Appendix

Tools to Monitor and Assess Health Status and Well-Being in Stress and Distress

The following pages contain ethograms, various types of scoring sheets, clinical assessments, and behavioral observations applicable to laboratory animals. As stated in the report, the interpretation of physiologic measurements as indicators of stress, distress, or welfare status is relative and does not always point to direct or straightforward links. Because little is known about behavioral changes directly attributed to stress and even less about distress, recognizing stress and distress in laboratory animals based on behavioral changes remains a significant challenge to investigators and animal care staff. Recognition of distress should be derived from intimate knowledge of the species’ or strain’s normal behavior and may be based on (1) clinical signs and/or (2) significant deviation from the expected behavioral repertoire. As a rule, when the expected repertoire of physiologic behaviors is absent or modified, an investigation into the reasons for the change is necessary.

Some clinical signs (e.g., changes in temperature, respiration, feeding behavior) indicate an abrupt onset of distress while others (e.g., weight loss) develop over a longer period of time and may serve as warnings. A thorough clinical examination with references to baseline effects of age, gender, genotype, etc., is necessary to establish the presence of distress, while an abrupt and marked change in behavior lasting more than a few days may also indicate a disease state. Although normal behaviors may sometimes be characterized simply by a lack of atypical behavior, such as stereotypic (i.e., repetitive) or self-injurious behavior, some species and strain differences are not always easy to discern, and further complications



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Appendix Tools to Monitor and Assess Health Status and Well-Being in Stress and Distress The following pages contain ethograms, various types of scoring sheets, clinical assessments, and behavioral observations applicable to laboratory animals. As stated in the report, the interpretation of physiologic measure- ments as indicators of stress, distress, or welfare status is relative and does not always point to direct or straightforward links. Because little is known about behavioral changes directly attributed to stress and even less about distress, recognizing stress and distress in laboratory animals based on behavioral changes remains a significant challenge to investigators and animal care staff. Recognition of distress should be derived from intimate knowledge of the species’ or strain’s normal behavior and may be based on (1) clinical signs and/or (2) significant deviation from the expected behav- ioral repertoire. As a rule, when the expected repertoire of physiologic behaviors is absent or modified, an investigation into the reasons for the change is necessary. Some clinical signs (e.g., changes in temperature, respiration, feeding behavior) indicate an abrupt onset of distress while others (e.g., weight loss) develop over a longer period of time and may serve as warnings. A thorough clinical examination with references to baseline effects of age, gender, genotype, etc., is necessary to establish the presence of distress, while an abrupt and marked change in behavior lasting more than a few days may also indicate a disease state. Although normal behaviors may sometimes be characterized simply by a lack of atypical behavior, such as stereotypic (i.e., repetitive) or self-injurious behavior, some species and strain differences are not always easy to discern, and further complications 

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6 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS are introduced by gender, age, physiological state, genetics, and genetic modification of the animals. The first three tables below contain behavioral categories and descrip- tions of physiologic activities in which rhesus macaques, common marmosets, and rabbits engage. In order to determine what kind of behavior it is that an animal exhibits, one needs to be knowledgeable in the ethology and hus- bandry of the species in question. For example, aggression may be a signal for fear or pain, but may also be observed in lactating mothers protecting their nest. Determining the variation of the behavior from normalcy is a matter of training, studying, and observation. TABLE A-1 An ethogram for Macaca mulatta (rhesus macaque) behavioral Categories Recorded behavior Definitions Aggressive behaviors Facial threat displays Open mouth face ± bared teeth or vocalization Aggressive approach Stiff approach, attacking run Physical aggression Slap, grab, biting, or wrestling Submissive behaviors Facial submissive display Bared teeth grin ± vocalization Avoidance Avoid, flee, leave, displaced Active appeasement Groom present, lip smacking Affiliative behaviors Affiliative contact Contact sit (within arms reach), embrace, touch Passive grooming Being groomed Active grooming Grooming other animal Sexual behaviors Sexual contact Genital present/inspection, mounting Appetitive behaviors Foraging Food search, eating, drinking Other activities Active Locomotion, enrichment use, self-grooming Abnormal behaviors Abnormal behaviors Stereotypies, autoaggression Inactive Inactive Lying, huddling, sitting, sleeping Vigilance Monitoring others Visually following other individuals Reprinted from Augustsson, A. and J. Hau. 1999. A simple ethological monitoring system to assess social stress in group-housed laboratory rhesus macaques. J Med Primatol 28:84-90.

