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Lab Animal Care (Lectures 1-4, AALAS 1-2)

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How do federal regulations address animal pain and distress in research?
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Animal pain and distress must be avoided or minimized when consistent with sound scientific practices.
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what is TRUE about pain and distress in experimental animals?
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Animal pain and distress may introduce sources of nonexperimental variation into a study
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What frequency of monitoring is most appropriate for animals following a procedure?
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Daily or more frequently if deterioration in the animal’s condition is anticipated
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What factors make the assessment of pain and distress difficult in rodents?
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Small body size Tendency to hide when disturbed Tendency to freeze when disturbed
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Which is TRUE regarding the recognition of pain and distress in rodents?
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Recognizing pain and distress is based on a composite of clinical signs and data.
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How does the clinical exam of a rodent differ from that of a larger animal species?
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Diagnostic methods and equipment are adapted to the small body size.
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No change in body weight in two weeks for an 8-week old rat may suggest which interpretation?
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Growth is inhibited
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What method of supportive care is most critical during a brief surgery with minimum blood loss?
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Maintenance of body temperature
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Which factors may cause or contribute to animal pain and distress related to a research procedure?
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Hypothermia Dehydration Surgical wounds
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Physiological changes caused by experimental procedures have a greater impact and develop faster in rodents than in larger species because:
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Rodents have a faster rate of metabolism.
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Which finding is a strong indication for pain or distress in a mouse?
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The fur around the eyes and nose is stained red
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What is TRUE about administering physiological fluids to rodents for hydration therapy?
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Physiological fluids may be administered subcutaneously.
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Which is TRUE about methods for clinical assessment of rodents with tumors?
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Clinical assessment methods are based on standardized clinical signs for all tumor types and specific signs related to the tumor type.
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Treating pain, distress and discomfort in rodents is best addressed by?
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A combination of therapies based on clinical assessments
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What should be incorporated into records management system for clinical assessments of rodents?
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A way to flag cages of animals to be monitored within an animal housing room should be incorporated into the system.
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Accreditation by AAALAC, International, provides you with an exemption from some of the mandates of the Guide when:
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Under no circumstances
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How is Hantavirus transmitted to humans?
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by exposure to the excretions of the animals by aerosol from the saliva of infected animals by contact with urine of infected animals
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What would be the recommended cage floor area to house a group of 5 mice with body weights in the range of 17 to 22 grams each?
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60 inches squared
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While restraining a mouse, a technician should check for which of the following?
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irregular respiratory movements skin discoloration around the nose and mouth feet turning blue
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Explain ear notching/punching in mice
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The marks on the animal’s left ear denote tens (10, 20…) The marks on the animal’s right ear denote units (ones). Rodent ear punching and notching can be used to identify to animals from numbers 1 to 99.
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Peri-procedural care includes:
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care of the animal during the procedure care of the animal after the procedure care of the animal before performing a procedure
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What is a tip for blood collection in mice?
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Sedation causes vasodilation which helps make the tail vein easier to find
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True or False: In the mouse, IP injection should proceed only after aspiration has shown that the needle is properly positioned.
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True
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Important things to note about oral gavage in mice
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Once the needle has reached the stomach, inject slowly. The procedure can cause respiratory distress. The needle should be long enough to reach the level of the last rib of the mouse.
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Typically, subcutaneous injections in mice:
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cause a bleb that can be palpated. are limited to 1 ml or less. are used to administer fluids or medications.
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What is an injectable anesthetic agent?
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Pentobarbital
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What is the easiest way to induce gas anesthesia for a mouse?
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anesthetic induction chamber
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What is true about rodents after surgery?
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Rodents are susceptible to subclinical infections after surgery
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What can correct respiratory depression in anesthetized mice?
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Immediately reducing the administration of gas anesthetic Chest compressions at the rate of 60 per minute Administration of doxapram on the tongue
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Which type of bedding is recommended for use in postoperative housing for mice?
