Fox P T and Denton D 2001 Brain responses associated with consciousness of breathlessness air hunger Proceedings of the National Academies of Science of the United States of America PNAS 98 4 2035 2040 Dripps R D and Comroe J H 1947 The respiratory and circulatory response of normal man to inhalation of 7 6 and 10 4 percent CO2 with a comparison of the maximal ventilation produced by severe muscular exercise inhalation of CO2 and maximal voluntary hyperventilation American Journal of Physiology 149 1 43 51 Demers G Griffin G De Vroey G Haywood J R Zurlo J and B dard M 2006 Harmonization of animal care and use guidance Science 312 5774 700 701 The American Veterinary Medical Association AVMA Guidelines on Euthanasia 2007 http www avma org issues animal_welfare euthanasia pdf Wong D Makowska I J and Weary D M 2013 Rat aversion to isoflurane versus carbon dioxide Biol Lett 2013 9 20121000 published 19 December 2012

August 2013 Animal Technology and Welfare POSTER PRESENTATIONS Originally presented at The Institute of Animal Technology Annual Congress March 2013 Winner of IAT LAVA Joint Congress 2013 Best Poster Award Sponsored by LBS formerly Lillico Biotechnology Improving animal welfare by providing extra environmental enrichment with play time NATALIE EDWARDS University of Cambridge School of Clinical Medicine Central Biomedical Resources Phenomics Laboratory Forvie Site Robinson Way Cambridge CB2 0SZ Correspondence ne247 cam ac uk Displayed at the Institute of Animal Technology Congress 2013 Introduction Rats are very social animals by nature and should not be housed singly unless it is an experimental requirement Single housing compromises their welfare and may cause them to become nervous and or aggressive and have permanent effects on their behaviour and physiology There are two particular periods during a rat s life when they are even more sensitive to being isolated These are isolation after maternal separation and during juvenile weeks five and six Studies have shown even brief social isolation during weeks 5 and 6 have a significant effect on adult social behaviour and reactions to stress These are important periods as they spend large amounts of time playing with cage mates establishing social bonds Figure 1 Rat enjoying time in the playpen 119

Poster Presentations on a daily basis and socialise with different family members These natural environments and behaviours are difficult to replicate within a laboratory and it is clear to see why a laboratory rat would become bored compared to its wild ancestors We have two main aims by providing rats with play time 1 To re group singly housed rats 2 To provide extra enrichment to long term study rats Playpen Our method to meet the above aims is by using playpens We are currently using two different playpens filled with plastic playpen balls Figure 2 Juvenile rats playing The rat model is extensively used within behavioural studies due to their ability to learn procedures and routines Therefore we can assume that long term held rats become accustomed to the husbandry routines taking place on a daily basis by the animal technician within the room Long term studies can be detrimental to a rat s well being as they can become stressed over their time within the laboratory This is likely to be due to boredom as they know what is occurring and there is no stimulation of new environments Some experiments have even shown that social isolation for a period of as little as two hours in adult rats can induce a state of anxiety This proves that any singly housed rat would not be able to display natural social behaviour such as grooming other cage mates fighting and wrestling and would become stressed Figure 4 The red Perspex box filled with plastic balls size 25 L x 18 W x 16 H Stressed rats are more difficult to handle and manipulate for the performance of experimental procedures A wild rat would live in an extensive burrow with a large mixed age group and sex family and would have opportunities to explore new areas and environments Figure 5 The Double Decker cage filled with plastic balls Figure 3 A wild rat in its natural environment 120 The advantages of having two different playpens are that it firstly provides variety and they can alternate between the two Secondly they have their own advantages The red Perspex box is a large area which appears dark to them and allows them to stand up tall

Poster Presentations The double decker cage has an inbuilt shelf which allows the rats to climb onto it this helps build strength in muscles they wouldn t normally use Using two playpens means that two groups of rats may be exercised simultaneously thus allowing animals to receive exercise more regularly aggressive fighting between the males This usually takes place on a Monday morning so the behaviour can be observed throughout the week What we have achieved Case 1 The table below shows the different materials we used to fill the play pens and the advantages and disadvantages of each material Strain Wistar Sex Male Age 7 months Problem Singly housed male to be grouped with a group of males There are problems when introducing a new male rat to another bonded group of male rats as the group already has their hierarchy established A new male entering the group would cause a new hierarchy to be established and this could cause aggressive fighting between the whole group of rats when housed together The play pen was successfully used to rehouse all the males together without any aggression being displayed Table 1 Advantages and Disadvantages of materials used For general play time a rota Table 2 is in place to try to ensure all rats receive play time on a regular basis I am currently using a marker on the cages which is moved along the rack of rat cages in turn A cage of rats will be placed in the play pen for at least twenty minutes When a new cage of rats is placed on a rack they will join the end of the line and be in the rota How many cages you have in the room will determine how often the rats can have play time The balls are cleaned between each cage of rats with ethanol or sent to cage wash Case 2 Strain Sex A general protocol for re housing two cages of male rats together if one or both rats has been left singly housed is followed 9 months Problem Re housing rats Male Age Table 2 Rota of play time WLD Two singly housed males to be housed together This case is not as difficult as Case 1 as there is no established group bond However in our experience WLD rats are a more aggressive strain of rat WLDs have also proved difficult to breed due to their highly stressed nature In this case two males were extremely stressed and very difficult to handle so this caused problems with rats being too aggressive towards each other However the play pen was used to successfully rehouse the two males together 1 Ensure the rats are identifiable use ear markings or mark their tail 2 Place all rats in the playpen for at least twenty minutes daily for at least a week 3 Monitor the behaviour in the playpen to ensure no aggressive behaviour is displayed 4 Re house the males in a new clean cage all together and monitor the cage closely to ensure no 121

Poster Presentations Observations When the rats first get placed into the playpen they first appear alert to the new unfamiliar surroundings The textures on their feet are new to them and they start to explore the area After a few minutes of being placed in the pen the rats get very excited and start exploring around with the other cage mates If they have experienced the playpen before then the rats have a much quicker excitement rate and start playing sooner as they are familiar with the playpen Observation of rat behaviour during time in the playpen indicates that our rats enjoy the extra stimulation it provides This is very important as the extra stimulation they are receiving is reducing their stress and boredom level which is improving their welfare standards Figure 7 Rats utilising cage shelf in double decker cage The playpens currently sit on the floor in the rat holding room and are not full IVCs individually ventilated cages meaning that this would not be suitable for a SPF specific pathogen free facility However the double decker cage is also supplied as an IVC so this version could be used in SPF facilities Laminar air flow cabinets or laminar air flow cabins can be used to place the rats in the playpens to overcome this issue Figure 6 Rat burrowing in playpen Rats secrete Porphyrin when stressed Porphyrin is a red pigment which is secreted by the Harderian gland which is located in the orbit of the eye We have not seen any Porphyrin staining in any of the rats that receive play time which suggests they are not stressed Discussion I feel the playpens are extremely beneficial as most importantly the welfare standard of the rats has been improved Being able to avoid social isolation as much as possible means that we are meeting the behavioural and social needs of the rat and reducing their stress and anxiety levels Also being able to provide extra stimulation to the long term study rats means we are improving their welfare as they receive some extra excitement on a regular basis to reduce their boredom Another advantage other than animal welfare of the playpen is that there is a financial benefit to the researcher and workload for the animal technician due to there being fewer cages The pictures on the right show the rats using the shelf in the double decker cage to climb up and down which helps display natural behaviour and build strength 122 Figure 8 Playpens filled with the polystyrene chips as an alternative to balls