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 APPENDIX TABLE A-2 An ethogram for Callithrix jacchus (common marmoset) behavior Code Definition Agonism Tufts-flick TF Rapid back-and-forth movement of ear tufts Frown FR Lower eyebrows, furl brow, and turn down corners of mouth while staring Cuff CU Swift, superficial blow or scratch performed aggressively Chase CH Pursue partner, with one or both animals exhibiting aggression and/or submission (not play) Fight FI Grapple aggressively with partner(s), involving biting, clawing, and wrestling Attack AT Lunge at or pounce on partner aggressively; may or may not result in fight Snap bite SB Direct a single short, sharp bite at partner Submit SU Flatten ear tufts and/or facial grimace (partially open mouth with corners of mouth retracted, exposing lower and sometimes upper teeth) and/or slit eyes (eyelids half closed) Continuous CS Continuous submit; start scoring after 5 sec submit Retreat RE Starting from a stationary position, move at least one body length away from another animal within 1 sec of the other animal establishing proximity (within 10 cm) Play Play PL Two or more animals lunge, grapple, wrestle, or chase for at least 1 sec in absence of aggression or intense submission; play face may or may not be present Solicit play SP Direct play face toward, pounce on, or initiate grapple with partner, in absence of ongoing play with partner Play face PF Open mouth without retraction of the lips Join play JP Join ongoing play bout between two or more partners Discontinue all social play for ≥ 3 sec End play EP Social play SO Social interactions involving non-aggressive physical contact with other individuals; high activity Infant-associated behaviors Climb on ON Climb onto any part of partner’s body so that all four limbs are on partner Solicit climb SC Position body directly above infant and/or pull infant onto on body; may or may not result in infant climbing onto partner’s body Climb off OF Voluntary climb off partner’s body after having all four limbs on partner Push off/reject PO Prevent juvenile from climbing onto body, or rub or otherwise force juvenile off body Have mouth on female’s nipple for ≥ 1 sec Nurse NU End nursing EN Discontinue nursing posture continued

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8 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS TABLE A-2 Continued behavior Code Definition Other social behaviors Sniff/nuzzle SN Orient face against or toward partner, excluding anogenital region Anogenital AI Orient face against or toward anogenital region of partner, inspect or use hands or mouth to investigate anogenital region of partner; includes anogenital groom Groom GR Use hands and/or mouth to pick through fur and/or mouth of partner, excluding anogenital region Sexual solicit SS Stare at partner with ear tufts flattened and eyes slit Mount MO Climb on partner’s back from behind and grip partner around waist and legs; may be accompanied by pelvic thrusting Initiate huddle IH Establish passive, torso-torso body contact with partner, with both animals remaining stationary and in passive contact for at least 3 sec Leave huddle LH Terminate huddle after at least 3 sec of passive, torso-torso body contact during which both partners remained stationary Object steal OS Take any nonfood object from hands or mouth of partner Attempt object AO Attempt but fail to take nonfood object from hands or mouth of steal partner Food-associated behaviors Food steal ST Take any food from hands or mouth of partner Attempt food AF Attempt but fail to take food from hands or mouth of partner steal Share food SH Eat from a food source from which partner is simultaneously eating or that partner is occupying without removing any food from partner’s mouth or hands New food NF Eat from a food source that no other animal is currently holding, eating from, or occupying Individual behaviors Bristle strut BS Arching posture and/or strut locomotion and/or general piloerection Scent mark SM Rub or drag anogenital, suprapubic, or sternal region along substrate, object, or partner Genital present GP Raise tail to expose genitals Object OM Sniff, bite, chew, gouge, handle, pounce on, grapple with, or manipulation otherwise manipulate inanimate object, excluding food items and water bottle, for at least 1 sec Written by Lissa Pabst. From Primate Info Net, Library and Information Service, National Primate Research Center, University of Wisconsin-Madison. Available at: http://pin.primate. wisc.edu/callicam/ethogram.html.