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paper bedding material
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What 3 things are helpful and provide valuable information in assessing pain and distress in mice
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systematic approach to observing clinical signs measurement of clinical parameters knowledge of normal mouse behavior
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What is the recommended method for the production of monoclonal antibodies?
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In vitro techniques
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True or False: The use of a conditionally acceptable method for euthanasia in your research protocol may require scientific justification and IACUC approval.
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True
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Your research must comply with the Guide for the Care and Use of Laboratory Animals if:
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your institution receives funds from the National Institutes of Health (NIH)
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lymphocytic choriomeningitis (LCM) – risk – what is it?
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There is a risk of transmission to the fetus of pregnant women exposed to LCM. It is an RNA arenavirus. The infection in mice is subclinical.
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factors that affect the routine sanitation schedule in animal facilities?
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Which of the factors below affect the routine sanitation schedule in animal facilities?
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When picking up a mouse from a filter top cage, putting the wire bar lid sideways on the cage is helpful to:
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Reposition your grasp as you take the mouse out of the cage as it will tend to hold on to the wire bar lid.
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Explain differentiation of anogenital distance in mice?
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The anogenital distance is greater in the male than in the female
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Which is true about mice in the age range of 6-10 days?
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Mammaries become visible on the females around 9 days.
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What is the age range when mice are weaned?
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21-28 days
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The goal of an occupational health and safety program is to
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prevent the transmission of diseases between and among laboratory animals assure the welfare of laboratory animals in research settings protect individuals who have frequent contact with animals in a research facility
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3 caging requirements for mice in the “Guide for the Care and Use of Laboratory Animals”
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Wire mesh flooring is preferred. Ventilated cages are required for all rodents. Minimum cage height is 5 inches.
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Which is true about mice in the age range of 11 to 14 days?
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Teeth are beginning to erupt at day 11.
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What is the maximum percentage of total blood that can be safely collected at one time from a mouse?
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10%
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The goal of appropriate animal care is to:
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minimize pain and distress where possible
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What group determines if a euthanasia method is acceptable for use in a particular species?
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American Veterinary Medical Association
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How are Salmonella spp. typically transmitted from animals to humans?
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fecal-oral route
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3 points regarding illumination in rooms housing rodents?
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Changes in light/dark cycles can affect body-weight gain. Photoperiod affects reproduction. Young albino mice prefer much lower illumination than the adults.
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mice in the age range of 1-5 days are what?
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They are born hairless
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The goal of an occupational health and safety program is to:
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protect individuals who have frequent contact with animals in a research facility
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According to the “Guide for the Care and Use of Laboratory Animals” what is the minimum cage height?
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Minimum cage height is 5 inches
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Where is IP injections for mice?
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The IP injection site is on the animal’s right side, close to the midline, in a caudal quadrant of the abdomen.
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True or False: A straight needle is preferred for oral gavage in a 18 gram mouse because it prevents trauma to the esophagus
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True
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What is the function of an IACUC?
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to make sure that all state and federal guidelines and mandates on animal welfare are followed to oversee all aspects of an animal care and use program to develop institutional policies regarding the care and use of laboratory animals
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What factors affect sanitation in animal facilities?
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the type of cage flooring the number of animals per cage the type of bedding used
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Which of the following are true when comparing a DecapiConeTM restrainer and a cylinder restrainer?
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Both can be used for tail vein injections. Mice tend to stay calmer in the DecapiConeTM restrainer. Both are appropriate for brief procedures on awake mice.
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What is true about the anogenital distance in mice?
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Which statement is true about the anogenital distance in mice?
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How are microchips administered to mice?
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A microchip is injected subcutaneously with a hypodermic needle.
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Who is responsible for the humane treatment of experimental animals?
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the research technician the animal technician the investigator
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Which of the following falls within the range of the total blood volume of adult mice? 12mL 60mL 6mL 1.2mL
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1.2 mL
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Why should an IP injection be made on the mouse’s right side?