August 2013 Animal Technology and Welfare Welfare for wobbly mice HELEN COOPER Research Animal Technician MRC Ares Building Babraham Research Campus Babraham Cambridge CB22 3AT Correspondence hcooper mrc lmb cam ac uk Figure 1 The MRC ARES building The place MRC Ares Building in Cambridgeshire is designed to house rodents primarily Genetically Modified mice in IVCs or flexible film isolators and has over 50 dedicated members of staff involved in operating the facility environmental conditions to vastly improve their welfare These environmental refinements ensure that mice are able to live as comfortably as possible until they are ready for research use The traffic light system The mice The facility houses mouse models of many diseases including Dementia and Parkinson s The P301S T43 is a transgenic mouse model whereby homozygous animals at 4 6 months of age develop a neurological phenotype including severe par tial paralysis of the lower limbs Labels are attached to the cage card of any animal which shows the phenotype There is one of each colour to make them easily distinguishable These colours also correspond to the strain protocol to make finding information as simple as possible The corresponding letter G A R is written next to each animal s individual ID on the card so those showing different stages of phenotype can be identified At ARES a traffic light system of green amber and red has been developed to identify the progressing stages of the phenotype This allows for close monitoring provision of necessary care and definition of human endpoints The refinements As these mice develop significant problems with locomotion it was necessar y to modify the Figure 2 Traffic light system cage cards 123

Poster Presentations 3 Unstable gait enhanced rear end wobble when mouse runs walks Coat condition may be poor 6 The mouse now appears to have some amount of muscle wastage especially around back end Unstable gait very wobbly with possible reduced speed May appear to have tremor shakes Coat condition may be poor Righting reflex still intact however may fall over more often Reduced leg strength cannot stand on hind legs to reach food hopper and reduced front leg grip 4 When suspended by the tail the hind legs stil stretch out as in mice without the phenotype Righting reflex is still intact 7 When suspended by the tail the hind legs are tucked under the body 5 Mouse is weighed and weight is written on back of cage card Green card is put on cage and G written next to animal s number Figures 3 5 Green stage phenotype criteria 124 8 The cage is moved to a dedicated rack The dedicated rack does not use automated watering as the phenotype mice may find reaching and using the nozzle difficult Instead water bottles are used with a long stem bottle top to enable the mice to reach the nozzle with ease

Poster Presentations 9 Bedding changed to Alpha dri this is preferable to sawdust as it is softer and at amber the mouse is unable to lift its body completely off the floor while moving round the cage Enviro dry nesting material is provided but may be substituted for a nestlet as sometimes the hind legs of the mice can become tangled Other enrichment such as a fun tunnel and chew stick are provided The fun tunnel does not inhibit thorough checking of the mice because at amber the the mice will be checked and handled daily 12 The mouse is weighed and Amber weight written on back of cage card The 20 23 weight loss is calculated using the green weight as a base line This is the weight that the animal cannot drop below as outlined in the PPL An A is written next to animal s ID number The mice are now checked daily and weighed weekly or more often if necessary Phenotype Health Record is filled out Green and Amber weights are recorded as well as visual condition of the mouse 10 Figures 6 12 Amber stage phenotype criteria 13 More noticeable weight loss may be near the 20 23 weight loss and muscle wastage especially around back end Mouse appears to be hunched 11 As the mouse can no longer reach the food hopper mash is provided daily along with a dietary supplement of pumpkin gel to try and reduce rapid weight loss Mouse appears to be paddling with hind legs to try and move around cage Alternatively feet may be tucked underneath body with very little movement if any 125

Poster Presentations 14 Very little or no righting reflex Mouse may be stuck on its side 16 Webcams are an invaluable tool for remote assessment of these wobbly mice and allow the LMB scientists to confirm that the appropriate required phenotypic stage has been reached The mice are then either used in behavioural studies or sent back to the LMB for analysis It is important that the mice are not sent out before they have reached the correct phenotypic stage so working closely with researchers and constantly refining techniques and practices is extremely important to ensure that no animals are wasted Figures 13 16 Red stage phenotype criteria 15 When suspended by the tail the hind limbs are tucked in and hind feet may be clasped Acknowledgements Animal is already being weighed daily so it should be clear if animal is close to the 23 20 weight loss Researcher scientist at the Laboratory of Molecular Biology LMB is contacted to confirm course of action 126 Thanks to the team in E32 for providing essential information and helping to take photographs Also thanks to Isabelle Lavenir for giving me background information about the phenotype mice

August 2013 Animal Technology and Welfare Webcams as a tool to aid communications diagnosis and treatment DARREN EGAN Training and Database Support Manager Biological Services Group MRC Ares Building Babraham Research Campus Babraham Cambridge CB22 3AT Correspondence degan mrc lmb cam ac uk The Medical Research Council Laboratory of Molecular Biology The LMB in Cambridge is one of the birthplaces of modern molecular biology Many techniques were pioneered at the laborator y most noticeably the sequencing of DNA Over the years the work of the LMB scientists has been awarded 9 Nobel prizes as well as numerous other scientific awards Quarantine geography and staffing Movement of staff between the LMB and Ares is controlled due to quarantine restrictions These restrictions are in place to prevent cross contamination between facilities and units This sometimes means that Researchers cannot view the animals first hand Due to the distance between Ares and the main research site research staff cannot just pop in Veterinary cover is shared with another Institute and can also restrict movement of the Named Veterinar y Surgeon between the animal units of each organisation Visual interpretation While there are no welfare issues surrounding these geographical and quarantine restrictions it can hamper data that needs to be collected by the researcher This is especially important when phenotypical behaviour or health status is border line and the opinion of the researcher is crucial in determining the fate of these highvalue animals These decisions are often time sensitive MRC Ares is part of the LMB and is situated 5 miles south east of the main MRC LMB site Ares is designed to house rodents primarily GM mice in IVCs or flexible film isolators and has over 50 dedicated members of staff involved in operating the facility It was therefore important that a way to visually interpret the behaviour and characteristics of the animal quickly and real time could be found The solution The solution appeared to be a live webcam link between the researchers work station and the animal room at Ares Both sites share a common web proxy so security and compatibility were not an issue Each of the animal rooms has at least one PC to log into the network and view the images from the webcam and so some hardware was already available The set up Our IT specialists identified an Axis 207 Webcam and Axis PoE Active splitter that could be used efficiently and simply Once the web address of the cameras was set up the webcam was plugged into a dedicated 127