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 APPENDIX TABLE A-3 An ethogram for rabbits Common rabbit postures, behaviors, and vocalizations Purring or teeth purring—A sound made by lightly and quickly grinding/vibrating the teeth as the whiskers quiver; a sign of contentment Oinking or honking—A sound made to gain food or attention or during courtship Clicking—A happy sound often made after a welcomed treat is given Wheezing or sniffing—Nasal sounds made by ‘talkative’ rabbits; can be distinguished from abnormal respiratory sounds because they are intermittent and stimulated by interaction with the rabbit Whimpering or low squealing—A fretting noise that is made when one picks up a rabbit that is reluctant to be handled; made often by pregnant and pseudopregnant does Chinning—Rubbing the secretions from the scent glands under the chin on inanimate objects and people to mark possession. Glands are more developed in males than females Nudging or nuzzling—The nose is used to nudge a person’s hand or foot, or the rabbit may pull on a pant leg to signal a desire for attention. When enough petting has been done the rabbit may push the hand away Head shaking, ear shaking, body shudder—A shake of the head or body in response to an annoying smell or unwanted handling; often occurs as the rabbit settles down and becomes relaxed enough to begin eating and grooming Courting or circling—A sexual or social behavior whereby a rabbit circles another rabbit or the feet of a human while softly honking Scratching at the floor—A rabbit may scratch at the floor with its forepaws in order to get a person’s attention or to be picked up Nipping—Not always done in anger, this can mean ‘move over’ or ‘put me down’ Presentation—The head is extended forward with the feet tucked under the body and the chin placed on the floor in order for the rabbit to present itself as subordinate for petting from humans or to be groomed by another rabbit Flattening—A fear response wherein the rabbit flattens its abdomen onto the floor with ears laid back against the head; the eyes may be bulging Thumping—A sharp drumming of the hind feet as a warning or an alert to other rabbits of danger; often accompanied by dilation of the pupils and seeking refuge Teeth grinding—A slower, louder teeth crunching, sometimes seen with bulging of the eyes and usually indicating discomfort, pain, or illness Snorting or growling—A warning sound, either hissing or a short barking growl, that occurs with aggression or fear and is often seen with the ears flattened against the head and the tail up and in the grunt-lunge-bite sequence Isolation—When a rabbit that normally seeks attention from its mates and human companions isolates itself and is less active. Such a rabbit should be checked for illness Kicking—If a rabbit feels insecure when being picked up it will kick violently in an effort to escape. The hindquarters must be supported to prevent trauma to the spine or legs. A rabbit should be placed hind-end first into a cage in order to help prevent injuries caused by kicking Aggression—Strained, upright stance with tail stretched out and ears laid back in defensive posture; the rabbit may also kick high and backwards Loud, piercing scream—Similar to a human baby crying; signaling pain and fear, as when the rabbit is caught by a predator Scanning—A rabbit with impaired vision may move its head from side to side to scan the area around it Reprinted with permission from Macmillan Publishers Ltd: [Lab Animal] (Mayer 2007), copy- right (2007). Mayer J. Use of behavioral analysis to recognize pain in small mammals. Lab Anim 36(6):43-48.