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It makes restraining the animal easier
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What best describes oral gavage?
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The administration of fluid into the stomach through a feeding needle that is inserted into the mouth and threaded down the esophagus.
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Explain appropriate technique for subcutaneous injections in mice
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Lift some skin on the back of the mouse, and insert the needle into the base of the tent.
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What does doxapram do?
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It combats respiratory depression.
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List 3 things when preparing a mouse for surgery.
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The skin should be disinfected around the incision site. The fur should be removed around the incision site. You should firmly pinch the mouse’s foot or toe to check for a pain reflex before beginning surgery.
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What would you do to correct respiratory depression in anesthetized mice?
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Administration of doxapram on the tongue Chest compressions at the rate of 60 per minute Immediately reducing the administration of gas anesthetic
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What is a recommendation for postoperative care of mice?
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Give additional fluids subcutaneously during the 24 hours following surgery. Deliver oxygen until respiration rate returns to normal. Continue to monitor and treat for pain.
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What can provide valuable information in assessing pain and distress in mice:
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systematic approach to observing clinical signs knowledge of normal mouse behavior measurement of clinical parameters
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When producing monoclonal antibodies in vivo, what parameter should be considered?
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The degree of abdominal distention The amount of pristane used The gauge of the needle used
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Which statement is true about mice in the age range of 11 to 14 days?
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Teeeth begin to erupt at day 11
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Why should an IP injection be made on the mouse’s right side?
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It avoids puncturing the cecum.
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Definition of Biomedical Research?
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the search for ways to heal living organisms such as humans and other animals.
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Examples of biomedical research
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stem cells for spinal, vision, and hearing repair Gene therapy for muscular dystrophy, cystic fibrosis, sickle cell disease Alzheimers vaccine
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New drug development process takes average of how long?
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avg of 12 yrs (from research lab to patient)
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What percent of drugs in preclinical testing actually make it to human testing?
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avg 10%
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How much money is spent on development of a new drug?
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avg. 359 million (from research lab to patient)
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according to international law, scientists are required to do what?
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conduct animal research before they test new medicines in people
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3 most common animals used in research according to CCAC
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1. fish 2. mice 3. rats 4. wildlife 5. domestic birds 6. amphibians 7. farm animals 8. other 9. dogs 10. cats 11. non-human primates
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CALAM stands for
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Canadian Association of Laboratory Animal Medicine
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CCAC stands for
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Canadian Council on Animal Care
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CALAS stands for
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Canadian Association for Laboratory Animal Science
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AALAS stands for
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American Association of Laboratory Animal Science
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CIHR stands for
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Canadian Institute for Health Research
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NSERC stands for
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National Sciences and Engineering Research Council
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Any animal facility receiveing grants must….
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adhere to the CCAC standards to continue receiving grants
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Use of animals in science is under what jurisdiction?
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Ontario Provincial Legislation
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who enforces the research act?
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OMAFRA – Ontario Ministry of Agriculture Food and Rural Affairs
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what does OMAFRA stand for?
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Ontario Ministry of Agriculture Food and Rural Affairs
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Which organization is accountable to the general public and sets and maintains standards for ethical care and use of animals in science (in Canada)
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CCAC
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GAP stands for
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Good Animal Practice (from CCAC)
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ACC ensures facility meets _____ standards
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CCAC
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3Rs
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Reduction Refinement Replacement
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3 Rs. Reducion means…..
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reducing number of animals used in research wherever possible examples: not ordering more animals than needed having good experimental designs
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3 Rs Refinement means…
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refining methods and procedures to enhance potential for good science Exmaples: keeping social animals in group housing providing enrichment for animals to express normal behaviours defining humane end-points CE
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3 Rs Replacement means….
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replace the use of animals wherever possible with suitable alternatives Absolute replacement – i.e. computer models Relative replacement – working with lower sentience. i.e. insects vs rats
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AUP stands for
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Animal Use Protocols
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AUPs are submitted to?
