CONTENTS Vol 14 No 2 August 2015 Editorial Jas Barley Chair of the Editorial Board ix The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury in group housed male Swiss mice Lewis Vaughan Monica Kloppers1 and Alexandar Whittaker2 77 The naked mole rat husbandry and maintenance Haley Forrest and Alison Robinson 85 PAPER SUMMARY TRANSLATIONS 91 TECH 2 TECH NACWO Guidelines 99 POSTER PRESENTATIONS The tail prick technique a refinement in blood sampling technique for a genetically modified mixed background strain of mouse Caroline Zverev and Vanessa Andrews 105 The development and refinement of bioluminescent fluorescent orthotopic xenografts models to reduce harm and improve animal welfare Alison Ritchie Pam Collier Phil Clarke and Anna Grabowska 108 The use of hypoxic chambers with large animals Craig Forest 111 Pruritic aged C57BL6 J mice Christine Marshall and Sally Carpenter 115 Supraglottic airway devices in rabbits how we implemented this technique to improve animal welfare and health and safety for staff Emma Tozer 118 Recommendations for environmental enrichment to enhance the welfare of mice in oncology research Gemma Willoughby Jason King Vicky Lacey Louise Wainwright James Finney Trudi Gee Ed Hale Will Tyler and Kelly Jones 121 3Rs Refinement use of hydrophobic sand in collection of analytical urine samples Hilary Lancaster Fiona McClure Debbie Ridley Jason Smith and Greg Whelan 125 Welfare and maintenance of the BIOZZI mice Sarah Bright 128 A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late stage Human African trypanosomiasis HAT Hollie Burrell Sawar Jean Rodgers Barbara Bradley Simon Croft and Theresa Ward 130 Evaluation of an automated CO2 euthanasia system Robert Stewart and Claire Booth 135 An updated road map towards ending severe suffering Elliot Lilley Penny Hawkins and Maggy Jennings 137 Evaluation of a novel rat gerbil nesting house with stereotypical behaviour body weights and food consumption Gordon Melville 139 Improving rat housing and enrichment a local initiative at the NIBSC Colette Mottram Hunt 142 Achieving and maintaining germ free status within an SPF facility Claire Rogerson Selina Hopkins Cordelia Brandt Silvia Hrnciarova Scott Kemp and Glenn Hazzard 145 Open or closed Reece Williams 150 Instructions to Authors 153 Index to Advertisers xviii ATW PROFILE Animal Technology and Welfare aims to be the medium for animal technologists and all those concerned with the care and welfare of animals used for research purposes to communicate best practice ATW especially aims to promote and develop the 3Rs particularly in respect of Refinement More importantly ATW promotes the generally accepted 4th R Responsibility The responsibility that all animal technologists have in ensuring dissemination of best practice to every institution using animals in research ATW enjoys a unique position as the scientific publication for the leading organisations IAT and EFAT for the welfare of animals in research Editor Jas Barley atweditor iat org uk

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August 2015 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 The mission statement printed at the beginning of the 3rd Edition of the Institutes of Animal Technology Guidelines for Named Animal Care and Welfare Officers NACWOs reads Our Mission Advancing and promoting excellence in the care and welfare of animals in Research It is important to remember that in our efforts to improve and promote our standards of welfare we must remind ourselves that not all improvements to welfare are in fact effective and must be carefully considered and tested before wholesale adoption of said improvements I am sure I am not alone in being able to think of various examples of new techniques and equipment that at first glance have appeared to be better than existing practices of the day but which have fallen by the wayside once they have been more widely tested and found wanting The first paper contributed by Lewis Vaughan and his team based in Australia presents just such a case where a surgical method initially appears to reduce injuries and loss of mice due to aggression proved at first glance to provide no significant reduction of the problem in the strain of mice the authors studied The surgical technique examined is relatively minor and has little if any long term effect on the mice in the study However it is difficult to accept that submitting an animal to a surgical process that does not clearly benefit either the animal or science as an advancement of welfare The author explains there may be strain differences and other factors influencing the anomalies in results between the original study and their own and that further investigations may clarify whether the surgery may prove beneficial in certain cases It is a reminder to us all that when adopting new techniques etc that we should proceed with caution until the pros and cons have been confirmed I am delighted to have been offered Lewis s contribution as it is the first from the Southern Hemisphere for some time and it is of great advantage to all of us to be able to share their findings Hopefully we will see an increase in overseas contributions in the future in fact it would be good to see an increase in contributions overall I know you are undertaking appropriate research I just need you to make it more widely available In addition to our Australian contribution we have in this issue a comprehensive article on the husbandry of the fascinating Naked Mole Rat Visually they are perhaps not the most attractive of creatures although it is obvious from the article that Haley Forrest and Alison Robinson the two young authors are passionate about them We also include in this issue the first of the Congress 2015 poster presentations These cover a wide range of topics and include a refined technique of blood sampling of mixed background Genetically Altered mice from Caroline Zverev and Vanessa Andrews the use of Hypoxic chambers for large animals by Craig Forest Sarah Bright s poster on care of the Biozzi mouse and an updated road map towards ending severe suffering from the team of the RSPCA Research Animals Department Science Group amongst others Developments in techniques and equipment are not the only issues that Animal Technologists need to consider Although those of you who are IAT members will have already seen the new edition of the Named Animal Care and Welfare Officer NACWO Guidelines which clarify the role and responsibilities of the NACWO under the revised Animal Scientific Procedures Act 1986 we have taken the opportunity to publish them in this issue Although intended for UK Animal Technologists they should provide helpful to anyone working under the EU Directive 2010 63 EU and those of our readers elsewhere who are working towards improving animal welfare and a Culture of Care ix

August 2015 Animal Technology and Welfare The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury in group housed male Swiss mice LEWIS VAUGHAN1 3 MONICA KLOPPERS1 and ALEXANDAR WHITTAKER2 1 2 3 Veterinary and Applied Science Centre Technical and Further Education South Australia TAFESA Gilles Plains South Australia Australia School of Animal and Veterinary Sciences The University of Adelaide Roseworthy Campus South Australia Australia Current address Animal Welfare Officer Flinders University GPO Box 2100 Adelaide 5001 South Australia Corresponding author lewis vaughan flinders edu au Summary Male mice are commonly group housed in biomedical research establishments and educational institutions However this form of housing may present a significant welfare cost to animals due to the elicitation of fear responses in subordinate animals and injury that results from actual fighting Preputialectomy has been proposed as one simple method to reduce aggression without altering reproductive hormonal profiles In this study 50 animals that had undergone preputialectomy and 50 preputially intact control animals were studied over a 9 week period to monitor aggression frequency and injury levels Under the conditions of this study there were no statistically significant differences in frequency of aggressive behaviour or injur y data between operated mice and controls It is concluded that preputialectomy of male mice may not reduce aggression nor contribute to improved welfare in a long term group housing situation Keywords Aggressive behaviour preputialectomy injury welfare mice Introduction The laboratory mouse is an important species used in biomedical research and teaching being the most used mammal in experimentation For example in 2011 in the EU it accounted for 61 of all animals used in research 1 The mapping of the mouse genome and the ability to create genetically modified strains has increased its significance as a laboratory species which has spawned further welfare challenges 2 3 In research and teaching the keeping of intact male laboratory mice in group housing has been identified as a significant welfare concern due to the aggression and consequent injury that frequently results from social instability 4 5 6 A number of workers have reported improvements to the welfare of group housed male mice by manipulating the structural cage environment and enrichment materials 7 8 adjusting group and cage sizes 9 or by castration 10 The castrated animal model is unlikely to receive widespread application since androgen reduction through castration may introduce confounding variables in research such as changes in tissue mass immunologic reactivity glandular secretory cells and plasma hormones such as insulin and leptin 11 12 13 Preputial gland secretions when applied to castrated male mice have been shown to be associated with increased attacks by intact males 14 15 It has also been demonstrated that dominant mice have heavier glands than subordinates 16 Subsequently Novotny et al 1990 identified two sesquiterpenes E E farnesene and E farnesene as constituents of mouse urine derived from the preputial glands 17 These farnesenes have been shown to provide information to mice regarding male social status and are par tially suppressed in subordinate males which show a concomitant reduction in the size of the preputial glands 17 18 In view of this physiological and anatomical awareness it could be assumed that preputial gland removal might lead to a reduction in male mouse aggressive behaviour Hayashi 1987 demonstrated that bilateral preputialectomy in Jcl ICR ICR JCL mice decreased aggression during the 14 day period after surgery when 77

The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury male mice pairs were separated by a wire net barrier and subsequently introduced to each other and obser ved for 26 minutes per day 19 Since the publication of these findings further research has elucidated the role of the preputial gland products urinar y volatiles and mouse urinar y proteins in physiology and social behaviour 18 20 21 In addition to these advances Nakamura et al 2007 has demonstrated the overriding influence of familiar urine odour in diminishing territorial aggression in a residentintruder paradigm 22 However there have been no fur ther studies investigating the effects of preputialectomy on aggressive behaviour in mice or whether such a procedure could contribute to enhanced group cohesion over a longer time course In this project we investigated the influence of bilateral preputialectomy on agonistic behaviour and injury rates for group housed male mice to ascertain if the procedure could be used as a tool to reduce aggression and hence improve welfare outcomes for mice kept in long term common husbandry conditions Methods Animals Outbred Swiss ARC ARC S mice were purchased from a SPF production facility University of Adelaide Laboratory Animal Services SA Australia These mice were free of common mouse pathogens including mouse hepatitis virus minute virus of mice mouse parvovirus and rotavirus The mice used in this study n 100 were bred in house from the purchased animals in open top conventional cages under barrier conditions At weaning the mice were randomly allocated into groups of 5 and placed into polycarbonate open top cages with dimensions of 415 mm x 260 mm x 145 mm Tecniplast NSW Australia Enrichment material consisted of tissues and one cardboard roll for each cage Enrichment materials were standardised between cages The housing facility was maintained with artificial lighting on a 12 12 hour light dark cycle humidity range of 40 to 70 temperature range of 20 C to 24 C and 15 to 20 air cycle changes hourly Recycled paper pellets Animal Bedding Fibrecycle Pty Ltd Yatala Qld Australia were used as bedding substrate Fixed formulation cubes Rat and Mouse Cubes Specialty Feeds Glen Forrest WA Australia were provided adlibitum from wire hoppers on cage tops All bedding and feed was autoclave sterilised prior to entry into the barrier facility Potable mains water was provided in glass bottles and was changed weekly The project was approved by the Institutional Animal Ethics Committees of Technical and Further Education South Australia TAFESA and the University of Adelaide according to the provisions of the Australian Code for the Care and Use of Animals for Scientific Purposes23 24 and the South Australian Animal Welfare Act 1985 78 Experimental design An investigator who was neither involved in the daily care nor the behavioural obser vations randomly assigned each of the 20 cages to one of two groups One group of fifty animals was subjected to surgical bilateral preputialectomy performed between the ages of 4 and 5 weeks The remaining fifty animals were left intact to form the control group One to two months after surgery when the animals were between 9 and 15 weeks of age behaviour obser vations were commenced These observations were conducted twice weekly over 9 weeks 18 observations Observations consisted of 7 minute periods during the animals light phase between 10 00 and 12 00 not in association with cage cleaning Previous studies have similarly conducted aggression tests during the light period 4 10 Veterinary technology students who were unaware of the animals treatment status conducted the observations from a distance of 2 m Students were supervised by an experienced technical staff member during all the observation periods Categories used to quantify the observed behaviours included characteristic patterns of aggression and defence such as offensive sideways chase bite defensive upright crouch freeze and flee behaviours 25 Observation data was recorded and quantified by an alloccurrences sampling method as used in a previous similar study 10 The number of behavioural occurrences was recorded and all mice were examined for injuries at the end of each observation period The data collected included the number of mice with bites in each cage the total number of bites per cage and the number of mice euthanised due to injur y Animals were euthanised according to strict endpoint criteria if there was evidence of body cavity penetration subcutaneous swelling three or more non healing bites or scab or skin excoriation of greater than one square centimetre Three classifications of minor moderate and severe injury were used in the injury scoring system Bites or scratches without skin penetration were recorded as minor injury Injuries consisting of skin penetration with slight redness swelling but without functional impedance were classified as moderate injuries Severe injur y included skin penetration with inflammation or discharge plus functional impedance Mice with minor to moderate wounds remained in the project without treatment We believed that removal of mice from all male groups for treatment and later reintroduction constituted a confounding variable by destabilising established group hierarchies In our experience superficial and moderate bite injuries heal rapidly whereas severe wounds do not To protect mouse welfare strict euthanasia criteria were adhered to so that all mice showing signs of severe wounds were euthanised Veterinar y examination was performed in such circumstances to confirm that euthanasia was justified

The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury Surgery and anasthesia Medetomidine hydrochloride 0 75 mg kg IP Domitor 1 mg mL Zoetis Australia West Ryde NSW Australia and Ketamine hydrochloride 100 mg kg IP Parnell Ketamine Injection 100 mg mL Parnell Laboratories Alexandria New South Wales Australia were administered to induce anaesthesia A non rebreathing modified T piece was used to supply oxygen throughout the procedures All procedures were undertaken using aseptic technique Bilateral preputialectomies were conducted via a midline longitudinal abdominal skin incision of 5 mm immediately over the preputial glands The preputial glands were separated from surrounding subcutaneous tissues and skin by blunt dissection as shown in Figure 1 Blood vessels were cauterised illustrated in Figure 2 and the skin repaired with Reflex 9 mm Wound Clips CellPoint Scientific Gaithersburg MD USA Perioperative analgesia was provided by Butorphanol tartrate 1 5 mg kg SC Torbugesic 10 mg mL Fort Dodge Australia Baulkham Hills New South Wales Australia delivered just prior to surgery and postoperative analgesia provided by Meloxicam 1 5 mg kg SC Ilium Meloxicam 5 mg mL Illium Veterinary Products Smithfield New South Wales Australia administered at the end of surgery Meloxicam therapy was continued for 3 days after the surgery by inclusion in the drinking water 0 0025 mg mL in water Ilium Meloxicam oral suspension 1 5 mg mL Ilium Veterinary Products Atipamezole hydrochloride 1 mg kg SC Antisedan 5 mg mL Zoetis Animal Health was given immediately after surgery to reverse the effects of medetomidine sedation During anaesthetic recovery mice were wrapped in air bubble packing as shown in Figure 3 and placed on thermostatically controlled heated pads until they regained their righting reflex and were then returned to their social groups Water soaked diet cubes were provided in the cage for 18 hours after surgery Mice were monitored daily for 7 days following surgery and veterinarians were available for consultation if problems occurred Wound clips were removed at 7 days Figure 1 An intra operative photograph that shows a mouse in supine position with the caudal ventrum visible through a fenestrated surgical drape with the scrotum on the right A preputial gland has been isolated after blunt dissection of the surrounding tissues Figure 3 A photograph that demonstrates postoperative wrapping of a mouse in air bubble packing in order to maintain body heat during recovery Statistical analysis Figure 2 An intra operative photograph with a mouse in supine position that shows excision of a preputial gland by clamping and cautery The behavioural and injury events were quantified for each cage over the 18 observations and means were ascertained for both groups Each cage was deemed to be a discrete experimental unit n 10 for each treatment Since data were not normally distributed non parametric statistical tests were used P values for the differences between the means of the treatment and non treatment control groups were calculated by using the Wilcoxon Mann Whitney test Analysis was performed by the Megastat Excel Add In version 10 2 McGraw Hill Higher Education New York NY 79

The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury Differences were determined to be significant when the P values were less than or equal to 0 05 between preputialectomised mice and control mice for all variables Results The serial time charts Figures 4 through 6 illustrate the lack of difference over the weeks of observations for the collated variables used to assess behaviour and injuries between cages of control and preputialectomised mice All mice in the preputialectomised group recovered from surgery without complications and required no additional rescue analgesia One cage n 1 of control mice was euthanised during the third week as a result of multiple severe injuries due to aggression The injury intervention criteria previously described were used in making the decision to euthanise these animals Data from this group except for the criterion number euthanised were discarded and not incorporated into the summary analysis of the study Statistical analysis of the behavioural and bite injury data Table 1 demonstrated no significant P 0 05 difference Behaviour criteria Preputialectomised mice Control mice Aggressive behaviours Offensivesideways 8 8 2 4 10 3 2 4 Chase 14 5 3 8 15 2 3 0 Bite 13 6 4 0 Figure 4 Number of aggressive events per cage mean SEM for each treatment group over the 3 month observation period Differences between groups are not significant P 0 05 but for data indicated by where P 0 05 12 6 2 0 Defensive avoidance behaviours Defensiveupright 11 4 2 8 9 3 2 3 Crouch or freeze 2 7 0 7 2 8 1 0 Flee 13 6 4 0 12 6 2 0 Bites injury observations Number of mice with bites 4 9 1 6 2 8 0 8 Total bites 3 6 1 3 2 4 0 6 Number with minor injury 1 1 0 6 0 4 0 2 Number with moderate injury 0 3 0 2 0 7 0 5 Figure 5 Number of defensive events per cage mean SEM for each treatment group over the 3 month observation period Differences between groups are not significant P 0 05 but for data indicated by where P 0 05 Number with severe injury Number euthanised Differences behaviours early in the cage were above between groups are not significant P 0 05 for all All animals from one control cage were euthanised study Data except number euthanised from this discarded and not incorporated into the analysis Table 1 Number of events of measured behaviours and bites injury observations per cage mean SEM for each treatment group over the 3 month observation period 80 Figure 6 Number of bite injuries per cage mean SEM for each treatment group over the 3 month observation period Differences between groups are not significant P 0 05