Poster Presentations network port housed in a socket station suspended from the ceiling The socket also provided power to the webcam unit allowing just a single standard metre Ethernet cable linking the webcam to the network This reduces on clutter and the need for an additonal cable or power points The length of the cable allowed for greater flexibility in positioning the camera while being connected to the dedicated socket Log in The camera has its own security which is provided by the webcam software There are different levels of access rights ranging from administrator to single use This way the tasking of the camera can be restricted to only those that have a specific need Once the address has been entered into the web browser and user id and passwords have been accepted the images from the webcam become visible on the sender PC The person viewing the images from the camera carries out a similar login process from the target location Focus and communication The camera did not have autofocus and so a focusing ring on the lens sharpens the image A very short focusing distance enables a high level of clarity on close ups Lastly a phone link between the operator of the camera and the viewer needs to be established This enables the operator to take direction on which specific features to focus on The rooms have cordless phones so the camera operator was unrestricted by telephone cabling Figure 2 Screen shot demonstrating the clarity and short focusing distance of the webcam Operation All the animal rooms in Ares are equipped with a webcam allowing use by anyone with user id and password Once all parties have logged into the network and established telephone communication the process is very simple to use and enables real time viewing of animals without the constraints of location or quarantine restrictions If an animal needs to be held to view specific areas of the body a second technician will hold the animal while the camera operator can concentrate on providing the image Figure 3 Camera in use showing HD set up and microphone headset Improvements Figure 1 Webcam showing the cabling yellow cable is the Ethernet connection 128 Two of the most widely used webcams have been replaced with a HD camera This camera also has twoway audio to enable improved communication and auto focus

Poster Presentations The cordless phones now have an earphone and microphone headset plugged into them to allow handsfree use of the camera by the operator It was found to be quite a task to cradle the phone between ear and neck in order to allow both hands free to hold and focus the camera As well as quickly becoming uncomfortable this position managed to cut off the phone link to the viewer on many occasions Acknowledgements Thanks to all the staff in Ares who provided feedback in the use of the webcam especially all in E32 and Annabelle Special thanks to Tom Pratt from IT for setting up everything in Ares 129

Animal Technology and Welfare August 2013 Single handed jugular blood sampling in conscious rats KATE HEATH GlaxoSmithKline Toxicology Support Safety Assessment Park Road Ware Hertfordshire SG12 0DP All animal studies were ethically reviewed and carried out in accordance with Animals Scientific Procedures Act 1986 and the GSK Policy on the Care Welfare and Treatment of Animals Method 2 Slanted table Introduction Animal studies are required during the safety testing of potential new medicines to check the amount of the compound that gets into the body and correlate it with any side effects observed Historically these were obtained post dose via caudal vena puncture after placing the rats in a whole body warming cabinet set at 39 C for up to 10 minutes We were asked to identify a refined method of collecting blood immediately post dose from animals that had been given compounds via either inhalation or the intravenous infusion route Method 3 Single handed jugular bleed This poster explains how we achieved our goal and how this was expanded for other toxicological studies Method 1 Caudal vena puncture Rats were removed from the inhalation tubes and post dose clinical signs were recorded Animals were then placed in a whole body warming cabinet and the samples were obtained approximately 10 minutes post dose A 25g 1 needle and 1ml syringe is used to obtain the sample Needle position is noted by the arrows Both sides of the neck are essential for multiple sampling 130

Poster Presentations Training Step 5 It is essential for success and must not be rushed Immediately the needle is removed firm but gentle pressure should be applied for approximately 15 30 seconds The neck is checked to ensure no damage to vessels and the rat has stopped bleeding There are 5 stages to the training process All 5 stages could take in excess of 12 hours in total to gain competency Step 1 For the initial hold the animal is held against the handler s chest The thumb and middle finger are placed over the forelegs The rat should be in a relaxed position before proceeding Step 2 Place the index finger on top of the animals head slide the index finger towards the animal s tail taking excess skin this will ensure the head remains parallel Comparison jugular verses caudal blood sampling Step 3 To ensure the right and left jugular vein are exposed the ventral cervical and upper thoracic region must be fully extended Once handling competency is gained the trainee is able to move onto the jugular blood sampling Step 4 Needle position is crucial for success and comfort of the animal The needle is inserted at the jugular subclavian junction When the vessel is located it is imperative the needle remains in position 131

Poster Presentations Comparison handling over slanted table method Conclusions G G Added values of technique G G G G G inhalation studies we can now obtain blood immediately after dosing IAD infusion studies IAD time points obtained multiple early time points i e 5 min 15 min 30 min most importantly Less stress to the animals larger volumes of blood obtained quicker reduced stress to both animals and staff more accurate in our timing able to adhere to study protocols IAD Opening our minds and embracing new ideas can really improve the welfare of laboratory rats Acknowledgements Safety Assessment and Laboratory Animal Science especially Sam List and Ali Robinson for supporting this initiative Peter Bacon and Keith Preston for taking the photographs 132

August 2013 Animal Technology and Welfare Determination of recovery period after intra abdominal transmitter and EEG electrode placement in common marmosets Callithrix jacchus LEO VAN GEEST1 MILENE RIJNBEEK2 HERBERT BROK1 JACO BAKKER1 RIANNE KLOMP1 INGRID PHILIPENS2 SASKIA ARNDT3 and JAN LANGERMANS1 1 2 3 Animal Science Department Biomedical Primate Research Centre Rijswijk The Netherlands Department of Immunobiology Division of Neuropathology Biomedical Primate Research Centre Rijswijk The Netherlands Department of Animals in Science and Society Division of Animal Welfare and Laboratory Animal Science Faculty of Veterinary Medicine Utrecht University The Netherlands Corresponding author Leo Van Geest geest bprc nl Introduction needed Transmitter insertion caused a significant increase in righting time in the hourglass test Recovery to mean values was observed within 7 days post surgery For the tower test the total amount of level changes declined 80 directly after surgery Recovery was seen after day 15 Until day 15 a significant decline in bodyweight was seen Subsequently there was an increase in bodyweight which recovered to baseline values 31 days after surgery In conclusion 15 days post surgery reliable results for research can be obtained in marmoset models with intra abdominal transmitters and EEG electrodes Materials and methods Animals In this study the recovery period of surgical placement of intra abdominal transmitters and EEG electrodes was determined in common marmosets To test whether the insertion of an intra abdominal transmitter and EEG electrodes causes physical discomfort two tests which are commonly used to quantify motor deficits in Parkinson s disease research were applied hourglass and tower test Additionally bodyweight and behaviour were monitored daily during 49 days post surgery During the entire study period the transmitters functioned properly and no additional surgery was Five female adult common marmosets Callithrix jacchus born at the BPRC were included 3 8 years and 367g Animals were housed in compliance with the Directive 2010 63EU Food was removed but water intake was never restricted Surgery One day prior to surgery 12 5 mg kg amoxicillin Synulox was given orally BID serving as prophylactic antibiotic One hour pre operatively 0 20mg kg meloxicam Metacam was administered orally and 0 02 mg kg buprenorphine Buprecare injected intramuscularly IM After surgery animals received oral meloxicam 0 10 mg kg once a day in combination with buprenorphine 0 02 mg kg IM twice 133