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100 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS Other useful references for common normal behaviors of various labo- ratory animals include: • Whishaw, I. Q. and B. Kolb, eds. 2005. The Behaior of the Labo- ratory Rat: A Handbook with Tests. New York: Oxford University Press. • Pellis, S. M. and V. C. Pellis. 1987. Play-fighting differs from serious fighting in both target of attack and tactics of fighting in the labora- tory rat Rattus noregicus. Aggressie Beha 13:227-242. • Bassett, L., H. M. Buchanan-Smith, and J. McKinley. 2003. Effects of training on stress-related behavior of the common marmoset (Callithrix jacchus) in relation to coping with routine husbandry procedures. J Appl Anim Welf Sci 6(3):221-233. • Stevenson, M. F. and T. B. Poole. 1976. An ethogram of the com- mon marmoset (Callithrix jacchus jacchus): General behavioural repertoire. Anim Beha 24:428-451. • Stone-Sade, D. 1973. An ethogram for rhesus monkeys. I. Anti- thetical contrasts in posture and movement. Am J Phys Anthropol 38(2):537-542. • Universities Federation for Animal Welfare. 1995. An ethogram for behavioural studies of the domestic cat (Felis silestris catus L.) of the UK. Cat Behaviour Working Group. Hertfordshire, England: Universities Federation for Animal Welfare. • Morton, D. B. 2002. Behaviour of rabbits and rodents. In The Ethology of Domestic Animals—An Introductory Text, P. Jensen, ed. Oxford: CABI Wallingford Oxford. 193-209 pp. • Bayne, K. A. L., J. A. Mench, B. V. Beaver, and D. B. Morton. 2002. Laboratory Animal Behavior. In Laboratory Animal Science, ACLAM series, 2nd ed. New York: Academic Press. 1240-1264 pp. • Latham, N. and G. Mason. 2004. From house mouse to mouse house: The behavioural biology of free-living Mus musculus and its implications in the laboratory. Appl Anim Beha Sci 86:261-289. • Bothe, G. M. W., V. J. Bolivar, M. J. Vedder, and J. G. Geistfeld. 2004. Genetic and behavioral differences among five inbred mouse strains commonly used in the production of transgenic and knock- out mice. Genes Brain Beha 3:149-157. • Fox, M. 1971. Behaiour of Woles, Dogs, and Related Canids. New York: Harper & Row. • Leyhausen, P. 1979. Cat Behaior: The Predatory and Social Beha- ior of Domestic and Wild Cats. Barbara A. Tonkin translation. New York: Garland STMP Press. • Foster, H. L., J. D. Small, and J. G. Fox, eds. 1981. The Mouse in Biomedical Research, ol. 1-4. New York: Academic Press.

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101 APPENDIX • Dixon, A. K. and H. U. Fisch. 1998. Animal models and ethological strategies for early drug testing in humans. Neurosci Biobeha Re 23(2):345-358. As stated in various places in the report, an effective assessment of distress is predicated upon solid knowledge of physiologic behaviors for each species and careful observation. In this respect, clinical and behavioral analysis of distress follows the investigative guidelines to determine the cause of any clinical symptomatology or pathology. Similarly, the goal of this exercise would be to remove, alleviate, or minimize the cause of dis- tress (if doing so does not conflict with the research protocol) and support the animal in order to help it recover (see decision-making algorithm at the end of Chapter 4). The approach should integrate information from mul- tiple behavioral and physiological parameters and should involve a team approach that includes researchers, veterinarians, and animal caretakers/ technicians, as distress levels will vary in relation to the species, husbandry conditions, and experimental protocol as well as with each individual ani- mal. The Committee points out that differential diagnosis of signs (clinical and behavioral) attributed to pain, sickness, or distress is quite difficult and requires careful observation and clinical skills. The following tables show- case the overlapping clinical signs and abnormal behaviors associated both with distress and/or pain in various animal species.

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102 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS TABLE A-4 Species-specific clinical signs indicating pain, distress, or discomfort in experimental animals Species Cardiovascular Respiratory Other Rat* Dark claws Shallow rapid Red starring around eyes and nose; and feet; breathing; cyanosis, congestion and jaundice in eyes bulge grunting on mucous membranes or non-pigmented and and pale expiration non-hairy areas; square fast (dehydration) Rabbit As rat White discharge from eyes, nose, and on inside of fore paws; cyanosis, congestion and jaundice in mucus membranes, or non-pigmented and non-hairy areas Guinea pig As rat Cyanosis, congestion, and jaundice in mucus membranes or non-pigmented and non-hairy areas Dog As rat Salivation and panting. As guinea pig. Raised body temperature; increase in specific gravity of urine and decrease in volume; sweaty paws, pupils dilate, eyes glazed Cat As dog As dog. Circumanal gland discharge: third eyelid may protrude Monkey As dog As dog * Many signs in rats may also be seen in mice. Reprinted from Morton, D. B. and P. H. M. Griffiths. 1985. Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet Record 116:431-436.