The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury Discussion The surgical procedure whilst not widely described in the mouse was simple to perform and easily learnt by competent surgeons The superficial position of the glands rendered the procedure minimally invasive and with good analgesic practice the surgical event itself is unlikely to present a significant welfare cost In this study no post operative complications were seen and the mice recovered quickly Hayashi 1987 showed that Jcl ICR male mice raised in groups of 5 or 6 isolated for 11 days between the ages of 16 and 20 weeks and then subjected to preputialectomy showed less aggression towards both familiar and unfamiliar male mice on a resident intruder model during the first 2 weeks after surgery 19 Our results in contrast showed no significant difference for aggressive and defensive behaviours or injury rates between the intact mice and those without preputial glands One cage of mice that had not received preputialectomy was lost to the study during the third week of observations as a result of our following the previously described euthanasia criteria for injury Whist this event may have marginally influenced the results obtained its effect is likely to have been minor given the overall lack of significance between the treatment and control groups In the study of Hayashi 1987 behavioural obser vations were begun by removing a wire net barrier between cage compar tments housing male mice pairs 19 Observations were undertaken for 26 minutes daily starting at 48 hours after surgery and continuing for 14 days In the current study we maintained all male groups of 5 mice from weaning for the entire period of the project Behavioural observations were not started until 4 to 8 weeks after surgery when animals were between 9 and 15 weeks of age Thus whilst observations were taken at similar mouse ages in both studies animals in the current study had undergone the surgery at a younger age and had been living in their static social groups for a period of several weeks before observations commenced It would have been expected that this experimental design in contrast to that of Hayashi 1987 19 would lead to reduced aggression and increased group stability The extended period of recovery time after surgery in our study should have allowed the animals to stabilise their social behaviour Due to the time between surgery and the commencement of observations we did not in contrast to Hayashi 198719 establish a sham surgery group This enabled us to refine animal usage in the study in line with the principles of the 3Rs of Refinement Reduction and Replacement promoted by Russell and Burch26 and as recommended by current guidelines27 and codes 23 24 We assert that our model for assessing aggression and injury is more realistic and representative of true husbandry conditions than that employed by Hayashi 1987 19 In that study observations started soon after surgery when recovery may have been still in progress Familiar and unfamiliar mice were maintained prior to and after surgery in separate cage compartments divided by wire partitions so that no opportunity for establishment or maintenance of stable social hierarchies could occur The results of the study of Tanabe and Kimura 1995 are also helpful in understanding the effect of preputialectomy on social behaviour 28 In their study male Jcl ICR mice pairs separated by wire net divisions showed greater violent attack behaviour towards an intruder and had the heaviest preputial glands and testes when compared with males kept isolated or kept in pairs without divisions This finding led to the assertion that a greater preputial gland size was associated with the attainment and or maintenance of a dominant social status The converse may also be true in that it has been demonstrated that mice subjected to repeated defeat are characterised by a decline in preputial gland weight 29 We used Swiss ARC ARC S mice which have a similar genetic origin to the Jcl ICR strain that Hayashi 1987 used 19 Both strains are classified as outbred mice although as described by Cheetham et al 2008 mitochondrial DNA evidence indicates the strains were derived from an extremely limited founder gene pool which has resulted in minimal mouse urinary protein MUP variation 20 This lack of MUP variation has an impact on the ability of Swiss derived strains to recognise individuals 30 Therefore if male Swiss mice are not constantly housed together but separated by a wire division as in the Hayashi 1987 study 19 they may not be able to easily recognise each other This could be due to their inability to associate changing urinary volatiles emanating from individuals with MUPs in urine scent marks 21 Perhaps in the latter case when the resident intruder test was used to assess aggression the presence or absence of preputial glands had significant roles in modifying aggression towards intruders It is possible that by establishing and keeping small group sizes from weaning we created conditions in which stable social hierarchies could be maintained and hence there was relatively little difference in aggression between the two experimental groups Although there appeared to be a low level of aggression in both groups in our study much higher levels of aggression have been observed when mice have been kept in larger groups 5 Van Loo et al 2003 endorse the strategy of keeping mice in small groups although they recommend an ideal size of 3 male mice 5 Such an environment may have fostered stable social conditions by ensuring that mice were able to recognise individuals from a distance by associating urinary volatiles which change with social status reproductive 81

The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury state health and food sources with their fixed MUPs emanating from the urine scent marks21 The moderating effects of preputialectomy on aggressive behaviour observed by Hayashi 1987 19 may not have been manifest in our project because of the stabilising effect of the factors previously discussed References 21 22 Although this blinded study controlled for variables by randomising animals and cages to treatment groups it was constrained by the use of student observers supervised by an experienced veterinary technician This resulted in the potential confounding effect of human observers monitoring behaviour from a two meter distance and recording the data in the mice s inactive phase light period In addition we did not establish a sham surgical control group of mice since we assumed that because observations commenced at least one month after surger y in the preputialectomised mice confounding affects associated with inflammation and healing would be minimal 23 24 25 26 Potential benefits of preputialectomy to mouse welfare could be further explored in future studies by housing one preputially intact male with preputialectomised cage companions In such future studies we aim to limit confounding variables by recording video of behaviour using a close in camera during all phases of the mice s circadian rhythm Perhaps such a strategy of group housing could foster the rapid development and maintenance of stable hierarchies and reduce welfare compromise It is questionable though whether such a scenario would entirely eliminate aggression and injuries since dominance is likely to be determined by numerous factors associated with androgen expression rather than farnesene production by the preputial glands alone This highlights the importance of the need to recognise the multi factorial causes of aggression and the significant role that social environment plays Unfortunately it seems likely that the complete elimination of aggression and injuries in group housed male mice is not likely to lie in just one simple surgical solution but will require substantial understanding of the complex interactions between housing and social environments that occur in laboratory animal housing It is concluded that based upon the observations of this study preputialectomy may not provide a benefit to group housed male mice in terms of aggression reduction nor improved welfare 27 28 29 10 11 12 13 14 Acknowledgements We express our gratitude to the students of the Diploma of Animal Technology 2012 programme and the staff of the Veterinary and Applied Science Centre We extend our thanks to Dr A Livingston for editorial support The study was funded by TAFESA 15 16 82 European Commission 2013 Commission Staff Working Document accompanying the Seventh Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member 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The influence of bilateral preputialectomy on aggressive behaviour and incidence of aggression related injury 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Novotny M Harvey S and Jemiolo B 1990 Chemistry of male dominance in the house mouse Mus domesticus Experimenta Vol 46 109 113 Harvey S Jemiolo B and Novotony M 1989 Pattern of volatile compounds in dominant and subordinate malemouse urine J Chem Ecol Vol 15 2061 2072 Hayashi S 1987 The effects of preputialectomy on aggression in male mice Zool Sci Vol 4 551 555 Cheetham S A Smith A L Armstrong S D Beynon R J and Hurst J L 2008 Limited variation in the major urinary proteins in laboratory mice Physiol Behav Vol 96 253 261 Hurst J and Beynon R J 2004 Scent wars the chemobiology of competitive signalling in mice Bioessays Vol 26 1288 1298 Nakamura K Kikusui T Takeuchi Y and Mori Y 2007 The critical role of familiar urine odor in diminishing territorial aggression toward a castrated intruder in mice Physiol Behav Vol 90 512 517 National Health and Medical Research Council 2004 Australian code of practice for the care and use of animals for scientific purposes Australian Government 7th ed Canberra National Health and Medical Research Council 2013 Australian code for the care and use of animals for scientific purposes Australian Government 8th ed Canberra Grant E C and Mackintosh J H 1963 A comparison of the social postures of some common laboratory rodents Behaviour Vol 21 246 259 Russell W M S and Burch R L 1959 The principles of humane experimental techniques Methuen London National Health and Medical Research Council 2008 Guidelines to promote the wellbeing of animals used for scientific purposes The assessment and alleviation of pain and distress in research animals Australian Government Canberra Tanabe M and Kimura T 1995 Aggression and preputial gland of male mice affected by the presence of other males J Ethol Vol 13 63 68 Brain P F Homady M H and Mainardi M 1983 Preputial glands dominance and aggressiveness in mice Bolletino di zoologia Vol 50 173 187 Cheetham S A Thom M D Jury F Ollier W E R Beynon R J and Hurst J L 2007 The genetic basis of individual recognition signals in the mouse Curr Biol Vol 17 1771 1777 83

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August 2015 Animal Technology and Welfare The naked mole rat husbandry and maintenance HALEY FORREST and ALISON ROBINSON Department of Psychology and Department of Physiology Developmental and Neuroscience Combined Facility University of Cambridge Downing Street Cambridge Cambridgeshire CB2 3EB Corresponding author ajr97 cam ac uk Abstract In an industry dominated by rats and mice Animal Technologists rarely get the opportunity to work with a species that is a little different The Naked Mole Rat Heterocephalus glaber is a fascinating animal which has very different husbandry requirements compared to most other rodents Native to East Africa it is a mostly hairless mammal which lives in colonies with a similar hierarchal structure to that of bees and ants It is one of only two species of mammals both mole rats that are eusocial As an Animal Technologist it is very important to understand the full requirements of the animal s maintenance and care in order to ensure its welfare The animals are very sensitive to their environment which complicates feeding and general husbandry of the species Breeding of Naked Mole Rats is also much more complicated due to their eusocial nature and their sensitivity to disturbances Although quite difficult to maintain they have special adaptations which make them good models for various scientific studies Keywords Naked Mole Rats eusocial environment sensitive husbandry Born to be wild The Naked Mole Rat also known as Heterocephalus glaber is a small rodent that is native to East Africa They are a mostly hairless mammal which live in colonies with a similar hierarchal structure to that of bees and ants They are one of only two species of mammals both mole rats which are eusocial The colony consists of anywhere between 20 300 animals but averages at around 80 It consists of one breeding female the queen who will breed with a few select males chosen from the rest of the workers These workers make up the bulk of the colony and consist of males and submissive females The queen rules her colony through aggression often biting others at any time When passing in a tunnel the queen will walk over the top of other colony members to show her dominance If the queen dies or is weakened the larger more dominant females will fight often to the death to become the queen Figure 1 Naked mole rat workers from different colonies in conflict Mendez R A n d Available at http www arkive org naked molerat heterocephalus glaber image G75282 html Accessed 29 January 2015 The worker mole rats have different jobs such as diggers whose role is to create new tunnels to find new food sources Also sweepers who keep the tunnels clean and clear of debris volcanoers that kick the Figure 2 Naked mole rat burrowing behaviour n d image online Available at http carnivoraforum com topic 9328742 1 Accessed 29 January 2015 85

The naked mole rat husbandry and maintenance debris out of the burrow opening and food collectors whose duty is to bring food to the other mole rats and especially the queen These underground burrows are maintained at a stable temperature of around 30 C regardless of the outside temperature There are approximately 30 species of mole rat of which the best known is the Naked Mole Rat All the other species of mole rat are furred and are larger than their naked relatives The different species of mole rat vary in size and they have a variety of different social structures ranging from completely solitary e g the Cape Mole Rat Georychus capensis to the social e g Damaraland Mole Rat and Naked Mole Rat chambers and tunnels which consist of sleeping areas toilets and feeding tunnels In our facility we tr y to replicate their natural environment by having a system of chambers all connected by tunnels A thin covering of substrate is provided which they then spend most of the day moving around leaving the plastic floor clear which encourages natural behaviour Paper wool is given as nesting material which the mole rats will move around depending on where they want their nest The room is maintained at around 28 C and there is a heat cable running under two of the chambers so that the mole rats have somewhere warmer to sleep if they wish The humidity needs to be kept at around 50 otherwise if it is too low they will start to develop dry skin Figure 5 and 6 Artificial burrows Figure 3 Colony of Naked Mole Rats Parsons L 2012 Digging the Underground Life Image online Available at http www the scientist com articles view articleNo 32136 title Underground Supermodels Accessed 29 January 2015 We do not provide them with a large amount of environmental enrichment as their daily activities of rearranging their housing and distributing their food keeps them busy for most of the day We do however provide them with a running wheel which may not be very natural to their environment but which they seem to love Some of them seem a little too big for the wheel but that does not stop them Housing In the wild mole rats live in underground burrows that can span up to a couple of miles depending on the size of the colony These are made up of interconnecting Figure 7 Naked Mole Rat using running wheel Figure 4 Structure of underground burrows Parsons L 2012 Digging the Underground Life Image online Available at http www thescientist com articles view articleNo 32136 title Underground Supermodels Accessed 29th January 2015 86 They are very clean animals and they will choose one chamber to be their toilet This gets very dirty so it is cleaned on a daily basis Apart from daily spot cleaning where any old food and soiled bedding is removed the

The naked mole rat husbandry and maintenance rest of the cages only need to be cleaned once a month This is achieved by completely changing the cage system making sure we keep some of the old substrate to scatter around to make it smell like their home The setup of chambers and tunnels is always the same and the position of items such as the running wheel is not changed Figure 9 Naked Mole Rats in feeding cage Figure 8 Latrine cage Another method of providing environmental enrichment and to aid the display of natural behaviour is to block various tunnels usually into the feeding chambers This encourages the mole rats to gnaw through the blockage as they would in the wild when creating new tunnels Feeding Naked Mole Rats are herbivores which in the wild consume mostly roots and tubers they find while excavating their tunnels Workers will gnaw these roots into manageable sized pieces and carry them to the DAY OF WEEK FOOD Monday Sweetcorn carrot green beans sweet potato Pronutro Tuesday Banana apple cucumber sweet potato Pronutro Wednesday Sweetcorn carrot green beans sweet potato Pronutro larger amount Thursday Sweet potato and Pronutro Friday Banana apple cucumber sweet potato Pronutro Saturday Sweet potato and Pronutro Sunday Sweet potato and Pronutro larger amount Figure 10 Naked Mole Rats manipulating food other colony members especially the queen These tubers can be used as a long term food source often lasting for many days as the mole rats will gnaw of chunks off the inside of the tubers leaving most of the outside intact so that it can continue to grow In our facility we try to give them a varied diet their staple food is sweet potato which is given ever y day Alongside this they get carrot cucumber green beans baby sweetcorn apple banana and Pronutro which is a high protein porridge like cereal Sufficient moisture is provided from the food they eat that they do not require a separate water source Handling Table 1 Food rotation table showing diversity of food Mole rats are docile animals who are easy to handle They can be picked up by the base of the tail if needed but it is easier to pick them up round the body and place them on your hand You do have to be careful when handling them as they will wriggle and they will try to walk backwards off your hand It is also possible to scruff them when required although because of their excess skin it can be difficult to get a good grip They are not vicious animals and are not likely to bite unless the handler does something to hurt or scare them 87

The naked mole rat husbandry and maintenance training Their sexual organs are actually internal but you can tell the difference by the presence or absence of a horizontal red line between the orifices Identification You can probably imagine that mole rats are not the easiest animals to identify for numerous reasons as they all look very similar Although once you get to know them you start to see differences in behaviour and appearance They also do not have ears which you could put a tag or ear notch in The mole rats in our colony are identified by microchips implanted under the skin on their backs so they all have their own individual number The queen is the easiest animal to identify by sight as she is noticeably longer than the others Figure 11 and 12 Handling naked mole rats Sexing It is difficult to determine the gender of each animal due to their external similarities but it is easy after Figure 15 Position of microchip Breeding Figure 13 Female Figure 14 Male 88 The queen mole rat can have a litter of anywhere up to 25 pups but generally averages between 10 19 Her first litter will be quite small but she will elongate her spine to accommodate larger litters thereafter She will breed throughout the year with 1 3 males and can have a litter every 75 85 days Post partum oestrus occurs 8 11 days after birth during which time behavioural oestrus lasts between 2 24 hours and repeated matings occurs Gestation then lasts between 66 to 76 days with the female becoming obviously pregnant from day 40 Just before the birth of the litter the nipples of the breeder female will become more prominent she will have difficulty with walking she will patrol the tunnels less and a nest will be created Once the litter is born the workers will move the pups into a nursing chamber where they will look after them and the queen will come to the chamber periodically to feed them The pups are born completely hairless with eyes closed no teeth yet present and weigh between 0 5 2g Their teeth start to emerge within a day of birth and start to grow in the first 2 weeks but no other major physiological changes occur At around 2 weeks of age the pups will start wandering around the enclosure but will often be picked up by the adults and returned to the