Poster Presentations a day for a period of 2 days This contributed to multimodal analgesia post operatively Sedation was done with 16mg kg alphaloxone Alfaxan Combined with the administration of meloxicam and buprenorphine this created with 75 minutes of surgical anaesthesia Surger y was performed according to Pearce et al 1998 with minor modifications Experimental design Time Schedule Time dimension in days Technique 14 0 0 1 2 3 49 Surgery Scoring of behaviour Body weight Hourglass test Tower Test EEG check Figure 2 Tower test setup Inside a cabinet composed of a transparent Plexiglas front five levels of horizontal situated crossbars with varying distances from first level to fifth level were present The marmoset was allowed to stay in the cabinet for 10 minutes after entering it by the bottom of the cabinet Simultaneously an observer sitting in another room scored each point of time the marmoset jumped from one to another level Figure 1 Hourglass setup A cylinder measuring 11cm x 27cm was used One trial consisted of the period from turning the cylinder 180 causing the marmoset to stand upside down to the moment was in upright position again righting time One test comprised of ten 180 turns of the cylinder Maximum correction time being 30 seconds 134

Poster Presentations Results Behaviour The marmoset Animal 1 showed a graph that did not correspond to the others Therefore this animal was not included in statistical analysis Although a drop in activity in the first week after surgery was seen no significant changes were measured when comparing before surgery to after surgery results Tower test Level of consciousness showed stable baseline measures in all animals but changed in three out of five marmosets after surgery They showed sopor during the first few days after surgery None of the marmosets showed abnormal consciousness after four days onwards Physical activity of the marmosets is shown using the total number of level changes in a 10 minute period Figure 4 All five marmosets showed a significant drop of 18 93 level changes in physical activity directly after surgery day 3 to day 8 when compared to their own baseline measurements The marmosets recovered during day 8 to day 15 with regard to physical activity In all marmosets faeces were firm droppings in baseline measures until amoxicillin Synulox was administered as prophylactic antibiotic From this day Day 1 marmosets excreted mushy to liquid faeces for eight days Bodyweight Fourteen days after surgery an average decrease of 10 9 g bodyweight was seen p

Animal Technology and Welfare August 2013 Maximising operational and research activities through the use of disposable caging REBECCA VARRALL DAVID SMITH and STEPHEN BAKER Worldwide Research Development Pfizer Inc Comparative Medicine 200 Cambridge Park Drive Cambridge Massachusetts 02140 USA Correspondence Rebecca Spice pfizer com Abstract In 2005 a novel approach for rodent housing was introduced in the form of the disposable cage The use of disposable caging has proven to simplify operational workflows and reduce the risk of personnel ergonomic issues In addition space for research activities can be maximised through a reduction in washroom and storage footprint and lower capital and operating costs achieved Pfizer has selected the disposable cage as the primary Individually Ventilated Caging IVC option for a new 52 000ft2 research facility with a 7 500 rodent housing capacity This poster describes our experiences using disposable cages Introduction Disposable cages are manufactured from recycled Polyethylene terephthalate PET BPA free material Cages are supplied ready for use irradiated prebedded and double bagged They are ideal for use with immune deficient animals and for work involving hazardous materials up to BSL3 In this application high risk cage processing activities are eliminated as the cage is bagged tagged and disposed of after use Cage Specs Mouse Disposable Rat Disposable Dimensions in 14 7 L x 9 2 W x 5 5 H 17L x 13 4W x 7 8H Floor surface area 81 in2 141 in2 Maximum Capacity 5 2 Change Frequency 14 days 7 days Image Table 1 Cage specifications for the mouse and rat disposable cage 136 Water bottles are irradiated and filled with reverse osmosis water treated by UV sterilisation Water testing has demonstrated bottles remain free from bacteria for at least one year after filling Disposable water bottles also remain microbiologically cleaner during seven days of use compared to conventional bottles1 Space and cost savings Vivarium planning using the disposable caging has permitted a reduction in washroom and cage storage footprint maximising the space available for animal holding procedure rooms and research activities It is not practical to entirely eliminate all washroom equipment however several standard pieces need not be included See Table 2 Conventional Washroom Disposable Washroom Rack Washer Rack Washer Bulk Autoclave Cabinet Autoclave Bottle Washer Glass Washer Bottle Filler Bottle Crusher Dumping Station Bedding Disposal System Tunnel Washer Bedding Dispenser Reverse Osmosis System Table 2 Comparison of equipment installed in a conventional versus disposable washroom

Poster Presentations A lower capital investment is required during facility construction due to the omission of a number of key pieces of equipment We estimate our savings to be in excess of 500 000 Capital savings ranging from tens of thousands up to multi million dollars for larger operations have been reported in the literature2 The reduction in washroom equipment and cage processing is predicted to yield annual savings of at least 100 000 in washroom utilities maintenance and supplies Furthermore both cage replacement and depreciation costs are avoided through implementing a disposable system Lower washroom labour costs and increases in workforce productivity are also expected but still to be evaluated Disposable cages permit a greatly increased storage density as the thin walled cages and cage components nest easily Up to 800 cage bottoms or 1200 filter lids can be stored on a single bulk truck Additional space savings are achieved as bedding materials do not need to be stored The high nesting destiny of the disposable cage coupled with the implementation of lean processes and a Just in Time ordering system has reduced storage requirements by 2 000ft2 Simplified workflows Considerable ergonomic benefits can be achieved using the disposable model Clean and dirty side cage processing activities are eliminated and operational workflows simplified Traditional cage processing methods have a high ergonomic risk due to the frequency of repetitive movements and high lifting factor The disposable system eliminates these tasks and allergen exposure associated with cage dumping The process chart below illustrates the workflows using disposable versus conventional cages G G G The average in use cage weight is 1039g for mice and 3259g for rats An animal technician changing 200 cages per day will lift 434lbs less for mice and 334lbs less for rats Weight lifting water bottles is reduced by 88 Our standard crate containing 36 full bottles weighs 30lbs compared to 3 5lbs for disposable bottles We have established a waste to energy program for processing disposable cages after use Published Life Cycle Analysis data shows that both the energy and material consumption of single use disposable cages is greater than that of re usable cages3 however recycling and waste to energy programs that reduce CO2 emissions were not considered Conclusions Working with disposable cages creates a highly flexible space and maximises the footprint for research activities The disposable model offers a low cost option for construction or renovation projects due to reduced capital expenditure and is most effective in facilities housing only rodents In addition faster turnover of research programs and equipment can be achieved whilst avoiding depreciation costs The implementation of a disposable system requires a considerable shift in perception to support operational process change Although visually ver y different internal validation studies show cages do not negatively impact animal welfare or experimental variables providing other best practices such as ensuring an enriched environment are maintained The disposable model establishes unidirectional workflows supporting efficiency and cost savings Literature suggests productivity increases up to 20 can be achieved during cage changing4 and reductions in washroom labour by 2 to 3 FTE are predicted Impor tantly the disposable cage reduces the ergonomic and animal allergen exposure risk associated with routine husbandry procedures The disposable cage is successfully in use within our smaller facilities The performance metrics on a large scale will need full evaluation when the new building is occupied in 2014 To date the disposable cage has proved to be a flexible and valuable addition to our vivarium planning and operations References 1 Figure 1 Process chart comparing workflows image taken from article Disposable Caging 2 Further ergonomic benefits are achieved during cage changing G Disposable cages are lighter than our conventional choice by 49 for mice and 19 for rats 2 3 4 Lin R 2010 Disposable Caging How it helped Our Lab Grow ALN Magazine Online Fallon J 2011 Disposable Caging ALN magazine Online D Incognito V 2010 Environmental impact of disposable and reusable cages Animal Technology and Welfare Vol 9 3 pp 147 150 Gagneron R 2011 Disposable vs Conventional Caging Poster AALAS 20th Educational Symposium 137