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103 APPENDIX TABLE A-5 Species-specific signs of behavior indicating pain, distress, or discomfort in experimental animals Species Posture vocalising Temperament Locomotion Other Rat* Persistent Squeals on May become Abdominal dormouse handling more docile or writhing in posture or pressure aggressive mice. on affected Eats bedding; area eats neonates Rabbit Looks Piercing Kicks and No spillage of anxious, squeal scratches or food or water; faces back dozey eats neonates of cage (hiding posture) Guinea Urgent Rarely vicious; Drags legs No spillage of pig repetitive usually quiet; back food or water squealing terrified, agitated Dog Anxious Howls, Aggression As guinea pig. Penile glances: distinctive or cringing Raised body protrusion; seeks cold bark and extreme temperature; frequent surfaces; submissiveness, increase in urination tail runs away specific gravity between of urine and legs; decrease hangdog in volume; look sweaty paws, pupils dilate, eyes glazed Cat Tucked- Distinctive Ears flattened; in limbs, cry or fear of being hunched hissing and handled; may head and spitting cringe neck Monkey Head Screams Facial grimace Arms across body * Many signs in rats may also be seen in mice. Reprinted from Morton, D. B. and P. H. M. Griffiths. 1985. Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Vet Record 116:431-436.

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104 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS TABLE A-6 Common clinical signs associated with pain in small mammals Production of fewer, smaller, or no fecal Reluctance to curl when sleeping (ferrets) pellets Tucked into abdomen Anorexia Strained facial expression, bulging eyes Half-closed, unfocused eyes Increased frequency and depth of respiration Aggression or shallow breathing Pushing abdomen on the floor Lameness/ataxia Stiff movements Polyuria/polydipsia (especially with GI pain) Immobility/lethargy/isolation Head extended and elevated Overgrooming/lack of grooming Piloerection Vocalization (squeal usually fear in rabbits) Porphyrin secretion Stretching with back arched Self-mutilation Stinting on palpation Squinting (especially ferrets) Hunched posture Absence of normal behavior Teeth grinding (bruxism) Reprinted with permission from Macmillan Publishers Ltd: [Lab Animal] (Mayer 2007), copy- right (2007). Mayer J. Use of behavioral analysis to recognize pain in small mammals. Lab Anim 36(6):43-48. On the following page is a score sheet that may be used for behavioral phenotyping in mutant mice. As stated in the report, genetically modified mice may exhibit abnormal behaviors, but those behaviors may be charac- teristic of the background strain or environmental factors rather than a result of genetic modification. Background strain effects are particularly important where new genetic lines are not completely inbred. In those cases, varia- tion should be expected as a result of different proportions of the progenitor background strains in each animal. Careful review of the characteristics of the background strains is necessary to avoid erroneously attributing differ- ences in test results to the genetic modification. The score sheet was devel- oped by Julie Watson, MA, VetMB, DACLAM, Johns Hopkins University Department of Molecular and Comparative Biology, adapted from Crawley and Paylor (1997).