The naked mole rat husbandry and maintenance nest Between 4 6 weeks these pups will become independent and take their place as working members of the colony Figure 16 Female naked mole rats with young Pacific Science Center Life Sciences Naked mole rats 2011 Image online Available at http pacscilife blogspot co uk 2011 12 festivusmiracle html Accessed 29th January 2015 Colony members show a heightened alarm response to noise and vibrations including routine noise such as feeding and cleaning This perhaps explains why they have quite a low litter success rate as any disturbances can cause the colony to scatter the pups which may result in them being left out of the nest and dying of starvation Baby mole rats also have to be quite robust as they will need to endure being trampled on swept away slept on by adults and crushed into things by their dam If the pups survive until weaning they will start eating solid food at 3 4 weeks of age as well as participating in allo coprophagy where they will beg for the faeces of adult colony members This is thought to help the pups to gain the gut flora they need to digest their food Behaviours Like all animals mole rats exhibit their own distinctive behaviours such as 1 Scent marking this involves wallowing in the toilet area to get the scent of the entire colony on their skin They will also scratch their head neck shoulders and mouth directly after urinating to help reinforce their own distinctive colony odour Scent is very important to mole rats as they are virtually blind so they will use smell to differentiate between friend and foe 2 Grooming mole rats will spend a portion of each day grooming themselves they clean their incisors and their face with their forefeet and groom everything else with their incisors In general mole rats will not groom each other but adults will groom the pups Although they will groom themselves each day it seems that this is more for maintaining their colony scent rather than keeping clean as they usually have a fairly grubby appearance 3 Vocalisation they have lots of different vocalisations which have different meanings They have alarm calls such as grunts and screams for when danger is near to alert the others to the danger The most common noise they make is a soft chirp which can have different meanings but is most often heard during physical contact with other animals They will also sometimes grind their teeth which is usually a sign of aggression between fighting animals but can also be exhibited by animals honing their teeth while reclining in the nest They also have various vocalisations for conflicts between colony mates and other colonies such as loud chirps and trilling 4 Anogenital nuzzling this is a common behaviour seen mostly between the breeding animals The queen and one of her mates will stop when passing each other to sniff and nuzzle at the genital region This is more commonly observed when the queen is in oestrus 5 Copraphagy all mole rats will consume their own faeces as well as the faeces of other colony members The breeding female is the only colony member aside from the pups that will beg for faeces from other animals whilst she is pregnant Conclusion Although very different from the usual species used in biomedical research Naked Mole Rats have multiple uses in research for example 1 Hypoxia resistance where they live and function normally in higher C02 concentrations this allows for them to be good models for research into sleep apnoea and strokes Research is being performed into which protein in mole rat nerves allows for or adapts to these higher C02 concentrations as neurons appear to resist damage Mole rats can be subjected to high levels of C02 with the appearance of being dead but once returned to normal air will come round and suffer no ill effects short or long term 2 Cancer studies they are also a good model for cancer studies due to their apparent resistance to it There have been no documented cases of cancer in mole rats this is thought to be due to a tumour supressing gene that creates a protein that stops cancer cells from multiplying 3 Extreme longevity mole rats are the only poikilothermic or cold blooded mammal known to man They are also incredibly long lived for a rodent living up to 30 years Their longevity is thought to be connected to their cold blooded nature and there 89

The naked mole rat husbandry and maintenance are studies being conducted to see if these two are linked 4 Insensitivity to acids like most animals mole rats feel pain from pressure and thermal stimuli however unlike most animals they do not feel pain from acids This adaptation allows them to live in their burrows which can have an acidic environment due to high carbon dioxide levels This is a potential opportunity for research into pain studies and creating painkillers Although the maintenance of this species presents initial challenges they adapt well to life in the research environment 90

August 2015 Animal Technology and Welfare PAPER SUMMARY TRANSLATIONS INHALTVERZEICHNIS Der einflus bilateraler pr putialektomie auf aggressives verhalten und auftreten aggressionsbedingter verletzungen bei in gruppen gehaltenen m nnlichen Swiss m usen LEWIS VAUGHAN1 3 MONICA KLOPPERS1 und ALEXANDAR WHITTAKER2 1 2 3 Veterinary and Applied Science Centre Technical and Further Education South Australia TAFESA Gilles Plains South Australia Australia School of Animal and Veterinary Sciences The University of Adelaide Roseworthy Campus South Australia Australia Animal Welfare Officer Flinders University GPO Box 2100 Adelaide 5001 South Australia Korrespondierende autorin lewis vaughan flinders edu au Abriss M nnliche M use werden in biomedizinischen Forschungs und Ausbildungseinrichtungen h ufig in Gruppen gehalten Diese Art der Haltung kann jedoch aufgrund von Angstreaktionen provozierter untergeordneter Tiere und Verletzungen infolge von K mpfen u erst negative Auswirkungen auf ihr Wohlbefinden haben Pr putialektomie wurde als einfache Methode zur Reduzierung von Aggressionen angeregt ohne dass Fortpflanzungshormon Profile ver ndert werden m ssten Bei dieser 9 Wochen dauernden Studie zur Beobachtung von Aggressionsh ufigkeit und Verletzungsausma wurden 50 Tiere die einer Pr putialektomie unterzogen wurden sowie 50 Kontrolltiere mit intaktem Pr putium untersucht Unter den Bedingungen dieser Studie wurden keine signifikanten Unterschiede bei aggressivem Verhalten oder Verletzungen zwischen den operierten M usen und den Kontrolltieren festgestellt Daraus wird geschlussfolgert dass Pr putialektomie m nnlicher M use wahrscheinlich weder zur Reduzierung von Aggressionen noch zur Verbesserung des Wohlbefindens von langfristig in Gruppen gehaltenen Tieren beitr gt Schlagworte Aggressives Verhalten Pr putialektomie Verletzungen Wohlbefinden M use 91

Paper Summary Translations Der Nacktmull haltung und pflege HALEY FORREST und ALISON ROBINSON Department of Psychology and Department of Physiology Developmental and Neuroscience Combined Facility University of Cambridge Downing Street Cambridge Cambridgeshire CB2 3EB Korrespondenzautor ajr97 cam ac uk Abstract In einer von Ratten und M usen dominierten Branche haben Tiertechniker selten Gelegenheit zur Arbeit mit einer Art die sich ein wenig von blichen Nagern unterscheidet Der faszinierende Nacktmull Heterocephalus glaber aus der Familie der Sandgr ber erfordert im Vergleich zu den meisten anderen Nagern v llig andere Haltungsbedingungen Der in Ostafrika beheimatete nahezu vollkommen haarlose S uger lebt in Kolonien mit einer hierarchischen Struktur die der von Bienen und Ameisen hnelt Neben ihm gibt es lediglich eine weitere gleichfalls zu den Sandgr bern geh rende S ugerart die eusoziales Verhalten zeigt Der Tiertechniker muss unbedingt mit s mtlichen Anforderungen an die Haltung und Pflege dieser Tiere vertraut sein um ihr Wohlbefinden zu gew hrleisten Die Art reagiert sehr empfindlich auf ihre Umwelt was sich generell erschwerend auf ihre F tterung und Haltung auswirkt Auch die Z chtung von Nacktmullen ist aufgrund ihres eusozialen Verhaltens und ihrer Empfindlichkeit gegen ber St reinfl ssen wesentlich komplizierter Doch trotz der generellen Schwierigkeiten ihrer Haltung verf gen sie ber spezielle Anpassungsmechanismen und eignen sich daher gut als Versuchstiere f r verschiedenste wissenschaftliche Untersuchungen Schlagworte Nacktmulle eusozial Umwelt empfindlich Haltung 92

August 2015 Animal Technology and Welfare CONTENU DE LA REVUE L influence de pr pucialectomie bilat rale sur le comportement agressif et l incidence de blessures li es l agression chez la souris suisse m le h berg e en groupe LEWIS VAUGHAN1 3 MONICA KLOPPERS1 et ALEXANDAR WHITTAKER2 1 2 3 Centre v t rinaire et des sciences appliqu es Enseignement technique et sup rieur d Australie du Sud TAFESA Gilles Plains Australie du Sud Australie cole de sciences animales et v t rinaires Universit d Ad la de Campus de Roseworthy Australie du Sud Australie Animal Welfare Officer Flinders University GPO Box 2100 Adelaide 5001 South Australia Auteur ressource lewis vaughan flinders edu au R sum Les souris m les sont couramment log es en groupe dans les tablissements de recherche biom dicale et les tablissements de formation Cependant cette forme d h bergement peut pr senter un co t social important pour les animaux en raison de l explicitation des r ponses de peur chez les animaux subordonn s et des blessures qui r sulte de combats r els La pr pucialectomie a t propos e comme m thode simple permettant de r duire l agressivit sans alt rer les profils hormonaux de reproduction Dans cette tude 50 animaux ayant subi une pr pucialectomie et 50 animaux t moins intacts ont t tudi s sur une p riode de 9 semaines pour surveiller la fr quence de l agression et la gravit des blessures Dans les conditions de cette tude aucune diff rence statistiquement significative n a t observ e au niveau de la fr quence des comportements agressifs ou des donn es sur les blessures entre les souris op r es et les souris t moins Il est par cons quent conclu que la pr pucialectomie des souris m les ne peut ni r duire leur agressivit ni contribuer am liorer leur bien tre dans une situation d h bergement de groupe long terme Mots clefs Comportement agressif pr pucialectomie blessures bien tre souris 93

Paper Summary Translations levage et maintenance du rat taupe nu HALEY FORR T et ALISON ROBINSON D partement de psychologie et D partement de physiologie de d veloppement et de neurosciences installation combin e Universit de Cambridge Downing Street Cambridge Cambridgeshire CB2 3EB Auteur ressource ajr97 cam ac uk R sum Dans une industrie domin e par les rats et les souris les zootechniciens ont rarement l occasion de travailler avec une esp ce un peu diff rente de celle des rongeurs standard Le rat taupe nu Heterocephalus glaber est un animal fascinant qui a des exigences d levage tr s diff rentes par rapport la plupart des autres rongeurs Originaire d Afrique de l Est il s agit d un mammif re essentiellement glabre qui vit dans des colonies dot es d une structure hi rarchique semblable celle des abeilles et des fourmis Ils sont l une des deux seules esp ces de mammif res les rats taupes qui sont eusociaux En tant que zootechnicien il est tr s important de comprendre toutes les exigences li es la maintenance et aux soins de l animal afin d assurer son bien tre Les animaux sont tr s sensibles leur environnement ce qui complique l alimentation en g n rale et l levage de l esp ce L levage de rats taupes nus est aussi beaucoup plus compliqu en raison de la nature eusociale de l animal et de sa sensibilit aux perturbations Bien qu ils soient tr s difficiles maintenir ils disposent de moyen d adaptation sp ciaux qui en font de bons mod les pour diverses tudes scientifiques Mots clefs Rat taupe nu eusocial environnement sensible levage 94

August 2015 Animal Technology and Welfare INDICE DE LA REVISTA La influencia de la extirpaci n bilateral del prepucio en el comportamiento agresivo y la incidencia de las lesiones relacionadas con la agresividad en ratones suizos macho alojados en grupo LEWIS VAUGHAN1 3 MONICA KLOPPERS1 y ALEXANDAR WHITTAKER3 1 2 3 Centro de Veterinaria y Ciencias Aplicadas Technical and Further Education South Australia TAFESA Gilles Plains South Australia Australia Escuela de Ciencias de los Animales y la Veterinaria The University of Adelaide Campus de Roseworthy South Australia Australia Animal Welfare Officer Flinders University GPO Box 2100 Adelaide 5001 South Australia Autor para correspondencia lewis vaughan flinders edu au Resumen Para tomar muestras de sangre a ratas mediante un sistema de tomas de muestras de sangre automatizado ABS por sus siglas en ingl s se les introduce una c nula en la vena yugular Estas muestras se utilizan para calcular los niveles de hormonas presentes en la sangre con el paso del tiempo En el protocolo original la c nula de la vena yugular se coloca fuera del cuerpo de la rata mediante una incisi n en la parte posterior de la cabeza Se le alimenta a trav s de un surtidor de metal protector y se conecta a un sistema ABS El surtidor de metal se ancla con dos tornillos met licos implantados en el cr neo de la rata La sofisticaci n que presenta este protocolo consiste en que la c nula se encuentra ahora en el exterior del lomo de la rata y un arn s de acceso vascular ancla el suministro protector en su sitio Los resultados demuestran que mediante la sofisticaci n del protocolo se ha mejorado el bienestar y la cr a de los animales y ha disminuido el n mero total de animales utilizados en el estudio Del mismo modo tambi n se ha demostrado que el nivel de la hormona del estr s la corticosterona en la sangre no aumenta con el uso de arneses de acceso vascular Palabras clave ratas canulaci n yugular corticosterona arn s de acceso sofisticaci n 95

Paper Summary Translations La rata topo lampi a cr a y cuidados HALEY FORREST y ALISON ROBINSON Departamento de Psicolog a y Departamento de Fisiolog a Desarrollo y Neurociencia Edificio de uso combinado University of Cambridge Downing Street Cambridge Cambridgeshire CB2 3EB Reino Unido Autora para correspondencia ajr97 cam ac uk Resumen En el contexto de un sector en el que predominan las ratas y los ratones los t cnicos especialistas en animales rara vez tienen la oportunidad de trabajar con especies que no sean los roedores convencionales La rata topo lampi a Heterocephalus glaber es un animal fascinante que requiere una cr a muy diferente de la de la mayor a de roedores Originaria de frica oriental es mayormente un mam fero sin pelo que vive en colonias con una estructura jer rquica semejante a la de las abejas y las hormigas Es una de las dos nicas especies de mam feros eusociales ambas ratas topo Como t cnico especialista en animales es muy importante comprender todos los requisitos de la atenci n y el cuidado de este animal para garantizar su bienestar Son unos animales muy sensibles a su entorno lo que complica su alimentaci n y la cr a general de la especie La reproducci n de la rata topo lampi a tambi n es considerablemente m s compleja debido a su naturaleza eusocial y su sensibilidad a la perturbaci n A pesar de ser dif ciles de cuidar tienen una adaptaci n especial que hace que sean buenos modelos para varios estudios cient ficos Palabras clave rata topo lampi a eusocial entorno sensible cr a 96

August 2015 Animal Technology and Welfare INDICE DELLA REVISTA L influenza della prepuziotomia bilaterale sul comportamento aggressivo e incidenza di lesioni di aggressione in topi Swiss maschi stabulati in gruppo LEWIS VAUGHAN1 3 MONICA KLOPPERS1 e ALEXANDAR WHITTAKER2 1 2 3 Veterinary and Applied Science Centre Technical and Further Education South Australia TAFESA Gilles Plains Australia del Sud Australia School of Animal and Veterinary Sciences Universit di Adelaide Roseworthy Campus Australia del Sud Australia Animal Welfare Officer Flinders University GPO Box 2100 Adelaide 5001 South Australia Autore corrispondente lewis vaughan flinders edu au Riassunto I topi di sesso maschile vengono comunemente stabulati in gruppo in centri di ricerca biomedica e in istituti educativi Tuttavia questa forma di stabulazione pu dar luogo a una perdita di benessere per gli animali dovuta all elicitazione di risposte alla paura in animali subordinati e a lesioni risultanti da effettivi combattimenti La prepuziotomia stata proposta come semplice metodo di riduzione dell aggressione senza tuttavia causare un alterazione dei profili ormonali riproduttivi In questo studio nell arco di 9 settimane sono stati analizzati 50 animali sottoposti a prepuziotomia e altri 50 animali di controllo con prepuzio intatto per monitorare la frequenza dell aggressione e i livelli delle lesioni In base alle condizioni del presente studio non sono state rilevate differenze statisticamente significative nella frequenza del comportamento aggressivo o nei dati sulle lesioni tra topi operati e topi di controllo Si concluso che la prepuziotomia dei topi maschi potrebbe non ridurre l aggressione n contribuire a un maggiore benessere nell ambito di una stabulazione in gruppo a lungo termine Parole chiave comportamento aggressivo prepuziotomia lesione benessere topi 97