Animal Technology and Welfare August 2013 Creation of a complex environment for NZW rabbits within the confines of an animal unit ASHLEY GRIMMER1 NATALIE EDWARDS KATE TANSLEY and all Phenomics Staff University of Cambridge School of Clinical Medicine Central Biomedical Resources Phenomics Laboratory Forvie Site Robinson Way Cambridge Cambridge CB2 0SZ 1 Corresponding author ag575 cam ac uk Introduction An important aspect of animal care is to encourage natural behaviour This can be achieved by making the animal s environment more complex A lack of stimulation and set routines could have a negative effect on the animal s wellbeing It is crucial that larger animals in this case NZW rabbits are given the space and variety of routine to flourish Meeting the animal s needs has both a positive effect on the animals natural behaviour exercise decreased aggression stimulation and the users easier handling quality research data Housing In this case a stimulating complex environment was provided to a colony of 20 female NZW rabbits The NZW rabbits were caged in pen style racking 2 3 animals were housed on each level One side of the cage had deep bedding and the other a gridded base The cages allow good visibility of the rabbits and provide flexibility as the sides can be closed off from each other Trays below the bases allow the faeces and urine to be observed for any abnormalities Within the cage hay balls Figure 1 were provided to encourage standing on hind legs The shelf enables a second level to climb and a safe environment underneath Deep bedding Figure 2 is used to prevent sore hocks and allow the rabbits to scratch and dig in the cage A radio on a timer is used to create a comfortable background noise during the day The rabbits were weighed every week and had a thorough health check each fortnight Figure 2 Caging with chew block and deep bedding Figure 1 Rabbit caging Toys hay balls shelving gridded base and closing window shown 138 A playpen Figures 3 6 allowing cage mates to exercise on the floor of the room was also provided The rabbits had space to run and jump and stand on hind legs Toys were supplied as well as bases to provide

Poster Presentations different levels The pen is temporary and can be cleared away after each use and the floor cleaned ready for the next group the following day The rabbits were placed in the playpen every 8 working days A variety of toys were given to the animals including i ii iii iv v vi Paper Houses i cardboard rat houses that the rabbits would chew scratch and rip apart Jingle Ball ii supplies a safe sanitisable object to help prevent boredom and promote psychological well being The ball satisfies rabbits nudging rolling and chewing instincts Little Red Houses LRH iii plastic mouse houses that could be cleaned Rabbits liked to chew and move about the cage Dumb bells iv sturdy lightweight dumb bells made of long lasting solid non toxic polypropylene provide interest and enjoyment Chew blocks v tough enough to chew and gnaw helping animals to satisfy their natural instincts and exercise teeth and gums promoting dental health Rat tubes vi cardboard tubes that can be chewed and pushed around the cage A toy rota was used over a two week period to give the rabbits frequent change of toys and objects to play with Changing toys regularly in the cage decreases boredom and aggression towards each other The toy rota is shown below Figures 3 6 Playpen showing tunnel toys and cage base WEEK 1 Provide Remove Mon Tues Weds Thurs Hay Hay Dumb bells Hay Balls Paper Houses Rat Tubes Dumb bells Paper Houses WEEK 2 Provide Mon Hay Jingle Balls Remove LRH Tues Weds Hay Hay Balls Chew Blocks Jingle Balls Thurs Fri Carrots LRH Hay Balls Fri Carrots Rat Tubes Hay Balls Chew Blocks Figure 5 Toy rota The rota could be adapted if rabbits began showing aggression towards each other extra toys and different combinations were used to provide stimulation The rabbits particularly enjoyed the paper houses and the little red mouse houses Diet was also subsidised with quality hay and carrots Hay can reduce enteropathies and hair chewing Conclusions Figure 4 i vi Examples of products used as toys The rabbits observed seemed more stimulated when given a rotation of toys and activities Aggression towards cage mates and users was decreased having a positive effect on handling and animal health Good animal health was seen from daily observations and from the regular health checks The stimulating environment aided to the weight gain shown in the 139

Poster Presentations rabbits on a weekly basis When exercised on the floor the rabbits made full use of the space jumping and running around promoting wellbeing and natural behaviour Based on regular obser vations and interaction the rabbits seemed much less stressed and consequently created a much happier animal Figure 6 In future studies a greater number of toys and diet restricted due to nature of project could be used Studies taking place over longer periods of time would require greater variation and imagination Figure 6 Happy rabbits 140

August 2013 Animal Technology and Welfare The chicken as a research animal LEJLA BEKTIC AVIAN Behavioural Genomics and Physiology Group Link ping University Sweden Correspondence to lejbe ifm liu se Why chickens G G G G G G egg laying and pre cocial young allow for breeding of new animals without mothers which decreases maternal effects in genetics studies ancestral species Red Junglefowl is available and relatively easy to house in laboratory conditions This makes the chicken a great model species for domestication studies sequenced genome gives good options for genomics studies as an impor tant food production species the chicken is an excellent model for animal welfare research well characterised behaviour spectrum relatively short generation time storing and incubating period eggs are placed on rotating trays with the blunt end up Incubation time is 21 days Day 1 routine includes health control weighing marking and vaccination of newly hatched chicks Figure 3 Rearing pen weeks 1 5 Figure 1 From Egg to Chick in 21 days The mating of birds is done either by ar tificial insemination or by pairing up males and females in pens or cages Thereafter eggs are collected for up to three weeks and stored at 15 C until the incubation When collecting eggs we can either collect from a group of animals or opt for a pedigree breeding During both The chicks spend first 5 weeks of their lives in the hatching facility The size of the pen is easily adjusted to follow the growth of young animals We use wood shavings as flooring material for all animals Our hen house is an adapted version of commercially available housing system For pedigree breeding and isolation of individual animals we use dog transport cages When working with smaller groups of animals in our laboratories we use dog pens for adult chickens and custom made cages for chicks Our routines include G G G G G Figure 2 Incubator with automatic turning daily supervision of animals regular health controls and vaccinations regular cleaning and sanitising of all animal facilities day to day work with animals through egg collecting handling and research experiments keeping thorough documentation Common behavioural problems and stereotypes are best countered with enrichment In addition to group keeping perches and hiding places available through commercial housing system we also use hay and seeds as enrichment 141