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10 APPENDIX SHEET A-1 Investigational screen for behavioral phenotyping Watson 1/06 Behavioral Phenotyping Level 1 Screen Accession #_________ Date________ Investigator ____________________ Genotype___________________________________ Background strain(s) ____________ Inbred / N#___ Tg /TM KO/KI/Cond Gene Name ____________ Key: 0 = zero; 1 = slow or reduced; 2 = normal; 3 = hyper Animal # WT Hemi -/- Animal # WT Hemi -/- DOB/Age Sex M F DOB/Age Sex M F Weight (g) Fur color Weight (g) Fur color Condition Score Condition Score Empty Cage 2 mins: Exploring 0..1..2..3 Empty Cage 2 mins: Exploring 0..1..2..3 N 0= <1 side; 1 =< 1 circuit; 0= <1 side; 1 =< 1 circuit; Gait abnormal Y Gait abnormal YN 2= multiple circuits; 3= frantic 2= multiple circuits; 3= frantic Posture abnormal Y N Posture abnormal Y N Freezing Y N Freezing YN Digging 0..1..2..3 Digging 0..1..2..3 Wild running Y N Wild running YN Grooming 0..1..2..3 Grooming 0..1..2..3 Stereotypies Y N Stereotypies YN Rearing 0..1..2..3 Rearing 0..1..2..3 Escape Y N Escape YN DORSAL VENTRAL DORSAL VENTRAL DRAW DRAW Bald patches/abnormalities Bald patches/abnormalities Y.. N Piloerection? Y.. N Piloerection? Bald patches? Y..N Bald patches? Y..N Physical abnormality Y..N Whisker damage Y..N Physical abnormality Y..N Whisker damage Y..N 0..1..2..3 Whisker response Body tone Body tone 0..1..2..3 Whisker response NA 0..1..2..3 NA 0..1..2..3 Petting escape 0..1..2..3 Ear twitch 0..1..2..3 Petting escape 0..1..2..3 Ear twitch 0..1..2..3 Passivity 0..1..2..3 Palpebral reflex 0..1..2..3 Passivity 0..1..2..3 Palpebral reflex 0..1..2..3 Trunk curl 0..1..2..3 Forelimb place 0 ..1...2...3 Trunk curl 0..1..2..3 Forelimb place 0 ..1..2..3 Righting 0..1..2..3 RL withdraw 0..1..2..3 Righting 0..1..2..3 RL withdraw 0..1..2..3 Visual placing 0..1..2..3 Biting 0..1..2..3 Visual placing 0..1..2..3 Biting 0..1..2..3 Reach c touch 0..1..2..3 Clicker 0..1..2..3 Reach c touch 0..1..2..3 Clicker 0..1..2..3 NA Grip: >60 Grip: >60 <60 time <60 time _______ _______ Notes: _______________________________________________________________________________ ____________________________________________________________________________________ Adapted from Crawley, J. N. and R. Paylor. 1997. A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Horm Beha 31:197-211. Sheet A-1 the mice were sent to Photoshop and we increased the contrast. and then we replaced the old mice on the form with the new mice. This was not very time consuming. Redrawning the mice is another matter.

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106 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS The following score sheets have been developed to assess animals in toxicology studies. This assessment is based on a detailed and systematic observation scheme that identifies and scores abnormalities according to a predetermined scale. The recorded symptomatology will determine the diagnosis and subsequent alleviatory actions. They can be adapted to any protocol or animal care facility system as long as the behavioral definitions are uniform across the same facility. SHEET A-2 Investigational screen for toxicology studies Step 1. Daily Cageside Observations This examination is typically performed with the animals in their cages and is designed to detect significant clinical abnormalities that are clearly visible upon a limited examina- tion and to monitor the general health of the animals. The animals are not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed include but are not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rup- ture), alterations in fecal consistency and fecal/urinary quantity. Clinical Observations Study personnel will conduct careful, hand-held, clinical examinations during the live phase of the study. The categorical observations made during this examination use a descrip- tion to record the severity. These observations can be made at any time during the study. a. Abnormal behavior: Description of unusual behaviors (e.g., circling, stereotypy) and changes in posture (e.g., arched back, splayed stance) not noted during the cageside portion of examination. b. Abnormalities of the eye: Any additional descriptive observations concerning the eye, including, but not limited to, cloudiness, opaqueness, overall size, ruptures, etc. c. Abnormal urine or feces: Description of animal excreta used to assess general health of animal, includes changes in color or quantity. d. Abnormalities of the gastrointestinal (GI) tract: Description of atypical visual finding related to the gastrointestinal tract (e.g., prolapsed rectum, decreased water or food intake, reflux of test material). e. Injury: Description of injury the animal has sustained. f. Missing extremity: Description of missing body part, includes tail, ears, limbs, etc. g. Abnormal muscle movements: Description of unusual movements (e.g., tremors or convulsion). h. Palpable mass/swellings: Description of unusual growths or swellings. Includes the location, onset, appearance, and progression of any finding. i. Abnormal posture: Description of unusual posture or stance. j. Abnormalities of the reproductive system: Description of atypical visual findings in the reproductive organs, including but not limited to: prolapsed vagina, unretracted penis, scrotum bluish, enlarged testicles. k. Abnormal respiration: Description of changes in respiration including shallow, slow, rapid, or mouth breathing. continued