Paper Summary Translations Talpa senza pelo zootecnia e mantenimento HALEY FORREST e ALISON ROBINSON Dipartimento di Psicologia e Dipartimento di Fisiologia Sviluppo e Neuroscienze Struttura combinata Universit di Cambridge Downing Street Cambridge Cambridgeshire CB2 3EB Autore corrispondente ajr97 cam ac uk Abstract In un industria dominata da ratti e topi gli stabularisti hanno raramente l opportunit di lavorare con una specie leggermente diversa dai normali roditori La talpa senza pelo o eterocefalo glabro Heterocephalus glaber un animale singolare con requisiti zootecnici molto diversi rispetto alla maggioranza degli altri roditori Nativo dell Africa orientale un mammifero quasi completamente privo di peli che vive in colonie aventi una struttura gerarchica simile a quella delle api e delle formiche Rappresenta una di sole due specie di mammiferi eusociali entrambe di ratti talpa Per poter garantire il benessere dell animale molto importante per uno stabularista comprendere i suoi requisiti completi di mantenimento e cura Gli animali sono molto sensibili al loro ambiente che complica l alimentazione e l allevamento in genere della specie L allevamento delle talpe senza pelo inoltre molto pi complicato a causa della loro natura eusociale e della loro sensibilit alle perturbazioni esterne Bench molto difficili da mantenere presentano adattamenti speciali che li rendono ottimi modelli per vari studi scientifici Parole chiave talpe senza pelo eusociale ambiente sensibile zootecnia 98

August 2015 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 NACWO Named Animal Care and Welfare Officer Guidelines 3rd Edition March 2015 www iat org uk Our Mission Advancing and promoting excellence in the care and welfare of animals in research GUIDANCE NOTES ON THE ROLE OF THE NAMED ANIMAL CARE AND WELFARE OFFICER NACWO IN ESTABLISHMENTS DESIGNATED UNDER THE ANIMALS SCIENTIFIC PROCEDURES ACT 1986 as amended Introduction The Animals Scientific Procedures Act ASPA as amended in 2012 has incorporated the specific requirements for personnel included in Article 20 24 and 25 of Directive 2010 63 EU As stated in ASPA section 2C 5 within each establishment as well as the person responsible for compliance with Establishment Licence conditions there needs to be one or more persons on site who will be responsible for overseeing the welfare and care of the animals in the establishment transposed in ASPA as the Named Animal Care Welfare Officer NACWO Summary These new NACWO guidance notes are designed to reflect the enhanced role of the NACWO and their additional responsibilities The guidelines will help both new and established NACWOs to meet the exacting standards required of those who breed keep or use animals for scientific purposes Please note that these guidelines provide the minimum knowledge required and further reading is recommended The NACWO is appointed and nominated by the Establishment Licence Holder ELH to carr y out functions under the Animals Scientific Procedures Act 1986 ASPA as amended for which the ELH is ultimately responsible to the Home Office Statutory duties performed by the NACWO on behalf of the ELH 99

Tech 2 Tech are directed towards minimising suffering and optimising the welfare of animals being bred kept for use or used for scientific procedures at the establishment The NACWO should be involved throughout the duration of the project licence including the early stages of planning The contributions of the NACWO towards refinement reduction and replacement 3Rs should be sought whenever required Improvements may include enhancements to care regimes husbandr y and housing standards Selection of a suitable person with the necessary qualifications and the desirable attitude to animals and colleagues is seen as key to the successful functioning of an individual in the NACWO role Overview of the NACWO role The NACWO is responsible for overseeing the welfare and care of the animals acting as a role model and promoting a culture of care at the establishment amongst both scientific and animal care staff The NACWO can contribute to a good culture of care in the establishment by supporting and endorsing staff training and reinforcing the importance of good animal welfare and promoting the implementation of the 3Rs The NACWO should have appropriate managerial authority and be regarded as the expert in their field Their advice on the welfare of animals during the design of procedures and their implementation should be sought by both personal and project licensees It is vital to establish good lines of communication with all people working within the legislation especially between the NACWO and ELH The success of the NACWO will in turn assist the ELH in the discharge of their responsibilities and obligations providing an important contribution to the 3Rs The NACWO should be an active member of the Animal Welfare and Ethical Review Body AWERB and advise applicants for licences and licence holders on oppor tunities for implementing the 3Rs Where establishments have large numbers of animals or facilities at different locations it may be necessary to appoint more than one NACWO to ensure this role is performed effectively The area of responsibility for each NACWO must be carefully defined and communication routes established to ensure a standard approach across the establishment NACWOs are often the point of contact for the Home Office Inspector and will often accompany them on their visits 100 The NACWO skills and knowledge requirement A suitable person may be a senior animal technologist with an animal technology qualification or an experienced stockperson with a qualification in agricultural science The Institute of Animal Technology IAT maintains a Register of Animal Technologists who may be appropriate to fill a NACWO post Further details are available at www iat org uk The skills and knowledge required to fulfil this vitally important animal welfare position are both numerous and varied and include An extensive knowledge of the welfare and husbandry requirements of the species housed at the establishment including the caging or housing needs environmental enrichment opportunities nutritional physiological and biological requirements A detailed understanding of ASPA as amended and able to advise other individuals working within it This will include the Establishment Licence Holder ELH Named Veterinary Surgeon NVS personal and project licence holders animal technologists and others working within the establishment Be familiar with the provisions of project licences particularly the adverse effects expected for each protocol the control measures and humane endpoints specified and the methods of killing specified in licences Appropriate personal authority to promote highstandards and good verbal written communication and diplomacy skills to champion a culture of care amongst both scientific and husbandry staff Appropriate managerial authority to enable them to ensure that high standards of husbandry and care are practised meeting or exceeding the minimum standards set out in the Code of Practice This responsibility extends into all areas named on the establishment licence The ability and knowledge to recognise any variance from normal animal health and behaviour determine the action that must be taken together with the degree of urgency and be conversant with all approved methods of euthanasia in use at that establishment Responsibilities of the NACWO NACWOs are responsible for overseeing the day to day husbandry care and welfare of the protected animals held at their establishment The NACWO role is key to establishment licence holders properly discharging their obligations and responsibilities

Tech 2 Tech NACWOs are a source of expert advice on the welfare of the protected animals Ever yone within the establishment must be aware the NACWO is acting on the establishment licence holder s delegated responsibility It is a pivotal position and must promote an appropriate caring attitude amongst all those in the establishment who come into contact with animals It is vital that the NACWO is not only appropriately trained and qualified e g to IAT Level 3 but that they constantly update this knowledge and skill base to remain at the forefront of new and emerging technologies In short the NACWO must be respected as a valued expert on animal husbandry care welfare and legislation The NACWO must have access to the project licences within their area of responsibility The NACWO must be able to advise and assist both personal and project licensees regarding the conduct of procedures helping them to maintain compliance Their knowledge should also extend to regulations controlling the transportation import and export of animals from and to establishments where these activities are undertaken The NACWO should establish an overarching system for continuous care of the animals ensuring the animals are checked daily and that an effective information decision chain is in place and known to all relevant staff This will ensure that any welfare concerns are recognised and dealt with promptly and appropriately If the health or welfare of an animal is giving cause for concern the NACWO must inform the personal licence holder who is responsible for the welfare of that animal If that person is unavailable NACWOs must ensure that the animal is cared for and if necessary that it is humanely killed using a Schedule 1 or other method approved in the establishment licence If necessary the NVS or the assigned Home Office Inspector should be contacted The NACWO should be familiar with the main provisions of ASPA have upto date knowledge and experience of relevant animal technology and a thorough knowledge of the husbandry and welfare needs of the species kept in the establishment be aware of the standards of care accommodation husbandry and welfare set out in the Code of Practice take appropriate steps to develop and maintain high standards of care and husbandry appropriate to the species NACWOs are required to complete training accredited by the IAT normally before taking up the position They must be prepared to initiate and progress improvements to the care and welfare of animals They should promote implementation of refinements in animal husbandry breeding and use The NACWO must keep abreast of developments and advances in the field of laboratory animal science technology and welfare particularly the 3Rs Continued professional development CPD is crucial to the role Where there are changes to the species models or types of work the NACWO should undertake relevant additional training Membership of professional laborator y science organisations such as the Institute of Animal Technology IAT and attainment of Registered Animal Technologist RAnTech status is strongly recommended The ELH must ensure that sufficient competent staff are available at all times to care for the animals know about relevant methods of humane killing listed in Schedule 1 to the Act together with any other approved methods listed in the establishment licence and either be competent in their use or be able to contact others named on a register maintained at the establishment The ELH must ensure that a register is maintained of those who are competent to humanely kill protected animals The NACWO must be familiar with standard methods of humane killing as they may be called to use them in an emergency be able to recognise the signs of pain suffering distress or lasting harm in the species for which they care and ensure that there is available expertise to monitor all animals to recognise any variation from normal health and behaviour NACWOs must have good knowledge of the animals under their care and be able to recognise indicators of poor welfare pain and other forms of suffering and the ways of alleviating them know which areas of their establishment are listed in the Schedule of Premises on the establishment licence Establishment licences contain a Schedule of Premises listing the specific areas within the establishment where protected animals may be bred held used in regulated procedures and or killed The NACWO should have a clear understanding of the specifics of the schedule of premises and be aware of the designated use for each area A project licence may be authorised to carry out 101

Tech 2 Tech procedures at a place other than a licensed establishment known as a POLE Although the role and responsibilities of the NACWO does not extend to POLEs a project licensee would be expected to take advice from the NACWO when planning such work In order to discharge fully their responsibilities the NACWO must have unlimited access to all areas listed as within their responsibility on the establishment licence establish a system to ensure that a competent person sees and checks every animal kept in an approved holding area at least once daily NACWOs are required to establish a system to ensure that a competent person sees and checks every animal kept in an approved holding area including designated rooms outside the facility at least once daily Checks may be carried out by the responsible licensee a competent person deputised by them a trained animal technologist or the NACWO The time date and name of the person who has per formed the inspection should be recorded know how to contact at any time the appropriate person s be familiar with the main provisions of project licences particularly the adverse effects expected for each protocol the control measures and humane end points specified and the methods of killing specified in licences NACWOs must know how to contact at any time the NVS or their deputy the ELH or their nominee and project and personal licence holders The NACWO must have access to the project licences operating within their area of responsibility A good working knowledge of each licence is required and in particular the 3Rs and the protocol sheets including the possible adverse effects and the humane endpoints specified It is the role of the NACWO to assist personal and project licensees to ensure that unnecessary pain suffering distress or lasting harm is prevented or alleviated The NACWO must be aware of any special care required by animals which are subject to regulated procedures pro actively working with the NVS as appropriate promote implementation of refinements in animal care husbandry and use The NACWO should work closely with the NVS to ensure that good practice is employed at all times with respect to animal care and husbandry help the establishment licence holder to keep suitable records of the health of the animals under 102 the supervision of the NVS of the environmental conditions in the approved areas in which animals are held and of the source and disposal of animals NACWOs must have a good appreciation and knowledge of record keeping systems Comprehensive health records should indicate whether there has been a deterioration in the condition of any animal These records should include details of the observation made together with the action s taken and may be kept as a paper record in for instance a day book or within a computer system Environmental records including ambient room temperatures and humidity should be kept as paper copy or within a computer system It is the responsibility of the NACWO to ensure environmental conditions are maintained as laid down in the HO Code of Practice Any deviations must be recorded with possible causes and the action taken to correct the problem and return conditions to acceptable levels Comprehensive records on behalf of the ELH of the source and fate of animals must be kept either as hard copy or within a computer system All records must be available to the HO Inspector on request champion a culture of care at your establishment acting as a role model for all those who care for and use animals The NACWO should be instrumental in establishing a culture of care ensuring animals are treated with compassion and respect An appropriate culture of care can only improve animal welfare Training CPD and good communication will help develop this culture of care be an active member of the Animal Welfare and Ethical Review Body AWERB at their establishment and advise applicants for licences and licence holders on practical opportunities for implementing the 3Rs The AWERB provides a forum for discussion and development of ethical advice to the ELH on all matters related to animal welfare care and use at their establishment which may impact on the lifetime experience of the animals At least one NACWO must be a full member of the AWERB NACWOs have the opportunity to contribute to the draft PPL ensuring that the 3Rs have been afforded full and proper consideration The NACWO is expected to monitor the progress of the project licence and to make suggestions to the project licence holder to improve the welfare of animals Competencies qualities of the NACWO NACWOs should have a good understanding of the local

Tech 2 Tech structure for management and responsibilities relating to animal use at the establishment including how key roles and related tasks are fulfilled The amount of time and effort necessary for this role may vary dependent on the size and complexity of the animal facility but the responsibilities of the NACWO remain constant relevant species as well as an understanding of the regulations governing the use of animals in research and their welfare In addition to a comprehensive knowledge of the legislation and the husbandry requirements of all species within the facility the following competencies are suggested as being key to the role of the NACWO ASPA as amended the associated Guidance and Codes of Practice standard conditions on licences and the NACWOs role in promoting compliance ethical issues relating to the use of animals relevant husbandry and care practices including methods of euthanasia ensuring compliance with CoP requirements the 3Rs relevant to the work at the establishment recognition of indicators of poor welfare pain and other forms of suffering and knowledge of ways of alleviating them record keeping systems Concern for standards only the highest standard is acceptable in every aspect of work with laboratory animals whether it is methods equipment systems procedures or outcomes They will act as a role model and should champion a culture of care at the establishment amongst both scientific and animal care staff CPD To maintain and continue to develop their professional knowledge and skills A NACWO who is a Registered Animal Technologist RAnTech will be obliged to undertake and record CPD Initiative a proactive approach seizing opportunities as and when they occur The NACWO will be expected to make suggestions for improvement in all aspects of laboratory animal science and welfare and seek and apply new ideas The NACWO should have a good appreciation and knowledge of The NACWO must keep up to date with technical advances within animal science and changes in legislation The Institute of Animal Technology offers a CPD programme providing opportunities to exchange ideas with peers and to acquire new knowledge Suggested Home Office Guidance of the Operation of the Animals Scientific Procedures Act 1986 March 2014 Conflicts of interest Where a NACWO is also a project licence holder or a personal licence holder another person should be nominated to fulfil the role of NACWO for providing advice on the welfare of animals being used under that project or personal licence Alternatively the arrangement may be overseen by the AWERB to ensure there is no conflict of interest Absence of the NACQO It is a condition on the establishment licence and a responsibility of the establishment licence holder to ensure that in the event of the absence of the NACWO suitable arrangements are made to ensure the animals are given adequate care standard condition 16 Training for the NACWO Code of Practice for the Housing and Care of Animals Bred Supplied or Used for Scientific Purposes December 2014 Directive 2010 63 EU On Protection of Animals Used for Scientific Purposes September 2010 IAT Registered Office 5 South Parade Summertown Oxford OX2 7JL E admin iat org uk T 0800 085 4380 An electronic copy of these Guidelines is available on the IAT website http www iat org uk doclibrary html NACWOs are required to complete training accredited by the Institute of Animal Technology normally before taking up the position If this is not possible then justification for the delay and the expected date of attendance at the course should be included within the application The inspector will consider each case individually The training of the NACWO should give sufficient understanding of the biology and husbandry of the 103

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August 2015 Animal Technology and Welfare POSTER PRESENTATIONS Originally presented at IAT Congress 2015 The tail prick technique a refinement in blood sampling technique for a genetically modified mixed background strain of mouse CAROLINE ZVEREV and VANESSA ANDREWS The Francis Crick Institute Lincoln s Inn Fields Laboratory 44 Lincoln s Inn Fields London WC2A 3LY Corresponding author caroline zverev cancer org uk Introduction For an ongoing study of ageing in a genetically modified mixed background mouse strain there is a need to per form a licensed procedure under the Animal Scientific Procedures Act 1986 to collect a small blood sample to determine if there is any variation in the components of blood between mutant heterozygous and wild type mice and to ascertain whether this is linked to their phenotype Figure 2 Wild type litter mate exhibiting normal tail The mutant mouse is clearly distinguishable to its wild type littermate The mutant animals for this particular strain exhibit fused vertebrate in their tails making the current tail vein method using a 25g needle to puncture the vein unsuitable as this often resulted in the production or little or no sample that can be analysed Figure 1 Mutant animal note deformed tail The Tail Prick technique shown on the NC3Rs website was then suggested as a solution After consultation 105