Poster Presentations Figure 4 Pedigree breeding isolation cages Figure 5 Chicken showing signs of feather pecking and chickens enjoying an enriched environment We work with four different breeds G G G G Red Junglefowl the wild ancestor RJF White Leghorn an egg laying breed WL Broiler a breed used for meat production B Crossbred animals IC Figure 6 Growth rate comparisons All our facilities are constructed and equipped according to the EU Directive 2010 63 EU Swedish Animal Welfare Act SFS 1988 534 and Swedish Animal Welfare Ordinance SFS 1988 539 All our research is approved by an ethical committee and conducted according to the aforementioned legislation 142

August 2013 Animal Technology and Welfare A study to assess the suitability of a stand alone aquatic system for housing Xenopus tropicalis frogs NICK HARMAN1 PETER THOMPSON DAVID MCDONALD NICOLA GOODWIN DIANE HAZLEHURST and LYNDA WESTALL Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Hinxton Cambridge CB10 1SA 1 Corresponding author nth sanger ac uk Introduction As part of a National Institute of Health NIH initiative to establish a wide variety of resources for Xenopus research Researchers at the Sanger Institute developed a high throughput method to identify mutations in Xenopus tropicalis Numerous mutant alleles have been identified in the coding sequence of individual animals and this has led to the development of the Xenopus Mutation Project website http www sanger ac uk Projects D rerio xmp where alleles are published and available on request to the scientific community A planned reduction in this work and an expanding Zebrafish programme led to a need to house frogs in a modern compact system in a smaller room A Zebrafish stand alone system SAS was assessed for its suitability to house X tropicalis Due to the smaller body size of X tropicalis it could potentially provide a suitable environment while providing flexibility of a multi species system of Tanks 3 Environmental Frogs Tanks Static of Enrichment 7 Lily System Pad Submerged 16L Tunnel Stand Alone 4 2 Lily System Pad Submerged 3 5L Tunnel Stand Alone System 8L 4 6 8 Lily Pad Submerged Tunnel Study details To assess the suitability we sought to compare the condition of frogs within our original system and the SAS using general body condition scoring and production parameters to define the systems suitability Fertile healthy frogs were selected for the trial Figure 1 Shows 16 litre tanks on the original system shelves 143

Poster Presentations The SAS has the capability to self siphon however due to concerns over high water level and frog escape modified siphons were fitted to 50 of both tank sizes This resulted in a lower water level but the self siphon capability was removed Figure 2 Shows the stand alone system with 3 5 and 8 litre tanks in place System preparation Clean reverse osmosis RO water was conditioned in the SAS by seeding volumes of water from the original system to the SAS reservoir Monitoring of Nitrate Nitrite and Ammonia levels were performed daily SAS and weekly Control tanks for the duration of the trial Daily pH conductivity and water temperature were performed for both systems prior to and throughout the trial Figure 4 Illustration of water levels achieved using standard and modified siphons standard to the left and right modified in the centre G husbandry procedures were timed e g manual siphoning and compared to the original system Husbandry procedures were mirrored between control and SAS groups Both groups were fed on Mondays Wednesdays and Fridays with the diet offered alternating between Reptomin a floating food stick made by Tetra and Trout Pellets small sinking pellets supplied by ZM systems Tanks were siphoned as required to remove waste or uneaten diet Standard Siphon Modified Siphon Overflow Overflow Figure 5 Tank baffles were tested to assess food debris post feeding The different types of baffle were Fry baffles which have a fine mesh window and fit tightly to the bottom of the tank and Adult baffles which are solid with a series of 1 5mm holes drilled across their face and leave a small gap between the base of the baffle and the bottom of the tank Figure 3 On the right a standard siphon high overflow point Water runs out of the tank and is siphoned into the drain trough On the left is the modified siphon which is a single tube with cut outs at a lower level The water overflows through these leading to lower water level in the tanks 144 The health status of the Control and SAS frogs was measured assessed in the following ways G G daily observation and condition scoring fertility

Poster Presentations trial 1 frog was scored as looking slightly thin for 2 consecutive days and 2 frogs were scored as slightly thin for 4 consecutive days These observations were made during a Bank Holiday weekend with an altered feeding frequency that may have contributed to the observation Fertility Proven frogs with a 100 fertility rate were trialled and mated in both control and SAS groups When re mated 6 weeks later frogs from both systems again achieved fertilisation rates of 100 with the embryos showing excellent quality as determined by firmness good division and no embryo death Use of Lily Pads Figure 6 Further observations and recordings were made to monitor the frogs use of the lily pads that were in each tank and to check that the frogs would still use them even when the water level was high The condition scoring system looked at 3 areas G G G skin condition behaviour body condition e g does the frog look thin The scoring was on a scale of 0 6 where 0 was perfectly healthy and 6 would indicate the animal was extremely sick All observations were calibrated between observers prior to the on set of the study Results Water Monitoring All parameters remained within range throughout the trial G G G G G Ammonia 0 00 0 50mg l Nitrate 0 50mg l Nitrite 0 00 0 50mg l Conductivity 750 950 s pH 7 0 0 9 Water temperature 24 C 2 C The lily pads were frequented more regularly by frogs in the larger higher populated tanks than the smaller tanks lily pad usage in the smaller tanks was rare This may be due to the water depth and is being investigated further A difference was also noted in lily pad usage between the different water levels in the tanks The frogs in the larger tanks with the higher water level used the lily pads far more commonly than those with the lower water level Conclusion For both criteria condition and fertility on which the frogs were monitored there were no significant differences between Control and SAS frogs The Stand Alone System provides an adequate and acceptable condition for the housing health and welfare of our frogs The choice of baffles siphon and diet buoyancy is critical in terms of supporting husbandry procedure timings and maintaining the quality of the water provided for the animals Environmental enrichment use was variable and an area that requires further development The study showed that the SAS appears to be suitable for the housing of X tropicalis frogs with no detriment to their health or breeding performance Siphoning Tanks fitted with the drilled adult baffles required significantly less manual siphoning than the tanks fitted with the mesh baffles On many occasions there were no pellets left at all to siphon from the tanks fitted with adult baffles It was also observed that more pellets remained to be siphoned when the floating diet was fed compared to when the sinking diet was used Condition scoring All of the control frogs 21 were scored at 0 throughout the course of the trial 33 of the 36 frogs on the SAS system were scored at 0 throughout the 145