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10 APPENDIX SHEET A-2 Continued l. Abnormal skin or hair-coat/mucous membranes: Description of atypical skin or mucous membrane color, changes in hair coat, loss of fur, etc. m. Excessive soiling: Description and location of increased body soiling. n. General abnormalities: Description of any other atypical finding not fitting any of the previous observation categories. Step 2. Detailed Clinical Observations (DCO) The purpose of the DCO examination is to provide information on the physical health of the animals for the duration of a study, as well as to document any changes in health status that may have occurred in response to chemical treatment of the animals. This examination, scheduled periodically during a study, is conducted in a careful and systematic manner. The examination begins at the head of the animal and works toward the rear of the animal. The observations are ranked according to severity. A. Cage-side observations. • Abnormal movements or behaviors: Unusual body movements (e.g., tremors, con- vulsions), abnormal behaviors (e.g., circling, stereotypy) and changes in posture (e.g., arched back, splayed stance). • Resistance to removal: The degree to which the animal attempts to escape capture is scored. The observer will slowly present a gloved hand into the cage and will grasp the animal over the shoulder area or by the tail. 1 = Decrease—clearly less resistance to capture than typical 2 = Typical—minimally to actively avoids capture and may be mildly aggressive 3 = Increase—clearly more resistance to capture than typical and is very aggressive (attempts to bite) b. Hand-held observations recorded while handling an animal. 1. Ranked observations—the following use a defined scale to rank the degree of severity: a. Eye obserations: Eyes are bilaterally examined; however, if a unilateral obser- vation is made, a concurrent observation is not made for the other eye if it is within typical limits. (1) Palpebral closure 1 = Closed (50% to completely closed) 2 = Open 3 = Protruding eyes (2) Pupil size (aided by penlight): Under typical examination conditions (white light), the typical appearance of the pupils in albino animals is complete constriction. Therefore a decrease in pupil size cannot be observed. 0 = Unable to evaluate 1 = Decrease—clearly decreased pupil size compared to typical 2 = Typical—completely constricted pupils 3 = Increase—clearly increased pupil size compared to typical (3) Lacrimation (noncolored periocular wetness) 1 = Decrease—extremely dry appearance of cornea 2 = Typical—glistening cornea (moderate dryness or wetness) 3 = Increase—extensive wetness around the eyes continued

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108 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS SHEET A-2 Continued b. Degree of saliation: 1 = Decrease—oral dryness 2 = Typical—limited to moderate perioral wetness, but lips and chin are dry 3 = Increase—extensive wetness around the mouth and lips c. Muscle tone: An assessment of muscle tone at the time of the hand-held observations. 1 = Decrease—clearly less muscle tone than typical 2 = Typical—animal is neither very relaxed nor very tense 3 = Increase—clearly more muscle tone than typical d. Extensor-thrust response: Extent of reflex response to brisk pushes (by finger) on the plantar surface of the hind feet. 1 = Decrease—clearly less response than typical 2 = Typical—clearly detectable extensor-thrust response 3 = Increase—clearly more response than typical e. Reactiity to stimuli: The degree to which an animal struggles to get free from hand-held restraint is ranked. 1 = Decrease—very slight or no struggling 2 = Typical—mild to moderate struggling, animal may vocalize 3 = Increase—aggressive escape behavior, may try to bite observer and usually vocalizes 2. Categorical observations—these are described in step 1 C. Open-Field Observations—Ranked observations made by placing the animal on a level surface. 1. Responsiveness to touch: The ventral aspect of the tail is lightly stroked using a fin- ger. Typically, the animal will lift its tail and wrap it around the finger when lightly touched. 1 = Decrease—does not lift tail, but may briefly hold tail in the air when manually lifted; no response to touch 2 = Typical—lifts tail when touched 3 = Increase—lifts tail and acts startled, may turn toward finger in an attack response 2. Gait evaluation: Open-field observations are used for gait evaluation. If the animal remains motionless in the open field, it may be forced to walk on its forelegs while the hindlegs are held off the floor. 1 = Unable to walk 2 = Clear knuckling, stumbling and poor coordination, may include falling and/or dragging of one or more limbs 3 = Typical—smooth and coordinated gait