Poster Presentations this technique was introduced to see if this could produce good quality samples for analysis as well as reducing overall stress levels for the animals Apparatus required for the procedure G G G G G G G a thermostatically controlled heat chamber a restraint tube 20ul EDTA lined capillary 3 per mouse transfer bulb 25G needle EDTA lined pot 1 per mouse tissue Method Restraint tube 1 Place the mice into the thermostatically controlled heating chamber set at 37 C for 3 minutes this will dilate the blood vessel sufficiently to collect the sample 2 Remove mouse and place into the restraint gently restraining by the base of the tail to avoid the mouse moving whilst taking the sample 3 Using a 25 gauge needle prick the tip of the tail getting as close to the tip as possible to avoid damaging it 4 Place 20ul EDTA lined capillary onto the spot of blood to collect the sample using capillary action 3 capillary tubes are required to achieve the 50ul blood sample required 5 Use the bulb to transfer the blood sample into an EDTA lined pot ready for analysis 6 Using a piece of tissue gently apply pressure to the tip tail for 15 seconds using a pinching action with your fingers to help stop the bleeding 7 Remove animal from restraint and return to their home cage 8 Check animal after 10 minutes to ensure the bleeding has stopped and the mouse is in good condition Bulb Capillary tube Figure 4 Thermostatically controlled heating chamber Figure 5 A small puncture is made on tail tip 25G needle Figure 3 Equipment required for collecting blood 106 Heating chamber Figure 6 Blood is collected via a capillary tube

Poster Presentations The animal does appear to experience minor discomfort from the prick on the tip of the tail but when compared to the other methods previously used this a significant reduction and is a major refinement Based on observational evidence we believe that stress levels have been reduced as animals are exposed to a defined temperature and restrained for a shorter period of time All of the refinements have been achieved without compromising the scientific results Consequently the tail prick method will now be rolled out across the site for use on all strains Figure 7 The mouse is safely returned to the cage How the technique has been further refined Acknowledgements NC3Rs website http www nc3rs org uk ourresources blood sampling Gary Childs and special thanks to Valerie Borel Vannier Cancer Research UK 1 Time in the heating chamber has been reduced from the suggested 7 minutes down to 3 minutes this has reduced stress in the mouse as heating the animal could result in discomfor t and dehydration 2 The mouse restraint time has been reduced due to a much faster and accurate sampling method 3 The amount of blood taken requires no further sampling to be taken from the mouse 4 Refinement of needle size has also taken place The first suggestion on the NC3Rs website was a 23 gauge needle once confident with this size of needle the 25 gauge needle was then introduced and it was still able to produce the same size sample of blood for analysis therefore not affecting the scientific results This technique leaves no internal or external wound damage and allows the wound to close quickly with minimal blood loss after the sample has been collected The animal will suffer minor discomfort from the prick on the tip of the tail but compared to the other methods previously used this has been greatly reduced Conclusion This method has been extremely successful and has been introduced as the method of choice for this strain of mouse This technique leaves no internal or external wound damage and allows the wound to close quickly with minimal blood loss after the sample has been collected The smaller needle size also creates a smaller wound which aids quick healing with no repeat samples being required The method also produces a uncontaminated sample as at no point does the capillary touch the tail 107

Animal Technology and Welfare August 2015 The development and refinement of bioluminescent fluorescent orthotopic xenografts models to reduce harm and improve animal welfare ALISON RITCHIE PAM COLLIER PHIL CLARKE and ANNA GRABOWSKA Cancer Biology Division of Cancer Stem Cells School of Medicine University of Nottingham Queen s Medical Centre Nottingham NG7 2UH Corresponding author alison ritchie nottingham ac uk Introduction As a pre clinical cancer research facility we constantly strive to improve our xenograft models to more accurately reflect the patient situation whilst also refining techniques and reducing animal numbers to improve welfare in line with the 3Rs principles To this end we have developed a number of singly and doublytransduced xenograft cell lines whose growth and development can be monitored bioluminescent and or fluorescently in real time using the IVIS Spectrum imaging system allowing us to monitor cell viability as well as specific biological parameters such as apoptosis and hypoxia and the development of metastases signals a 3D reconstruction of the tumour can be created showing placement in relation to chosen organs on the Living Image Mouse Atlas thus allowing accurate calculation of the tumour volume including areas of necrosis or cavities Materials and methods Human tumour cells are tranduced using a viral vector with e g firefly luciferase and or mCherry to display a bioluminescent and or fluorescent signal which can be monitored using the IVIS Spectrum system The cells are injected or surgically implanted orthotopically in the corresponding organ in immunodeficient mice with placement aided by the Optilia HD camera Tumours are monitored for growth and metastatic spread and areas of necrosis or hypoxia Using the measured Figure 1 Bioluminescent human tumour cells imaged in the IVIS Spectrum 108 Figure 2 Surgical hood with HD camera Figure 3 HD image of mouse skull showing drill hole for brain tumour cell injection

Poster Presentations Figure 4 IVIS Spectrum imaging system Results Orthotopic models These pictures demonstrate or thotopic models showing bioluminescent signal in target organ Figure 7 A549 Lung tumour Metastatic models These pictures show tumours in target organs and associated metastatic spread Figure 5 LnCap Prostate tumour Figure 8 MED 1 Brain tumour with spinal metastases Figure 6 EJ28 Bladder tumour Figure 9 C170HM2 Intraperitoneal tumour with liver metastases 109

Poster Presentations Figure 10 MCF 7 Mammary fat pad tumour with liver metastases Figure 13 3D reconstruction using Living Image Mouse Atlas Figure 14 3D reconstruction using Living Image Mouse Atlas 3D Reconstruction These pictures show the bioluminescent signal and the associated 3D reconstruction demonstrating position and volume Discussion By monitoring growth in each animal in real time G G we eliminate the need for time point sampling thereby reducing numbers required per study we are able to identify scientifically appropriate endpoints at an earlier stage before the onset of adverse effects thus improving welfare while still providing valid experimental data We also constantly strive to refine our orthotopic models by for example G G developing a less invasive method for priming the bladder lining when setting up a bladder model intranasally loading the lungs with cells rather than directly injecting via thoracotomy Using the HD camera has improved consistency and reproducibility and therefore allows a reduction in animal numbers Figure 11 MCF 7 Bone metastases from breast cells We believe our approach of using bioluminescent fluorescent cells wherever possible and constantly working to improve and refine our models to reduce both numbers and the harm to the animal is a major development in improving welfare in line with the 3Rs while delivering solid experimental results Figure 12 3D reconstruction using Living Image Mouse Atlas 110 Acknowledgements Thanks to Marian Meakin and Alison Mackie for their technical assistance

August 2015 Animal Technology and Welfare The use of hypoxic chambers with large animals CRAIG FOREST University of Cambridge Barcroft Building 307 Huntingdon Road Cambridge CB3 0JX Correspondence caf49 cam ac uk Abstract Hypoxic Chambers are common within animal units these days all of varying sizes and capabilities but most being made to house only rodents and which are easily maintained However there are a few that have been custom made and purpose built for large animals such as sheep These have all the same configuration and adjustability if not more so than the standard but trying to adapt a simple idea into a fully working model throws up a few problems which need to be overcome There are now four fully working and operational large animal chambers within the University of Cambridge Although like all hypoxic chambers they are not without problems The size of sheep and the ability to carry a single foetus to term makes them a very good model for the research into human heart disease and the role of parental influences Main problems with scaling up hypoxic chambers Size is the main issue the volume of air inside the chambers dramatically increases with size This requires the air pumps and regulator to deal with a higher hourly air flow in order to keep the chambers at a positive pressure and controlled hypoxia Chambers used at Cambridge University have been specially designed so that all air used is generated and mixed on site They can be altered to suit researchers needs by the user and still handle 12 5 air changes per hour All air is vented out into the external atmosphere As the air coming into the chambers is very dry due to the air generators there has been a standalone humidifier system added to ensure the best quality of air Sheep are social animals and always need to be in sight of another sheep so these chambers have been made from a clear Perspex in order for the sheep to see through and be happier These chambers have also been made to be able to accommodate two sheep in each chamber and there is enough food trough space and water to cope with two sheep allowing the user to house up to eight sheep at any one time Feeding large quantities of food such as hay and pellets would not be viable if using pass through hatches Also as the sheep are standing on a false floor with holes in sprinkling the pellets would be impractical Consequently each chamber has its own feeding station where the food troughs can be spun around sealing off any air leaks while at the same time keeping all food contained These troughs also allow the researcher to weigh the food and monitor intake Figure 2 Close up of feed trough Figure 1 Hypoxic chamber showing feed and water troughs Water buckets cannot be used due to the fact if the bucket is tipped over all the water will drain through the perforated floor and sheep would be left without water To combat this problem there is a permanent water 111

Poster Presentations supply plumbed in with a stop cock ball valve on a raised drinking trough thus allowing the sheep access to constant fresh water chamber This avoids the need for gloved access around the entire chamber and having to chase the sheep around Using the three gloves situated at the front of the chambers the users have easy access to the pass hatch and sheep in order to take their samples Figure 3 Close up of water trough Usually animal faeces and urine produced by the sheep and left to mix in with any bedding and to be removed during the weekly or more frequently cleaning out schedule The Cambridge chambers have a raised false floor which is perforated allowing all waste material to fall through and be caught in the base of the chamber keeping the living compartment clean and dry The bottom of the chambers are fitted with a butterfly valve which allows the staff to drain out all liquid waste together with some of the solids Once the study period has finished and the sheep have completed their time in the chamber the floor can be lifted and thoroughly cleaned Figure 5 Position of sheep for sampling Emergency Access to fix the chambers is rarely needed but to keep the animals in hypoxia while repairs are made there is a transfer tunnel which clamps between two chambers to allow one sheep to enter another chamber and remain in hypoxia the whole time So that the original chamber can be turned back to normoxia to allow user access Cleaning is easy once the sheep has been removed either into a different chamber or has been removed after the study period has elapsed In the latter case sheep are allowed to give birth lamb down in the barn under normal large animal conditions The chamber floor can be taken up and all waste can be removed A high pressure washer is then used to blast clean the entire chamber including all sprockets and chain that work the moveable restraining gate Once surface cleaning is achieved the chamber is then chemically disinfected and reassembled ready for the next sheep Figure 4 Detail of water supply and flooring Due to their size sampling rats and mice whilst in hypoxic chambers is relatively easy as is handling Sheep are very much the opposite and therefore there has to be a restraining gate installed to the chambers There is a simple sprocket and chain system under the floor and once the handle is turned the gate moves forward forcing the sheep to come to the front of the 112 Figure 6 Floor removed for cleaning

Poster Presentations An alarm system constantly measures all of the set parameters including oxygen nitrogen carbon dioxide humidity and temperatures for each of the four chambers The entire chamber system has its own backup generator in case of a mains electrical failure and the main standby generator does not kick in this way we can ensure the sheep will ALWAYS be in the required conditions Figure 7 Showing detail of drainage system and waste collection Air flow in all chambers has to be monitored very closely Each of our chambers has its very own air flow system and Figures 8 and 9 shows the complex series of pipes and flow regulators that are used Each of the gases nitrogen and compressed air can be independently adjusted to allow a stable 10 oxygen level This is sustained by adding more nitrogen to the air so instead of having low levels of oxygen pumped in there is a lack of space for 21 oxygen due to the increase of nitrogen Reducing oxygen levels has to be done with the sheep present in the chamber as the only way for the sheep to enter the chamber is to remove the front panel and lift sheep in This would of course would allow all the hypoxic air to escape and fill the room so this is not possible The sheep are placed in the chambers under normal air condition for 1 day for habituation then once the air is adjusted the oxygen level slowly drops over a period of 6 minutes this is as fast as the air can be adjusted and is slightly different from rats that are placed directly into hypoxia using a double door system Figure 10 Alarm system Uses in research Figure 8 Air monitoring system Figure 9 Close up of monitoring system Oxidative stress in the foetal heart and vasculature underlies the mechanism via which prenatal hypoxia programmes cardiovascular dysfunction in later life Developmental hypoxia independent of changes in maternal nutrition promotes foetal growth restriction and induces changes in the cardiovascular metabolic and endocrine systems of the adult offspring which are normally associated with disease states during ageing Treatment with antioxidants of animal pregnancies complicated with reduced oxygen delivery to the foetus prevents the alterations in foetal growth and the cardiovascular metabolic and endocrine dysfunction in the foetal and adult offspring The work reviewed offers both insight into mechanisms and possible therapeutic targets for clinical intervention against the early origin of cardiometabolic disease in pregnancy complicated by foetal chronic hypoxia Giussani DA et al 113

Poster Presentations Figure 11 Hypoxic chambers in situ References 1 Giuassani D A et al Pubmed 2014 Heart disease link to foetal hypoxia and oxidative stress Online Available from http www ncbi nlm nih gov pubmed 25015802 Accessed 27th Feb 2015 114

August 2015 Animal Technology and Welfare Pruritic aged C57BL6 J mice CHRISTINE MARSHALL and SALLY CARPENTER Biological Resource Facility BRF The Roslin Institute The University of Edinburgh Easter Bush Midlothian Scotland EH25 9RG Corresponding author christine marshall ed ac uk Abstract Mice may start to itch for many different reasons Scratching will cause even more skin irritation than the initial problem thus causing mice to scratch even more In turn this causes more irritation and more scratching This itch scratch cycle is a never ending cycle that is hard to break and it quickly becomes a habit sometimes even if the initial problem is gone Once this habit has taken effect it is very hard to break Figure 3 Stage 3 Figure 1 Stage 1 Figure 4 Stage 4 Figure 2 Stage 2 It is our job as Animal Technologists to introduce a different variety of enrichment into the environment of the mouse to try and take their mind off the itching We have introduced nail clipping resulting in a reduction of mice having the ulcerative dermatitis The 1st stage is to have a mouse in the early stages of ulcerative dermatitis This becomes apparent as the fur on the mouse s dorsal side becomes ever so slightly thin This then leads to the 2nd stage where the fur loss is more obvious It is from the 2nd stage we really need to monitor the mice closely as the 3rd stage is where the skin gets very inflamed and red All the mice have 115

Poster Presentations already received extra enrichment so it is now becoming habitual Lastly the 4th stage is where the skin becomes broken and lesions form This sadly becomes the end point for that mouse Method 24 aged mice born in September 2013 arrived in the BRF in April 2014 at 7 months old By June 2014 it was noticed that there was hair loss beginning to appear on some of the mice Shortly after they began scratching excessively with some resulting in broken skin ulcerative dermatitis The scratching was apparent in around 94 of the aged mice Naturally the Named Veterinary Surgeon NVS was informed and a course of action was agreed Figure 7 Non trimmed The hind nails would be clipped in all remaining mice with the nail growth to be monitored closely along with the ulcerative dermatitis The mice are also being checked on a daily basis and are on a Weight and Body Condition scoring system Figure 8 Trimmed Figure 5 Non trimmed where the mice are being scored on the same day every week by the same technicians This helps to maintain consistency The scoring system involves visually checking the condition of the aged animals and scoring them on a point s scale of 0 to 2 as follows on our database system 0 no Abnormalities Detected NAD 1 mouse has Fur Loss 2 mouse has Broken Skin Results Figure 6 Trimmed 116 By February 2015 the mice were now 17 months old and since they have been having their nails cut there have been no more mice developing ulcerative dermatitis There has been no further cases of broken skin no red or inflamed skin nor has the fur loss worsened After the initial treatment the mice with stage 3 ulcerative dermatitis had a reduction in inflammation and redness They continued to have noticeably improved skin over the following weeks returning back to stage 2

Poster Presentations It must be noted however that the fur loss that developed on the mice back in June has not regrown but plans are in place to investigate this further Another husbandry practice we have introduced is any aged mice over 1 year old are to have their nails clipped and their body weights recorded every three months on our database Recently tissue samples have also been submitted to the laborator y for cytology analysis and we are interested to hear that the tissue samples did not have any disease on either the healthy skin or on the skin with the ulcerative dermatitis Finally we have some other exciting plans and ideas which we have now implemented and are compiling data to further our research Acknowledgements Leslie Penny Named Veterinary Surgeon Named Veterinary Surgeons The Roslin Institute Gordon Melville BRF Deputy Unit Manager Dave Davies BRF Unit Manager Lorraine Blackford BRF Deputy Unit Manager Reference 1 Bilton Jan 2013 Treatment of ulcerative dermatitis at a transgenic mouse facility in Leeds https circabc europa eu sd d 5aeabc9b 81ad 4a81 08bilton pdf 117