Animal Technology and Welfare August 2013 Challenging taboos in animal breeding and welfare JACKIE HARRISON1 CAROL COPLEY and NICHOLA CHROBOT MRC Harwell Mary Lyon Centre MLC Harwell Science and Innovation Centre Oxfordshire OX11 0RD 1 Correspondence to j harrison har mrc ac uk As an Animal Technologist you are trained by your peers to conform to a set of standards which have been defined from many years experience in the field but are all these standards still relevant today When there has been so many new developments concerning rodent welfare particularly over the last fifty years for example increased barrier protection Since the Mary Lyon Centre opened in 2004 we have kept comprehensive electronic records for all the animals under our care Over the past two years we have used this data and set up breeding experiments to challenge some of the standards previously set such as mating age litter sizes to name a few and have found that actually breaking these industry taboos has a positive effect on the animals in our care Mice breed whatever Conventional Breeding Unit Old Standards 1 Environmental Enrichment Kleenex tissue Tunnel 2 Dietary Supplement Ground RM3 3 Diet RM3 4 Water manually chlorinated 5 Cage floor space 370cm2 6 Technicians Only Lab coat and gloves No wet dry showering 7 No pet policy New Standards 1 Environmental Enrichment Shred bed tunnel 2 Dietary Supplement Transbreed irradiated 3 Diet RM3 irradiated 4 Water Filtered reverse osmosis Chlorinated 7 15ppm 5 Cage floor space 542cm2 6 Technicians Full PPE at least 1 type of shower 7 Pet policy enforced and 48 hours quarantine True but environmental changes influence breeding and mice that are not compromised by a poor health status produce better scientific results One breeding plan suits all We retrospectively analysed breeding data from 18 strains in 2010 As a rule we bred each inbred strain using the same batch breeding template The planned duration of the mating was 9 months We took data from 723 matings in this study and looked at the following criteria Mary Lyon Centre 146 1 Average litter size for each litter 2 Mating duration NB Matings were culled if there had been no litter for two months 3 Number of litters produced per mating

Poster Presentations Males were housed in boxes of 5 prior to mating and were not exposed to any females as they aged We trio mated the male cohorts at 12 18 24 30 and 36 weeks of age The females were plug checked every day before 10am for signs of copulation For each mating we looked at G G how many plugs were observed before successful copulation how many matings had litters Figure 1 Figure 1 Successful copulation Mated at three weeks just good friends As a general rule we had always set up inbred matings when the animals were considered to be sexually mature i e 6 7 weeks of age but we found this to have its disadvantages G G 1 There is a correlation between mortality and litter number 2 The optimum duration of the matings varied between strains As a result we now have individual templates for each strain and each year as the colony ages the data is reanalysed and the batch system is updated as required I m plugged I m pregnant For this research we used the following inbred strains C57BL 6J C3H HeH and CBA CaH in cohorts of 10 males of per age group We decided to ask the questions Is it true that animals mated at 3 weeks take longer to litter down Would there be any welfare concerns The potential advantages would be G G G cage space became available on the stock racks the risk of cross contamination would be minimised incidence of aggression would be reduced particularly in male stock cages Using C3H HeH we set up and analysed the data from trio matings 20 matings were set up at weaning the remaining 20 were set up at 6 weeks of age Age days We found that our previous assumption to be False and that one breeding plan did not suit all Productivity of a mating is affected by many factors including a mortality of an adult in the cage From the data retrieved it indicated that stock racks were being taken up by adolescence future breeding stock which can lead to the potential for cross contamination between strains 70 60 50 40 30 20 10 0 3 weeks 6 weeks Dam mating age days Figure 2 Age of dams when littering 147

Poster Presentations Again the conclusion was false G we found there to be no delay in the arrival of the first litter when animals were mated at 3 weeks of age Figure 2 G we had no welfare concerns produced as an effect of setting the animals up before puberty G this is now our standard procedure when mating inbred mice Experienced is always better As a production ward we often get requests for experienced females to mate with experimental breeding stock The general idea is that females rearing pups for the first time do not in some cases do well and the size and mortality of the litter suffers as a result To test this theory we retrospectively looked back at the breeding data for 1250 C57BL 6J matings G A long running debate has always been whether we should be sexing pups at birth 12 years ago this was common practice now it is frowned upon The view being that if you touch the animals at birth the mother will cannibalise or neglect the pups because a strange scent has been given to the pups leading to higher mortality rates We conducted a 9 month study with over 5000 mice looking into the effects of handling at birth using C57BL 6J firstly looking at average litter size Figure 4 secondly analysing mortality rates Figure 5 using 1208 litters G 8 7 Number of pups No touchy touchy 6 5 4 3 2 1 Procedure We set up two batches of 56 trio matings G batch 1 Handled normally batch 2 No Touchy Touchy Cage checked daily but not opened unless necessar y i e low diet Litters were recorded on day 3 10 0 Disturbed Not disturbed Figure 3 Average litter size at weaning 1 2 3 4 5 6 7 8 9 10 11 12 Litter number Figure 4 Average litter size 50 0 Mortality G 0 40 0 30 0 20 0 10 0 0 0 1 2 3 4 5 6 7 8 9 Litter number Results Primarily we found that there was no significant difference between the average litter size of animals handled at birth and animals which were not handled for 3 days after birth Figure 3 We found that there were more unrecorded litters recorded for the no touchy touchy matings which would suggest that animals were born but culled by their parents prior to the litters being recorded Our research found the answer to be False G G G 50 of the C57BL 6J 42 of the CBA CaH 26 of the C3H HeH Matings required more than one plug for a successful pregnancy to be achieved Except for the C57BL 6J strain age played a critical role in whether the animals sired litters at all 148 Figure 5 Neonatal mortality Again we found this idea to be false from the data we analysed Using naive females was not detrimental to the mortality of first litters Figure 5 Average litters sizes were also comparable if not better than later litter sizes and did not show any significant reduction in size Figure 4 Conclusion Inbred strains are not all the same and we cannot assume that one rule fits all Do not be afraid to challenge pre conceived ideas in a computerised industry we have the ability at our fingertips to analyse years of data and redefine breeding procedures and in doing so improve the welfare of the animals in our care As animal technologists we are in a unique position to be able to make a change after all no one knows your stocks better than you The subtle changes you notice can make a big difference to your stock and should be discussed

Poster Presentations Acknowledgements Thank you to the Mary Lyon Centre Production team past and present and to Dr Sara Wells for her input into this poster Understanding that high quality mice leads to high quality science 149

Animal Technology and Welfare August 2013 The changing face of flexible film isolators at the Mary Lyon Centre Quarantine Suite MARK HARRISON1 and ALISON GALLOP Medical Research Council Mary Lyon Centre Harwell Science and Innovation Centre Oxfordshire OX11 0RD 1 Corresponding author m harrison har mrc ac uk Introduction Disadvantages of Quadro Locks The Mary Lyon Centre s quarantine MLC Q suite supports a large programme specialising in cleansing mouse stocks before transfer to the SPF barrier facility In this capacity the MLC Q operates high and low health status flexible film and semi rigid walled isolators Since 2004 there have been numerous design changes to the isolators designed to improve the efficiency and bio security of the MLC Q This poster goes through some of the modifications that have been implemented to flexible film isolators and some of the ongoing modifications 1 Awkward access for racking cages 2 Once chipped started corroding 3 Expensive to replace if damaged Original isolator designs The original isolators in the MLC Q were 50 cage flexible film isolators Each isolator was fitted with a floor port fitted which was sealed with a Quadro Lock Each port had an internal and external Quardro Lock fitted 1 Port fitted with Quadro Lock at either end 2 Floor ports to hold a clinical waste bag with QuadroLock door 3 Clinical waste bag attached to floor port Advantages of floor port clinical waste bag 1 Reduced number of port procedure 2 Easy access to rubbish disposal 3 Reduced need for smaller rubbish bags Disadvantages of floor port clinical waste bag 1 2 3 4 5 Reduced working area Lost use of one cage Extra entry point to the isolator Once chipped started corroding Time consuming to change Initial isolator modifications Advantages of Quadro Locks 1 Easy access to port 2 Simple to use 150 1 Floor port totally removed 2 Internal port Quadro Lock replaced by a hinged door 3 External Quadro Lock fitted to an acrylic housing rather than powder coated 4 External hinged door housing Quadro Lock fitted