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10 APPENDIX An alternative example is the following score sheet developed at the University of Birmingham Biomedical Sciences Unit, courtesy of David B. Morton, BVSc, PhD. SHEET A-3 General screening and applicability Strain: Start weight Mouse ID: Date: DAy TIME Appearance Inactive, Less active (L) Ataxic Stary coat Dull eyes Huddling Isolated Pinched face Eyes half closed Discharge eyes/nose Not grooming Scratching *Abnormal breathing **Type of breathing Feces pellets soft/hard Not eating Not drinking continued

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110 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS SHEET A-3 Continued Hunched posture Boarding of abdomen Tiptoe walking Handling Aggressive Not inquisitive/alert Crusty eyes/nose Reluctant to move Dehydration/skin tone Vocalisation Hyperactive Bodyweight % weight change from start % weight change previous day Body temperature Treatment Prolapse Overgrown teeth Other NAD Vet/PI contacted Signature: Scoring details * Abnormal breathing: Breaths/min ** Type of breathing: R: Rapid, S: Shallow, L: Labored, N: Normal

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111 APPENDIX As stated in Chapter 4, establishing surrogate or humane endpoints as part of the experimental protocol and before experiments commence is one of the ways to minimize and alleviate distress in laboratory animals. The following is an example of a tiered scoring system of defined humane end- points specifically developed for an arthritis mouse model. In this system the levels range from 0-5. When the arthritic wound is judged to be between levels 0-3, the animals are evaluated weekly by the investigator/veterinarian/ animal care team. When the wound advances to level 4, the animals are evaluated daily. All animals whose wounds reach level 5 on any day or that remain at level 4 for ten consecutive days are euthanized. Because more than one person evaluates the animals, some variation among the animal care staff does exist, a fact that should be taken under consideration. SHEET A-4 Establishing humane or surrogate humane endpoints Collagen Mouse Scoring Sheet 0 No reaction 1 Small scab +/- Reddening at tail base 2 Moderate scab +/- Swelling at tail base 3 Extreme scab +/- Swelling up to one third of tail +/- small or superficial ulcer (ligaments not visible) 4 Tail ligaments visible Tail ligament visible and one or more signs of poor health below: → Constriction at tail base with swelling/discoloration of most of the tail 5 → Agitation → Subdued behaviour An animal that scores 4 for 10 consecutive days or a score of 5 on any day is euthanized. Humane endpoints for the Collagen-induced Arthritis Mouse Model. Developed by Ghislaine Poirier, DVM, PhD, GlaxoSmithKline Pharmaceuticals. Sheet A-4

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112 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS Finally, the Committee acknowledges that to date there is lack of con- sensus on the best way to achieve “normal species-specific behavior” within the conditions most commonly provided for laboratory animals. To this effect, a pair testing record from the Wisconsin National Primate Research Center is included (courtesy of Joseph Kemnitz, PhD), which is used to document the process of social acclimation and housing of nonhuman primates. The animals are paired and their interactions are observed. Pri- mates with undesirable behaviors are identified and appropriate measures are taken. SHEET A-5 Nonhuman primate pair testing record Sheet A-5. Non human primate pair testing record. Wisconsin National Primate Center Pair Testing Record Species Animal IDs Date Project# Initials BHAV: Test#: Time In: Time Out: Test Conclusion: Compatible – will pair Incompatible – will not pair Inconclusive – another test needed Successfully paired / Date paired Shared food: Yes No Aggression demonstrated: Yes No If yes, describe below and note which animal performed/initiated aggressive actions. Example: threatening, grabbing, biting, fighting Affiliation demonstrated:Yes No If yes, describe below. Examples: sitting in proximity/near each other, sitting in contact/together, grooming, mounting Comments: Initials