Animal Technology and Welfare August 2015 Supraglottic airway devices in rabbits how we implemented this technique to improve animal welfare and health and safety for staff EMMA TOZER Research Centre Smith Nephew York Science Park Heslington York YO10 5DF Correspondence emma tozer smith nephew com Introduction In the past rabbit anaesthesia at our establishment has been delivered via a facemask scavenging system this type of system is not only uncomfortable for the animals but also presents an exposure risk to the theatre technician surgeon due to poor scavenging We have made several attempts intubating rabbits using a variety of methods however none have been successful and or provided confidence that the tube is inserted correctly This poster looks at the steps we took for the successful implementation of the supraglottic airway device and the issues we encountered during the development of this technique V gel The V gel device was developed by Docsinnovent after its technology was proven within human anaesthesia known as the i gel airway device V gel uses a noninflatable anatomically shaped cuff made of a soft gellike material which creates an airway seal around the pharyngeal laryngeal perilaryngeal and the upper esophageal structures The benefits of V gel Anatomical matching features combined with a soft gellike material to give a high quality pressure seal that avoids laryngeal and tracheal trauma which means safer anaesthetics and superb comfortable patient recoveries Fast easy safe and stress free device insertions No post operative coughing or gagging Low airway breathing resistance due to the large airway channel within the device High quality pressure seal restricting leakage of volatile anaesthetic agents thus improving health and safety in anaesthesia and overcoming patient sensitisation to smell a common problem in rabbits Super soft contoured tip for a highly effective upper oesophagus seal to prevent potential aspiration of reflux fluid Integral gas sampling port to reduce re breathing dead space and making high quality monitoring easier Integral bite block to stop patient damaging device and occluding the airway Materials validated for autoclave sterilisation to eliminate cross infection Millpledge Veterinary 2014 Implementation Figure 1 The V gel device 118 Small animal surgery within our unit was very limited and the anaesthetic method of choice was via a facemask However due to staff pregnancies and the uncertainty of the amount of anaesthetic the rabbit was inhaling other options were investigated The first and most obvious method was that of intubation so that the delivery of anaesthetic could be controlled monitored and savaged more effectively A number of different techniques were tried including

Poster Presentations Observation at a local veterinary surgery Use of the Flecknell small animal laryngoscope Both of these methods proved difficult as under the Animals Scientific Procedures Act 1986 practising techniques on live animals is not permitted When the technique was put in to practice we found that the premedication given did not give adequate sedation to allow intubation Rabbits are notorious for been difficult to intubate Tran et al 2001 state that the unique anatomy of the rabbit contributes to these difficulties they have small narrow oropharynx with restrictive mandibular excursion a relatively large tongue for their size and large primary incisors Due to the size of a rabbits tongue it tends to cover the epiglottis from view the laryngeal opening is small and situated ventrally therefore making it difficult to locate with a laryngoscope Our NVS brought the V gel technology to our attention after discussions concerning the limitations of the current method and difficulties intubating and invited us along to the local practice where a demonstration was taking place on a model rabbits This device is designed to mirror the anatomical airway structure of the rabbit therefore providing an excellent alternative to using endotracheal tubes face masks as both of these methods can be uncomfortable for the animal and unsafe for the staff Figure 2 The V gel device is supplied in a variety of sizes dependent on the animal weight with an autoclavable cradle marked with 40 sterilisation cycles Figure 3 The V gel must be lubricated prior to application within the theatre As it can be seen from the pictures to the left demonstrating the V gel device in use the preparation and application are very simple Figure 4 To help with insertion and visualisation the rabbits mouth can be held open and tongue pulled aside Figure 5 The V gel device is simply pushed into the mouth over the tongue and will stop when appropriately placed condensation within the tube aids in confirmation of correct placement Figure 6 An appropriately sized lace cord is used and tied around the back of the head to keep the device insitu 119

Poster Presentations Conclusions We believe that the refinements put into place have improved animal welfare and promoted a safer environment for theatre staff As this method offers a more efficient mode of anaesthesia it has enabled us to change the premedication regime providing a more successful survival rate and fewer complications during recovery Acknowledgements Katie Blackwell and Lisa Griffin References Millpledge Veterinary 2014 V gel Advanced veterinary airway management system Tran H S Puc M M Tran J L V Del Rossi A J and Hewitt C W 2001 A method of endoscopic endotracheal intubation in rabbits Laboratory Animals 35 pp249 252 120

August 2015 Animal Technology and Welfare Recommendations for environmental enrichment to enhance the welfare of mice in oncology research GEMMA WILLOUGHBY JASON KING VICKY LACEY LOUISE WAINWRIGHT JAMES FINNEY TRUDI GEE ED HALE WILL TYLER and KELLY JONES Crown Bioscience UK Ltd Hillcrest Dodgeford Lane Belton Loughborough LE12 9TE Corresponding author gemma willoughby precos co uk Introduction A major objective of Crown Bioscience UK is to promote and develop the highest standards of animal care management and welfare in oncology research Continuing trials of environmental enrichment are an important component in this strategy Environmental enrichment is the process of providing stimulating environments for research animals in order for them to demonstrate their species typical behaviour to allow them to exercise control or choice over their environment and to enhance their well being Enrichment provides a number of positive refinements to promote natural behaviours and interaction with the environment such as nesting gnawing exploring and finding cover Evidence suggests that environmental enrichment may reduce or prevent stereotypical behaviours such as bar mouthing cage circling back flipping and barbering It has also been noted that environmental enrichment may reduce conflict situations and fighting in male mice 1 2 Mice were checked twice a day AM PM as standard practice The animals were also observed during cage cleaning and other procedures An initial review of current enrichment products was carried out Currently these include ECO Pure 6 flake bedding Sizzlenest Aspen Bricks and play tunnels Animals were observed upon welfare checks and evidence of interaction noted with these products i e Chewing marks on Aspen bricks For the trials of the new products were randomly placed in cages with the same husbandry and environmental conditions detailed previously The animals were observed upon introduction of the product and during the observational checks detailed earlier Observations of interaction were noted and pictures collated The products were then rotated to different groups A questionnaire was carried out to gain animal technologist feedback on each product questions included Did the mice interact with the product How much interaction was observed What behaviour did the mice exhibit and finally the pros and cons of use Results The objectives of this project were G review of current environmental enrichment used at Crown Bioscience UK G investigate new products available G trial use of products Standard Enrichment Experimental design These trials were conducted with both male and female mice Strains included MF1 nudes Nod SCID and Balb C nudes Animals were housed in groups of five using both IVC s and standard flexible film isolators The mice were kept on a 12 hour light dark cycle with temperature and humidity consistent within the ranges stipulated by Home Office guidelines Figure 1 Sizzlenest 121

Poster Presentations quickly and soiled easily therefore the animals were cleaned out weekly on this bedding Nesting Materials Figure 2 Play tunnels Aspen blocks and ECO Pure 6 bedding Our standard bedding and environmental enrichment products given to stock and study mice are ECO Pure 6 bedding Sizzlenest play tunnels and Aspen bricks Sizzlenest Figure 1 is a dust free nesting material made from paper and is suitable for sterilisation We decided to continue using the Sizzlenest as it encourages natural behaviours such as nesting and group interaction Obser vation indicated that this product has high interaction value with all strains Play tunnels Figure 2 are given to provide an ideal refuge and hiding place for mice it provides a 3 dimensional environment as the mice can move over and through the tunnels These had ver y high interaction observations and are a multi use product which encourage not only cover but are also chewable Aspen Bricks Figure 2 were chosen as they provide a reusable and transferable material that enables the mice to express their natural gnawing behaviours ECO Pure 6 Figure 2 was used as the standard bedding material Foraging and digging was observed with this bedding This bedding was found to get wet 122 Figure 3 Showing Happy Mats chewed into nesting material Happy Mat Happy mats have been chosen as they are reusable and easily transferable material Made from natural materials and suitable for sterilisation and immunocompromised mice Obser vation showed high interaction including exhibition of nesting and foraging behaviours Bedding Materials Bed o Cobs 1 8 Bedding was found to stay clean for longer than a week with good bottom up absorption The mice on this bedding did not need to be cleaned out weekly therefore it is cost and time effective A variety of nesting behaviours were observed as well as an increase in foraging and digging

Poster Presentations Smart homes provided mice with shelter allowing them to nest within the house enhancing group interaction The animals were observed to sleep in groups in the Smart home Smart homes enable flexible use as they can be split into two smaller houses Figure 4 Cage containing Bed o Cobs 1 8 Premium Bedding This bedding was shown to be very clean for and had very good bottom up absorption This bedding has shown to be highly effective to reduce the onset of urine scald associated with oestrogen pelleted studies of breast and ovarian cancer models Observation noted positive interaction with the bedding including foraging digging and chewing Figure 6 Smart homes with occupants Mouse Igloo The mouse igloo provided a safe refuge for hiding but also enabled good visibility for welfare checks as the plastic is transparent Mouse igloos are reusable which is a cost saving and easy transferable during cage change Figure 5 Cage containing Premium Bedding Housing Shelters Smart Home Observation of interaction with the smart homes exhibited gnawing and climbing in and on the product Figure 7 Mouse igloos in use 123

Poster Presentations Mouse Hut Mouse huts provided mice with an ideal shelter the flat top of this hut also offered another dimension to the cage with mice climbing in and on this product Tinted plastic enabled good visibility at welfare checks without having to disturb the mice unnecessarily Easily transferable at cage change and re usable which is a cost saving When observing the mice interact with this enrichment it appears to be very popular as all mice observed used this product These tunnels were placed on the cage floor and suspended from the grid top Observations showed high interaction in both positions providing shelter on the floor and promoting climbing behaviours whilst suspended from the grid top The tunnels provided shelter for the animals whilst allowing good visibility for the technician It was noted that these tunnels allowed easy access to the animals for handling Figure 8 Mouse hut in situ with occupants Clipper home Clipper homes provide mice with an additional dimension to their environment Figure 10 Cage with polycarbonate tunnel suspended from cage lid These homes have proven effective for mice that are fighting as the suspended home enables a safe retreat Summary The products detailed in the results have been recommended from each different category bedding nesting materials shelter provisions and chewable for use in the Crown Bioscience UK animal unit A number of products were removed from the trials due to lack of interaction impracticality of use and size of some products The use of these products will be implemented in the animal unit on a rotational basis Environmental enrichment is a developing focus and new products and ideas will be reviewed in the future to promote and maintain Crown Bioscience UK high standards in animal care and welfare References 1 2 Figure 9 Cage with Clipper Home attached to cage lid Polycarbonate Tunnels 124 NC3Rs website www nc3rs org uk Wolfer D P Litvin O Morf D Nitsch R M Lipp H P and Wurbel H 2004 Laboratory animal welfare Cage enrichment and mouse behaviour Nature 432 821 822

August 2015 Animal Technology and Welfare 3Rs Refinement use of hydrophobic sand in collection of analytical urine samples HILARY LANCASTER FIONA McCLURE DEBBIE RIDLEY JASON C SMITH and GREG WHELAN GlaxoSmithKline Laboratory Animal Science and Safety Assessment Gunnels Wood Road Stevenage Hertfordshire SG1 2NY Corresponding author jason c smith gsk com Introduction Hydrophobic sand is a commercial product that is used in veterinary surgeries to collect cat urine samples for analysis The sand has a hydrophobic coating and the urine forms as droplets on the top allowing it to be easily collected and stored Currently rodent urine collection for scientific purposes requires the use of metabolism cages The disadvantages of the metabolism cages are Animal Welfare mice are housed in metabolism cages for 16 hours Regulated procedure under the Animals Scientific Procedures Act Costs buying the metabolic cages maintenance of equipment and consumables Takes a lot of technician time to set up dismantle wash and reassemble point Using the results from this preliminary study a comparison study was undertaken A cross over study design was used over 2 weeks comparing metabolism cages and hydrophobic sand Environmental conditions were the same as the preliminary study but mice were sampled in North Kent Plastic Cages Ltd type 1 mouse cage with 150g Kit4Cat hydrophobic sand for 3 hours of collection starting at 18 00 hrs For this study 10 male and 10 female C57Bl6 J mice were used due to historical data from metabolism studies Triple A Trading Solid Drink was placed in the hopper as a substitute for food and water see Figure 1 picture A Urine samples were still taken at 30 minute intervals to reduce the risk of urine being lost to evaporation or Hydrophobic sand has the potential to reduce time costs animal stress remove the regulation and improve animal welfare Method A preliminary study was performed investigating the optimum time for urine collection The times chosen to assess were 06 00 12 00 18 00 hrs and 24 00 hrs A Latin square study design was used to reduce the number of animals required This also allowed us to determine whether habituation and or the time of day had an effect on sample volume 8 male and 8 female C57Bl6 J mice were used They were held in a standard stock room with a 07 00 19 00hrs light cycle 22oC 2 and 50 RH 10 The mice were housed in Tecniplast 1145 cages containing 200g of Kit4Cat hydrophobic sand for 1 hour with food and water withheld They were checked at 30 minute intervals and any urine present was collected with a 1mL syringe and decanted into plastic vials These were stored at 80 C After 1 hour the mice were returned to their home cages and any remaining urine collected This process was repeated weekly for 4 weeks until all mice had samples taken at each time Figure 1 Pictures of caging and urine droplets A Cage set up used for 3 hour urine collection including Solid Drink 125

Poster Presentations contamination Tecniplast rodent metabolism cages were used overnight on a 16 hour duration starting at 15 00 hrs At the conclusion of the study the animals were euthanased using a Schedule 1 method followed by an appropriate confirmatory method a necropsy was performed and the stomach contents were examined using a dissection microscope for evidence of the presence of hydrophobic sand Routine urine chemistry parameters were assayed using standard methodology on the Siemens ADVIA 1800 automated chemistry analyser results were corrected for creatinine 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 Results For standard parameters in transgenic phenotyping we require a minimum sample volume of 0 2mL In the preliminary study we identified that the peak sample volume was obtained at 18 00 hrs At this time point samples meeting the 0 2mL requirement were obtained from only 69 of animals Using 3 hour collection 85 of the hydrophobic sand samples reached the 0 2mL minimum volume compared to 65 of the metabolism cage samples see Table 1 for the combined results Hydrophobic sand 0 2ml 0 2ml no sample 1 hour 18 19 00hrs 69 25 8 3 hours 18 21 00hrs 85 10 5 Met cages 16 hours 15 07 00hrs 65 10 25 0 2ml could be measured with a 1 in 4 dilution Table 1 Usable urine samples B Example of uncollectable urine deposited on curved edge of the cage Post mortem examination of stomach contents showed only 2 grains of hydrophobic sand in 1 of the 10 mice The stomachs mainly contained Solid Drink In the preliminary study it was observed that a proportion of the urine was deposited on the curved corners of the Tecniplast 1145 cage and therefore not touching the hydrophobic sand see Figure 1 picture B This prevented the urine from forming a droplet and made it hard to collect In the second study switching to the North Kent Plastic cages enabled all the urine to be collected see Figure 1 pictures C D Table 2 Measure urine parameters C and D Example droplets of urine that can be collected 126 In the preliminary study it was observed that a proportion of the urine was deposited on the curved corners of the Tecniplast 1145 cage and therefore not touching the hydrophobic sand see Figure 1 picture B

Poster Presentations This prevented the urine from forming a droplet and made it hard to collect In the second study switching to the North Kent Plastic cages enabled all the urine to be collected see Figure 1 pictures C D Conclusions Collection periods of 1 hour did not provide enough urine to give a robust sample for each mouse In the second part of the study we increased the collection time to 3 hours with the addition of Solid Drink to provide food and water The 3 hour collection increased the samples that could be measured without dilution from 69 to 85 planning to investigate the use of this product to perform their own pilot studies to determine the optimal approach for their facility Although we recognise that hydrophobic sand will never completely replace metabolism cages for urine collection for example using radiolabelled drugs when collection of the total urine output is required we believe that this method of urine collection should be introduced widely Acknowledgements Leigh Bailey Michael Fulleylove Mark Lennon The habituation to the cages had no significant effect on urine volumes but there was an increasing trend towards greater volumes of urine after habituation data not reported It was also observed that repeating collections did not reduce the amount of urine the mice excreted This opens up the possibility of increased frequency of urine collection without an impact on rodent welfare Comparison of 3 hour hydrophobic sand and 16 hour metabolism cage urine chemistry results corrected for creatinine did not identify any differences that would preclude the use of hydrophobic sand for standard urinalysis collection Generally the data points were closer together on the hydrophobic sand samples This could be due to a reduction of circadian variation as collection was over a 3 hour period as opposed to 16 hours for the metabolism cage samples see Figure 2 Hydrophobic sand samples showed no evidence of faecal contamination This may be due to the way faecal pellets can stick to the cones under the metabolism cages which then contaminate the urine as it flows over the pellet When collecting with hydrophobic sand there is little to no contact between urine and faeces Despite the apparent increase in technician time to collect the samples overall the time taken to perform the full study using hydrophobic sand was significantly less compared to metabolism cages when cleaning and set up time was taking into account At post mortem there was no significant ingestion of the hydrophobic sand This removes the issue of any chemicals entering the rodent s body and possibly causing interference The use of hydrophobic sand has the potential to significantly reduce the amount of times metabolism cages are required and so have a positive impact on animal welfare As noted above the studies were completed on C57BL6 J mice other strains might respond differently requiring longer or shor tened collection periods We would advise any organisation 127