Poster Presentations 1 Front panel is flexible but the top sides rear are all 15mm acrylic 2 Base is 10mm white Perspex Black was avoided for aesthetic reasons 3 Sleeves are removable to allow us to just replace sleeves due to wear and tear Advantages of hinged port doors 1 Easy access to port 2 Simple to use 3 Removal of racking and cages easier Disadvantages of hinged port doors 1 Lock loosened breaking the seal and increasing need for regular checks 2 Rack impinges on internal door restricting opening Advantages of no floor port 1 Less entry points to the isolator 2 Working surface not inhibited 3 Don t lose the use of a cage Disadvantages of no floor port Increased use of smaller rubbish bags Latest isolator modifications The latest round of canopy replacement has enabled us to move away from flexible film isolators and on to semi rigid walled isolators This has led to further gains in efficiency and biosecurity Advantages of semi rigid isolator canopy 1 Improved biosecurity 2 Longevity 3 Only need to replace front panel due to wear and tear 4 Easier to clean when broken down 5 Better all round viewing of cages Disadvantages of semi rigid isolator canopy 1 Less range of movement for user 2 Expensive 3 Need to ensure front panel gasket fitted correctly 151

Animal Technology and Welfare August 2013 Our experience as GSK apprentices JACK ARMES1 and BEN JAGGS GlaxoSmithKline Safety Assessment Research Development Park Road Ware Hertfordshire SG12 0DP 1 Corresponding author jack x armes gsk com Introduction Apprenticeships allow students to work whilst gaining not only education but valuable real life skills that enable them to excel in a career which could have previously been unavailable to them In the UK alone there are more than 250 different types of apprenticeships available offering over 1 400 job roles expanding over a huge variety of sectors We were part of the pilot apprenticeship scheme to launch at Ware R D site We began as a group of four which has now evolved into a number of groups spread throughout the UK sites across a range of different departments Objectives G G complete college courses at BTEC level 3 build functional capability within each department G rotation between a number of departments G understand and learn about working in a pharmaceutical company become multiskilled and able to work in different departments gain Personal Licences G G G become competent in licenced procedures The programme The apprentices began working in different departments working for four months at a time before rotating to work in another area of the business Within this time it would enable us to gain knowledge and develop the skills which would begin to increase our value as staff members and broaden our sights to the bigger picture Whilst working apprentices also gain a qualification relating to the particular sector that they choose Apprentices attend College to study a BTEC gaining qualifications equivalent to GCSE A level and Degree level This is all made available to them by the Company which invests not only money but time into seeing that apprentices achieve recognised qualifications It is a great opportunity for young adults to be immersed into a working environment obtaining the capabilities necessary to do the job from seasoned professionals This benefits both the trainer and trainee as they create new ways of thinking which inspires new methods of training whilst leaving existing methods being questioned and refined 152 Figure 1 Dog acclimatisation training in preparation for regulated procedure Dog facility This department is very labour intensive which requires around the clock maintenance from appropriately trained staff as the dog s welfare and health are the main focus of the work we conduct This cannot be emphasised enough We adhere to strict rules and regulations that ensure that we perform to a high standard

Poster Presentations Personal licences The main objective of having our licences was to be able to work as part of the Toxicology Support Group and carry out important work on studies such as blood sampling and dosing This lead to our opportunity to go to the Royal Veterinary College where we gained our Home Office Licence in modules 1 2 and 3 obtaining theoretical training in regulated procedures We also passed an exam relating to ethics and knowledge of the subjects Once the licences came through we were mentored and super vised while we gained competency Figure 2 Dog play time Small animal facility We work in a new building which houses rats mice and rabbits which are used for toxicology testing This means we have to house them in a highly regulated environment and work to GLP The animals are given environmental enrichment which is reviewed regularly and all of us have the opportunity to source new enrichment items Our roles in the facilities The primary focus of our team is animal welfare We carry out husbandry including cage changing and pen cleaning as well as health checks Once qualified to use our Home Office licence we then performed regulated procedures including blood sampling via the tail vein for rats or jugular vein with dogs and oral gavage dosing with both We have set up studies under supervision and are training to become the main point of contact Figure 4 Pipetting samples under a fume hood whilst working in Clinical Pathology Future objectives G G G G G achieve BTEC Level 3 continue education to Foundation Degree acquire competency in licenced procedures train as Responsible Licencees further multiskilling abilities across departments including Histology and Clinical Pathology Career prospects After completing this apprenticeship we will hopefully be granted a job proposal to become full time within the company This would be a huge opportunity for us as we would have had three years experience as technicians and will be able to continue progression as staff members working in a team Our training will still be ongoing and we will be given further opportunities to expand our knowledge and capabilities Figure 3 Mouse environmental enrichment 153

Poster Presentations Acknowledgements We would like to say a big thank you to each Safety Assessment staff member who took the time and effort to train and support us thoroughly and shape us into the technologists we are today We would also like to thank LAS where we worked whilst in the animal facilities We would like to extend a special thank you to our manager Matthew L Affineur for being a truly inspirational figure in our lives and giving us the guidance and support that is required when starting out in this fast paced highly pressurised industry It has been a wonderful experience 154

August 2013 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 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 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 Articles for submission should be sent to Journal Editorial Board Chairman 5 South Parade Summertown Oxford OX2 7JL Submission No responsibility will be accepted for loss or damage to such articles 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 Format 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 to the address below together with a copy on disk CD or DVD 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 155

Instructions to Authors 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 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 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 Illustrations charts and photographs supplied on disk should be in JPEG TIFF or EPS formats and have a resolution of no less than 300dpi The captions for illustrations charts and photographs should be typed in double spacing in numerical order on a separate sheet of paper 5 References to other literature should be indicated in the text by superior figures12 and should be typed in numerical order in the order in which they appear in the text on a separate sheet of paper 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 Content 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 Animals 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 microorganisms 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 e g Saigeman S 1998 Environmental enhancement of cats what why how Animal Technology Vol 49 No 3 145 154 Number of animals and any pretreatment Time of day of procedure s Quantity and frequency of any samples Books Surname and initials of author s date title of book Name of publisher Town of publisher Statistics e g Flecknell P A 1987 Laborator y Animal Anaesthesia Academic Press London Reprints 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 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 87 105 156 Tests used should be named 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

INDEX TO ADVERTISERS August 2013 Allentown Inc iv Avid Plc xiv Bell Isolation Systems xiii Charles River Laboratories IFC Edstrom Industries Inc vii Harlan Laboratories xi Institute of Animal Technology vi xii xv xvii xviii IPS Product Supplies Ltd IBC LBS viii PFI Systems iv Special Diets Services v Sychem Ltd x Tecniplast UK OBC VetTech Solutions xvi Wiley xv