Animal Technology and Welfare August 2015 Welfare and maintenance of the BIOZZI mice SARAH BRIGHT Harlan Laboratories UK Station Road Blackthorn Bicester Oxfordshire OX25 1TP Correspondence aw harlan com Abstract The Biozzi AB H mouse was first developed in 1970 by Guido Biozzi In 1972 he selectively bred these mice to study the immunopathological mechanisms underlying polygenic diseases This strain of mouse became key for the study of human inflammatory neurological diseases It was first brought to Harlan Laboratories UK in 1994 and has been bred within the company ever since Whilst working with this strain it has became apparent that they require substantially more care and attention than that of most colonies we house within our barriers They are extremely temperamental and do not tolerate change well Any change to the personnel and the environment has detrimental effect on their breeding performance and welfare The purpose of this project was to investigate ways to reduce the stress these mice experience in order to improve their welfare and breeding performance During this project we looked at several areas where we felt improvements could be made in order to achieve this Study detail We identified 5 areas that we could change to improve welfare and productivity Provide a constant approach for both environment and technologist The same technologist where possible Do not move the colony from one area to another Reduce the cleaning to every other week and on the same day if possible If animals are in the last third trimester or they have a litter less than 5 days old do not disturb Add extra bedding and nesting to their home cage to provide more privacy Add an area to allow the mice to hide if they want to this can be in the form of a mouse house or tunnel preferably the red polycarbonate variety These changes were applied to our production colony and using the JD Edwards system recorded parameters prior Introduction The Biozzi mouse colony at Harlan Laboratories is housed under full barrier conditions under the standards set in the Animals Scientific Procedures Act 1986 Code of Practice for the Housing and Care of Animals Bred Supplied or Used for Scientific Purposes 1 Conventional cages are used and they are fed on a standard diet and are on an automatic watering system There are two colonies which consist of a foundation and production colony The foundation colony are bred monogamously and supply the production colony with new breeding stock whereas the production animals are bred in trios and offspring from these are supplied to the scientific community 128

Poster Presentations to making the changes Red columns and then taking the same parameters post changes Green columns Conclusions As you can see from the results the changes we applied have made significant improvements to the colonies welfare and productivity This project has helped us for fill our obligations to promote and use the highest standards of care and welfare possible Refining the techniques we use for breeding these mice has G G G reduced the number of animals required for breeding by increasing productivity shown us that each strain has it own quirks and can require adjustments in the way we look after them reduced the number of pre weaned losses We can measure the welfare improvements by correlating the increased productivity and the mice certainly do look and seem happier Breeding performance and welfare was monitored using JD Edwards reporting This is a computerised data capture system which holds all of the breeding data and performance for all our colonies Reference 1 Home Office 2014 Code of practice for the housing and care of animals bred supplied or used for scientific purposes This publication is available at www gov uk government publications Results By applying these changes we have been able to improve the welfare and productivity of the Biozzi mice G G G increased litters produced decreased litter losses increased average litter size improved PEI PEI Performance efficiency index Number of pups weaned number of breeding females PEI Per formance efficiency index Number of pups weaned number of breeding females 129

Animal Technology and Welfare August 2015 A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late stage Human African trypanosomiasis HAT HOLLIE BURRELL SAWARD1 JEAN RODGERS2 BARBARA BRADLEY2 SIMON CROFT1 and THERESA WARD1 1 2 London School of Hygiene Tropical Medicine Keppel Street London WC1E 7HT University of Glasgow Urquhart Building Glasgow G61 1QH Corresponding author hollie burrell saward Ishtm ac uk Introduction Human African trypanosomiasis HAT is caused by a vector borne protozoan parasite that caused fewer than 8000 reported cases in 2013 T b brucei GVR35 is a pleiomorphic strain which is commonly used to study late stage HAT and assess drug therapies using the standard drug relapse method of 180 day protocol2 Bioluminescence imaging BLI can provide a highly sensitive non invasive detection of parasite distribution in a single mouse for the entirety of an experiment A bioluminescenct GVR35 strain utilising red shifted luciferase with a 1000 fold higher bioluminescence than previous green strain GVR35 LUC2 has been validated to produce a drug model capable of determining relapse in 90 days and evaluate dose response in 35 days post infection p i 3 4 D28 All mice blood filmed and imaged D35 Mice blood filmed and imaged Blood and perfused brains collected for qPCR analysis and ex vivo imaging D35 180 relapse study For relapse mice imaged and filmed every 7 days p i Results 1 Fexinidazole D21 Methods All experiments were carried out under UK Home Office Animals Scientific Procedures Act 1986 Using a T b brucei GVR35 strain transfected with humanised red shifted luciferase GVR35 VSL 2 3 the following Protocol was used Drug Efficacy and Relapse D0 CD1 females infected with 5 x 103 trypanosomes intravenously from donor mouse 22mg kg D21 All mice blood filmed and imaged using IVIS Lumina Perkin Elmer UK Mice were then treated with drug 130 66 7mg kg 200mg kg

Poster Presentations D24 D35 22mg kg 66 7mg kg 200mg kg 22mg kg 66 7mg kg 200mg kg 3mg kg 6mg kg D28 Melarsoprol D21 22mg kg 66 7mg kg 200mg kg 1mg kg 131

Poster Presentations D25 D35 1mg kg 3mg kg 6mg kg 1mg kg D28 2 Fexinidazole 1mg kg 132 3mg kg 6mg kg 3mg kg 6mg kg

Poster Presentations Melarsoprol 4 Melarsoprol 10mg kg 3 Melarsoprol 6mg kg 1 and 2 Dose response is detected in both whole animal imaging and excised ex vivo brain 1 For fexinidazole clearance is not detected for 22mg kg with signal evidence in the nose Relapse occurs at D35 p i for 66 7mg kg with disseminated signal throughout animal In contrast low doses of melarsoprol clear infection with relapse not occurring until D35 in 3mg kg and early signs of relapse head region occurring in 6mg kg mice 2 Ex vivo imaging and qPCR supports dose response as increased bioluminescence is mirrored in the average parasite burden of the brain in both the fexinidazole and melarsoprol studies 3 and 4 Drug relapse after treatment with melarsoprol at 6 and 10mg kg x 3 days intravenously After treatment infection is cleared as evident by clearance of both bioluminescence and parasitaemia 3 6mg kg caused relapse as early as D49 p i as measured by bioluminescence 4 Mice remained cleared of infection with relapse occurring only in Mouse 2 The relapse 133

Poster Presentations was detected 21 days earlier through bioluminescence than in peripheral parasitaemia Conclusions VSL 2 model is capable of providing a sensitive drug assessment system using noninvasive imaging Low parasite burden verified by qPCR can still produce a strong bioluminescence allowing early detection of low level infection to be possible After treatment with late stage drugs parasitaemia is cleared which is evident by the undetectable bioluminescence and periphery parasitaemia The highly sensitive model was able to detect relapse and recover y via bioluminescence approximately 21 days earlier than peripheral parasitaemia counts A dose response effect can be determined through qualitative bioluminescence as early as 35 days VSL 2 is able to reduce the current 180 day model to 90 days reducing the time required to assess preclinical efficacy of new anti trypanosomal drugs References 1 2 3 4 WHO 2011 Human African Tr ypanosomiasis Retrieved 21st March 2012 2012 from http www who int trypanosomiasis_african en Jennings F W Whitelaw D D and Urquhart G M 1977 The relationship between duration of infection with Trypanosoma brucei in mice and the efficacy of chemotherapy Parasitology 75 143 153 McLatchie A P Burrell Saward H Myburgh E Lewis M D Ward T H Mottram J C Croft S L Kelly J M and Taylor M C 2013 Highly sensitive in vivo imaging of Tr ypanosoma brucei expressing red shifted luciferase PLoS Negl Trop Dis 7 e2571 Burrell Saward H Rodgers J Bradley B Croft S L and Ward T H 2014 A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late stage human African trypanosomiasis Journal of Antimicrobial Chemotherapy in press 134

August 2015 Animal Technology and Welfare Evaluation of an automated CO2 euthanasia system ROBERT STEWART1 and CLAIRE BOOTH2 1 2 Comparative Biology Centre The Medical School University of Newcastle Framlington Place Newcastle upon Tyne NE2 4HH Plexx PO Box 86 6660 AB Elst The Netherlands Corresponding author robert stewart ncl ac uk Background Carbon Dioxide CO2 is widely used as a method of laboratory animal euthanasia in rodents Fixed flow chambers offer a method of home cage euthanasia that can improve animal welfare and user safety CO2 is introduced through a manifold which ensures even gas distribution followed by pre set gradual rising concentrations of CO2 then a pre defined dwell time and finally evacuation of all gas at the end of each cycle which ensures the chamber has no traces of CO2 which would adversely affect subsequent groups We evaluated the Euthanex Fixed Flow Chamber System which because it allows euthanasia of rats and mice in their home cage is much less stressful for the animal and less time consuming for the operator Once the animals have been placed in the chamber the system automatically locks the doors until final CO2 evacuation is complete The chamber is charged with a low flow of CO2 and when it reaches euthanisation level the gas shuts off and the chamber remains fully charged at levels sufficient to ensure proper euthanasia of all animals The chamber is then evacuated of CO2 before allowing the doors to be unlocked Figure 1 The fixed flow chamber was evaluated for several factors over a number of weeks by a range of users including research staff technologists and veterinary surgeons As well as scoring a range of factors from 15 the users were also encouraged to record any comments they may have had Method Animal technologists and researchers were asked to complete the score sheet Figure 2 when they used the system to euthanise cages of mice and rats They were asked to rate ease of use of the equipment and to provide any additional comments on their experiences Figure 1 Cages being loaded into chamber and doors locked before starting a euthanasia cycle Figure 2 Score sheet 135

Poster Presentations The overall findings were then recorded Figure 3 Any automated system such as the one evaluated here that provides a pre set rising concentration and ensures adequate dwell time reduces operator error and addresses some of the concerns relating to CO2 euthanasia Overall the scores and comments were very positive with users particularly noticing how stress free it was for both animals and operator Acknowledgements The authors would like to thank the technologists researchers and veterinar y surgeons at the Comparative Biology Centre University of Newcastle who took part in the evaluation Figure 3 Overall scores Comments Clicking noise before the CO2 starts affects the mice This can be prevented by reducing the inlet pressure into the chamber The timing after the mice have died is slightly too long The dwell time provides a safety margin to ensure all animals are properly euthanised Variable chamber size would reduce the amount of CO2 Varying chamber sizes are available to meet required cage capacities A viewing window in the top would increase visibility There is no way to ensure that enough CO2 is available in cylinder before starting The Intelliswitch II is an optional safety accessory to monitor CO2 levels Very stress free for the animals and the operator It would be better if it was able to be moved easily Transport trolleys are available for easy mobility of chambers Conclusion The advantages of CO2 euthanasia particularly when used for large numbers of rodents is well documented However since studies have highlighted concerns that rodents find even relatively low levels of CO2 aversive it is very important that we carefully consider the concentration of CO2 introduced into the chamber 136

August 2015 Animal Technology and Welfare An updated road map towards ending severe suffering ELLIOT LILLEY PENNY HAWKINS and MAGGY JENNINGS Research Animals Department Science Group RSPCA Wilberforce Way Southwater West Sussex RH13 9RS Corresponding author penny hawkins rspca org uk Abstract The road map process Revision of the EU Directive controlling experiments on animals has focussed attention on the need to reduce animal suffering in scientific procedures Classification of levels of suffering into mild moderate and severe and the need to report actual levels of severity has provided added impetus to the drive to refine the most severe models and procedures as has greater recognition that high levels of suffering impact on an animal s physiological responses increasing variability of experimental data So ending severe suffering is a desirable goal for scientific ethical and legal reasons Every establishment should ensure there is a process to achieve the following for severe models or procedures This is therefore an excellent time to look at the sources and nature of suffering within the research context to perform a severity audit to evaluate the effectiveness of current refinement practices and to seek more effective ways of avoiding or minimising all unnecessar y pain and psychological distress experienced by animals Central to the success of such an initiative is a receptive institutional culture and a robust and challenging ethical review process This poster will outline the key questions and practical considerations that establishments need to address in order to reduce suffering for all animals and to work towards ending severe suffering Culture Establish and maintain a progressive open minded and caring research culture 1 Analysis Establish to what extent severe suffering occurs 2 Evaluation Look at why severe suffering occurs and what current approaches are used to avoid it 3 Define obstacles Establish what the impact of ending severe suffering would be 4 Overcome obstacles Set out a plan to overcome issues and to end severe suffering An institutional culture of care an essential prerequisite of effective implementation of the Road Map Components of such a culture include 1 A collective responsibility and accountability for the welfare of animals shared by all staff 2 Demonstrable commitment to high standards of housing care and welfare above the legal minimum from senior management 3 Internal openness including the ability to raise share and resolve concerns 4 Support for Named Persons such as Animal Care and Welfare Officers Veterinar y Surgeons Information and Training and Competency Officers 5 A robust framework for training assessment of 137

Poster Presentations competence and CPD of all staff 6 Effective and well supported institutional ethical review of scientific work 7 An effective ethics or animal care and use committee e g the Animal Welfare and Ethical Review Body AWERB in the UK Use a mechanism based approach rather than a disease model approach Apply Refinement e g Refine every element of the lifetime experience of the animal Establish validate and implement humane endpoints Analysis Perform an in house severity audit of all protocols procedures and models Establish where there is the potential for severe suffering prospective assessment and then what actual severity is experienced by individual animals retrospective assessment Evaluation For procedures where severe suffering occurs ask 1 Why the procedure is used and what factors contribute to it being severe 2 Is severe suffering really necessary to achieve the scientific objective 3 What proportion of animals in each protocol procedure or model experienced severe suffering 4 What refinements are already in place how effective these are and whether there is potential for further application of the 3Rs Define obstacles What are the scientific obstacles to ending severe suffering Set these out clearly and assess the genuine impact of stopping severe protocols procedures or models Overcome obstacles Take an alternative approach e g use a non severe model Re frame the research question to avoid a severe model 138

August 2015 Animal Technology and Welfare Evaluation of a novel rat gerbil nesting house with stereotypical behaviour body weights and food consumption GORDON MELVILLE The Roslin Institute and R D SVS University of Edinburgh Midlothian EH25 9RG Correspondence gordon melville roslin ed ac uk Introduction The object of this study was to evaluate the availability of a purpose built nesting box in the home cage as a means of enriching the quality of life for rats The nest box was made of stainless steel with a polypropylene top both materials being autoclavable Quality of life was to be determined by close monitoring and recording of body weights food consumption and observation of any changes in the rats stereotypical behaviour Confidence of a positive finding is high as a similar study conducted at the Roslin Institute has already tested this design of nesting house on gerbil breeding pairs and found that there was a noticeable size difference in weanlings that had use of the nesting house The Gerbil weanlings that had the nesting house were larger than the weanlings without the nesting house Coat condition was also markedly better on the adults as well as the weanlings and were less agitated Method Six randomly selected ex stock female Sprague Dawley rats weighing between 88 9g to 95 5g were placed on study to examine environmental enrichment improvement namely the effect of presence or absence in the home cage of a novel nesting house observing bodyweight BWT food consumption and observations on activity and behaviour The rats were separated into two groups of three house or no house and individually ear marked for identification purpose and set up in identical home cages Tecniplast Blue Line IVC 1500 cm floor area for an acclimatisation period of a week before being monitored three times per week Monday Wednesday Friday at a set time of 16 15 over a period of four weeks Figure 1 Prototype nesting house and schematic showing dimensions The animals were only handled by the one technologist for the duration of the study Stereotypical behaviour observations were made from the IVC rack for ten minutes per rat per study day and BWT and food consumption were recorded in a laminar flow changing station Whilst recording their BWTs and observations the rats were handled for an equal amount of time Personal Protective Equipment PPE including scrubs and gown were kept the same and only worn by the one technician handling the rats to mainatin the same scent This was to ensure minimum variability as too many variables would have been detrimental to the study outcome 139

Poster Presentations more relaxed and vocalised less than the rats with no nesting house By the end of the study the rats with the nesting house allowed themselves to be picked up without running away or vocalising and became extremely placid The rats without the house were still more relaxed than in the beginning of the study but still vocalised occasionally and ran away when an attempt was made to catch them Without any external stimulus the rats with the nesting house were nearly always running through the burrows and playing whilst the rats without the nesting house were often sleeping or sedentary Figure 2 Cage with nesting house in situ and cage without nesting house Figure 3 Rats using nesting house Behaviour During the acclimatisation period the same volumes of bedding and nesting materials were added to both cages In the rat cage that had the nesting house bedding was deliberately placed outside the nesting house to see if the rats made their nest inside or outside the nesting house On the first day of the study it was clear to see that the rats had shred the bedding material and taken it through the burrow and into their secluded nesting house which was a highly positive sign that they already felt secure in there In the first week of the study the rats vocalised when being handled but later in the second week the rats with the nesting house seemed a little easier to catch 140 Body weights and food consumption The other very positive result which indicated that the nesting box is a refinement is reflected by the BWT s which demonstrated that the rats with the use of the nesting house actually gained less weight but consumed more food than the rats without the nesting house It is concluded that the rats in the cage with the nesting house were more active throughout the whole study duration whereas the rats without the nesting house appeared to only eat and sleep with minimal activity

Poster Presentations The raw data collected are presented by a number of figures illustrating any changes whilst comparing the two different types of housing conditions Discussion The addition of the nesting house to the rats home cage has altered the stereotypical behaviour of standard laboratory rats The availability of this design of nesting house appears to increase activity in the sample observed n 3 thus enriching their lives The animals studied whilst more active still gained weight in a comparable way to rats housed without the nesting box Rats also displayed an improvement in behaviour during the study s duration It must be noted that the degree of handling the rats experienced on study was higher than usual by taking regular body weights which may have had an effect on the rats by making them more docile for the weight recording process but it had no effect on their behaviour at any other time The extra activity that was observed of the rats with the nesting house without any external stimulus was from their own free choice to utilise the nesting house It is recognised that there is a financial cost to buying sterilising washing and maintaining nesting houses if they were to be added to all rat and gerbil home cages but the refined improved and more stimulated lifestyle experienced by the rats should go a long way to justify this additional expense Further planned studies of the nesting box are suggested to evaluate long term benefits Acknowledgements Biomedical Research Resources Dr Graham Thomas Director of Biomedical Research Resources for his assistance and support Vince Ranaldi Operations Manager School of Biological Sciences John Verth Chief Superintendent of The School of Biological Sciences for the initial financial outlay and support for the nesting house design to be built and allowing the testing to continue within Biomedical Research Resources 141

Animal Technology and Welfare August 2015 Improving rat housing and enrichment a local initiative at the NIBSC COLETTE MOTTRAM HUNT National Institute for Biological Standards and Control NIBSC Blanche Lane South Mimms Potters Bar Hertfordshire EN6 3QG Correspondence colette mottram nibsc org Introduction Whilst much effort is put into improving conditions for larger laboratory animals such as primates dogs and rabbits small rodents often miss out The goal at NIBSC was to create a whole new environment including new style caging and environmental enrichment that would significantly improve the welfare of our rats by providing them with more space and the opportunity to practise a wider range of natural behaviours Figure 2 Old style conventional caging Materials and methods Figure 1 Rat using new style cage Conventional caging at NIBSC Natural behaviours of rats include nesting digging climbing and gnawing which are extremely restricted if not impossible under standard laboratory housing conditions Providing animals with larger and more complex housing is known to reduce stress improve welfare and even buffer against anxiety responses Initial planning involved calculating the minimum size we wanted large enough to house our largest rats in groups of 5 without needing to split test groups over multiple rooms Once designs were drawn up complete with sketches and measurements the job was put out to tender 142 One company took up the challenge turning amateur sketches first into professional plans and then manufacturing amazing new caging The cages use a battery system rather than conventional shoe box cages with a combination of wire sides for climbing and to allow the addition of enrichment items and solid opaque sides for privacy The cages have an internal floor space of 4088cm2 730x560mm and a volume of 220 752cm3 height of 540mm As such the cages provide more than double the minimum cage size for our standard rats 5 x female at 120g Light and easy to use plastic trays were originally fitted but unfor tunately the animals quickly chewed holes in them and escaped Plastic has now been replaced by anodised aluminium trays which are a significant improvement Food hoppers and water bottles are located on the outside of the cage so that the maximum internal space is available to the occupants The cages have a shelf and nest box built in in order to utilise the available vertical space Cleaning out is done primarily by tray changing the shelf and nest box

Poster Presentations are simply wiped down Although cleaning takes longer than with conventional caging the larger size has enabled us to reduce cleaning from twice a week to once decreasing overall workload We continued to provide nesting material as before as well as fun tunnels one on the cage floor and one mounted as we had done in our old caging wider range of enrichment not only improving the complexity of the cage but in order to provide for a wider range of natural behaviours Figure 4 Rats utilising built in nest box Enriched cages Wall mounted fun tunnels have been replaced with reusable red plastic tunnels to provide variety and reduce costs long term Large aspen blocks are mounted to the cage front using bag ties so that they can be climbed as well as chewed To allow for digging behaviour and provide vertical barriers a small mouse box filled with nesting material paper wool or EnviroDri is provided Disposable socks stuffed with shredded paper or EnviroDri and forage treats are also hung from the side of the cage Figure 3 Rack of 3 cages complete with built in enrichment Figure 5 New style cage showing environmental enrichment Built in nest box Once the cages were in use it was clear that the rats utilised the space as much as possible but that the cage still looked empty and the space at the front of the cage was not being utilised effectively Further consideration of this led to opportunities in providing a Wall mounted enrichment In addition to improving the physical environment we have also implemented a programme of feeding enrichment At cleanout new trays have forage mix seeds cereals primate treats etc scattered 143

Poster Presentations throughout the clean bedding to encourage foraging Rats are also given Bonio dog biscuits once a week which encourage chewing gnawing behaviours New caging in use The caging is not only better for the rats it is an improvement for the technologists too Cleaning out is now only a weekly task improved visibility makes it easier to check animals in their cages and removing animals from cages is easier Figure 6 Environmental enrichment to encourage chewing and gnawing behaviour Feeding enrichment Figure 8 New style caging with occupants The effect on the rats has been clearly visible to the technologists working with them The animals are more active and make full use of all the space and enrichment Acknowledgements Obser vations suggest that aggression low level fighting and bar biting have been reduced Footage taken throughout the night shows the rats at their most active climbing and jumping in ways not possible or even attempted in conventional caging Finally we have noticed a marked reduction in incidence of porphyrin staining previously this was common when rats were given intra muscular injections on study now this particular sign of stress and distress is rarely if ever seen Figure 7 Example of feeding enrichment 144 Arrowmight Design and build of the new caging Pete Gerson Former Head of Department

August 2015 Animal Technology and Welfare Achieving and maintaining germ free status within an SPF facility CLAIRE ROGERSON SELINA HOPKINS CORDELIA BRANDT SILVIA HRNCIAROVA SCOTT KEMP and GLENN HAZZARD Wellcome Trust Sanger Institute Cambridgeshire CB10 1SA Corresponding author cr4 sanger ac uk Introduction The Research Support Facility is a large barrier facility maintaining mice to a high Specific Pathogen Free SPF health status Having the capacity to support up to 24 000 individually ventilated cages it is well established as a large production facility with a structured layout to maintain the health status of the animals it houses The science of our Institute is continuously evolving in part driven by new studies such as the interaction of our microbiota and genes In recent years there has been a resurgence in the need to maintain Germ Free GF mice which possess the advantage of a similar gene complement to ourselves while having a completely na ve intestinal tract Limited space required careful planning of layouts with entry working areas and equipment storage being critical Restricted entry and limited staff transition makes provision of consumables and their use a major consideration Close attention was paid to the initial cleaning and sterilisation of the area Deep cleaning and the use of Vaporised Hydrogen Peroxide VHP was used throughout the preparation and stocking of the room Defined and strict cleaning regimes ensure the maintenance of the area Peracetic Acid for port and isolator sterilisation and 1 Virkon for sur face disinfection are routine Environmental swabbing to ensure successful sterility is enhanced by in house and external microbiological testing To provide this capability requires the use of positive pressure isolators and a strictly managed and clean environment The challenge was to identify a suitable location within our barrier to breed and maintain the health status of this vulnerable mouse model Visits to Bern and Nor wich Universities assisted in our understanding and development of ideas and processes to combine best practises The limitations of pre set locations for services such as autoclaves and end destination of the germ free mice have driven the development Here we describe the hurdles and approaches we have taken to establish our Germ Free status and considerations for others wishing to undertake this approach Defining layouts and location The location of the Germ Free room was critical and accounted for the shortest routes between autoclaving and the external barrier This allowed for supply of cleaned and sterilised materials while reducing the transport routes for experimental animals Figure 1 Schematic of area layout 145

Poster Presentations Minimising transfer of contaminants Structured process for entry into the Germ Free Facility to protect from potentially harmful contaminants Figure 2 4 Views of germ free area Establishing of a germ free set up Entrance of equipment and animals This presents the greatest risk when establishing and maintaining the Germ Free colonies Any period where entry is required to stock the isolators or room presents an opportunity for breakdown Well designed transfer isolators and service equipment is paramount Ensuring ergonomics and staff wellbeing is balanced against the needs of what is to be achieved requires detailed planning Once established the training and adherence to clear Standard Operating Procedures SOP is critical Short cuts and variations will lead to break down and loss of the GF status The use of autoclaving VHP and irradiation is based on the potential exposure to the animal and the most suitable method of sterilisation We established our first colony with the help of Norwich University who kindly provided breeding pairs Breeding and movement into secondary and tertiary isolators ensures that Continuity planning is upheld 146 Figure 5 a k Process for entry

Poster Presentations Figure 6 Diagrams of 4 and 6 cage transfer isolator Figure 8 Autoclave Drums Autoclave cycles are run at 121 C cycle Adjustable platform allows entry of consumables at various heights Figure 9 Autoclave drum ready for transfer of sterile contents Into isolator Maintaining the germ free status Figure 7a and b Photograph and diagram of 90 cage double tier rigid isolator Germ Free status in the mice is tested three ways Culturing 16S PCR and Microscopy This is confirmed by lack of growth from culturing no product from 16S PCR and no bacterial staining under microscopy Faecal pellets are collected from the mice fortnightly and mashed up in some sterile water to create a faecal suspension This is then treated the following ways 147

Poster Presentations Microscopy Faecal suspension is placed onto a microscope slide Stains are added that colour bacterial cells The slide is then viewed to determine the presence or lack of bacteria 16S PCR will copy the gene millions of times so it can be seen using a process called gel electrophoresis The PCR product is loaded into an agarose gel and an electric current is passed across it Any 16S PCR product will then move through the gel and can be seen using UV light If a bright band appears on the gel then bacterial DNA was present in the faecal suspension This would indicate bacterial contamination in the mice Figure 10 Na ve faeces Figure 12 Culturing Faecal suspension is added to liquid broth or spread over the surface of an agar plate Broth and agar plates are specially designed to contain the nutrients any bacteria may need to grow They are placed into aerobic or anaerobic incubators at 37 C Anaerobic incubators are important as many gut bacteria are strict anaerobes and cannot grow in the presence of oxygen Signs of growth are then checked for in the broth and on the agar plates over the following days Figure 11 Germ free faeces 16s Polymerase Chain Reaction PCR 16S PCR is used to check for bacterial DNA in faecal suspension Bacteria present are broken open using a mechanical lysis method to release their DNA A PCR is done using universal bacterial 16S PCR primers as the gene is present in all bacteria 148 Figure 13 Broth and agar olates in anaerobic chamber

Poster Presentations Summary Careful planning and training have allowed us to implement a Germ Free capability within the SPF barrier of our facility Through a combination of working practices supported by strict SOPs faecal monitoring and technologist diligence we have maintained Germ Free status and reached our 1 year anniversary with wild type mice Moving forward we are looking to introduce Knock Out mice and build up a successful breeding colony This will involve a new set of skills in developing transport into the facility and or a defined rederivation process to maintain the Germ Free status Acknowledgements Trevor Lawley and Simon Clare Scientific direction Mark Stares Provision and exper t advice on Microbiology Lynda Westall and James Bussell Establishment and support Kathleen Macoy Bern University Early advice on set up Norwich University Supply of Germ Free mice Bell Isolators Datesand and Surrey Diagnostics Supply of equipment and advice 149

Animal Technology and Welfare August 2015 Open or closed REECE WILLIAMS Imperial College London Central Biological Services Hammersmith Campus The Hammersmith Hospital Du Cane Road London W12 0NN Correspondence r williams imperial ac uk Introduction Due to animal allergens in our facility open cages are no longer used to house rodent species Following scientific concerns about the growth weight differences during an ongoing study of Myocardial Infarction MI in Sprague Dawley SD rats which became apparent after the animals were moved from traditional open top cages to ventilated double decker cage environment Method For this study a 1500U Tecniplast cage was used This cage in essence was similar in size to the open cages that the rats had been previously housed except now this is an Individual Ventilated Cage It was agreed to keep the same twice weekly cleaning regime for the 1500U as that of the previous open type cage The idea was to simulate the rats being in an open cage with the same amount of enrichment food and handling The other cage used was a Tecniplast GR1800 Double Decker with the added red shelf within the cage Sprague Dawley and Wistar rats were both studied to see if any differences occurred between the two strains The Wistar rat is currently one of the most popular used for laboratory research It is known for its wide head long ears and having a tail length that is always less than its body The Sprague Dawley strain was developed from the Wistar rat and is known to be less active than Wistar rats Figure 1 Tecniplast 1500 513 x 381 x 256 mm Figure 3 Wistar Rat Figure 4 Sprague Dawley Rat Figure 2 Tecniplast GR1800 Double Decker 462 x 403 x 404 mm 150 The rat s weight in the Double Decker cages affected on the results of the MI study and it was believed the

Poster Presentations rats were too comfortable in the Double Decker cages and were becoming less active It was possible the cleaning schedules of the IVC cages which are now only once a week not twice would mean less handling time by the staff This could also cause the rats to become more stressed with prolonged interaction NC3Rs 1 more enrichment has been added to IVC cages in order to improve the quality of life for the laborator y animals Having different levels of enrichment should have an impact on the behaviour of the animals and the overall food intake The 1500U cage was set up with less enrichment and food to mirror the set up of an open cage Following guidelines from the National Centre for Replacement Refinement and Reduction of Animals in Research Variables CURRENT NORM TRIAL CHANGE Animal Charles River Charles River Sprague Dawley Wistar Sprague Dawley Wistar Male Male Weight start 120 240g 120 240g Population 3 animals 3 animals IVC Housing Double floored cage Single floored cage Position Lower on rack Higher on rack Diet 100 ad lib 85 restricted Cleaned 1x per week 2x per week Handled 1x per week 2x per week Enrichment Tunnel sizzle nest Sizzle nest Figure 7 Sprague Dawley Rat weight gain in the two study groups Figure 5 Method for study Figure 8 Combined data for Sprague Dawley Rats courtesy Harlan Laboratories UK Figure 6 Combined data for Sprague Dawley Rats courtesy Harlan Laboratories UK Figure 9 Sprague Dawley Rat weight gain in the two study groups 151

Poster Presentations The rats weight in the Double Decker cages had an effect on the results of the MI study It was believed the rats were too comfortable in the Double Decker cages and becoming less active It was possible the cleaning schedules of the IVC cages which are now only once a week not twice would mean less handling time from the staff This could also cause the rats to become more stressed with prolonged interactions Conclusions The rats gained slightly more weight within the 1500U cages for both sets of strains This could be for a number of reasons Being placed in the upper tiers of the rack may lead to animals being less active due to higher levels of light in the 1500Us Having a shelf in the Double Decker allows for more comfortable resting periods This in turn could potentially cause less stress The smell of fresh food and it being added more frequently may cause the rats to be more interested in the smell of food and may result in an increased appetite If this study was to be performed again it would be ideal to use more cages with different parameters Also studying more strains to get a broader spectrum of weights would allow us to see if there are strain differences 152

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INDEX TO ADVERTISERS August 2015 Allentown Inc OBC Arrowmight xv Bell Isolation Systems iv Contec xi Datesand Ltd viii Getinge UK Ltd iii Harlan Laboratories IFC Institute of Animal Technology vii xv xviii IPS Product Supplies Ltd IBC LBS xii North Kent Plastics vi PFI Systems xiii Plexx BV v Surrey Diagnostics xvi Tecniplast UK x Vet Tech Solutions xiv