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ACC
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How does a researcher select an animal model?
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– Some animal models are better for different areas of research (e.g. diabetic rat models) – Researchers review other study material and published papers to see what other models are currently being used in that area of research – Life span of the animal – Select the lowest possible level of sentience
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How many humane endpoints does an animal need to display for the vet and PI to be contacted?
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1 or more
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Examples of humane endpoints
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– changes in BW – measurable clinical changes (HR, RR etc.) – Changes in unprovoked behaviour (lethargy etc.)
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non-experimental factors
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– animal factors (including disease) – Physical factors – environmental factors – husbandry – animal care and handeling – research manipulation factors (ideally no variation)
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Needle size for SC administration in mice
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23G
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Needle size for IP administrationin mice
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25G
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over _____ percent of mouse genome is similar to ______
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95% our own genome
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What are the 3 strains of mice
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outbred strain inbred strain transgenic strain
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outbred strains of mice are a result of _______
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random mating
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outbred strains have________
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genetic variation
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Inbred strains of mice are the result of _______
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brother/sister mating father/daughter mating mother/son mating for MINIMUM of 20 generations
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Example of the inbred mouse strain is _____
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BALB/c mouse
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how are transgenic mice made?
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injecting DNA into mouse eggs altering the genome.
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How are knockout mice made?
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gene is removed or blocked by replacing section of DNA or disrupting it.
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List ways strains are defined be ecological characteristics
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Axenic or germ-free Gnotobiotic Specific Pathogen Free (SPF) Viral Antibody Free (VAF) Barrier-sustained Conventional animals Sentinel animals
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Mouse lifespan
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1-2 yrs
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mouse blood volume
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70-80mL/kg
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Mouse food consumption percentage of food consumption that is protein
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3-6g 12%
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Mouse rectal temp
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36.5-38 C
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Mouse litter size
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6-12
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Mice weight what at birth?
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1g
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Mice are weaned at what age?
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21 days
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Mice estrus cycle
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4-5 days
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Mice breeding age
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6 weeks
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Mice gestation period
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19-21 days
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mice water consumption
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3-7mL
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Mice communicate by_________
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ultrasonic frequencies to their pups (and vice versa)
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what is avoidance of open space called?
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thigmotaxis
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What t is called when other mice in a cage have trimmed the whiskers of a mouse
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barbering
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What is a potential risk of bright light when it comes to mice?
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It can damage their retinas causing formation of cataracs especially true for albino mice
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What light is used to observe mice without disturbing them?
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red light
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which sex of mice can be housed together
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female mice
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What do mice not have?
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sweat glands
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what are mice susceptible to?
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temperature fluctuations
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In mice, what is the blind sac located between the small and large intestine?
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cecum
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what should be avoided during IP injection?
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cecum
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Cecum in mice is located where…. Meaning IP injections will be given where?
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left of abdomen IP injections given on MOUSE’s right side
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Female mice have how many pairs of nipples and mammary glands?
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5 pairs
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Do male mice have nipples?
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No
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Males have _______ anogenital distance
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long (twice that of females)
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Females have _______ anogenital distance
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short
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how many hours of light compared to dark should a mice have?
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14 hrs light 10 hrs dark
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are pups typically born during light or dark time?
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dark
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what time frame after birthing should you avoid disturbing the mum, pups, nest, and cage?
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at least 3 days
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explain the Bruce effect
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pregnancy block that happens when female is exposed to an unknown male within 48hrs of copulation. (unfamiliar male scent on bedding can do the same)
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explain Whitten effect
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majority of group of females will be in estrus the third night of exposure to male
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Explain postpartum estrus
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happens within 24hrs of parturition female can be in gestation at same time as having litter
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plug equals what?
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copulation
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Plug can mean, or cannot mean……
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pregnancy
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are plugs always visible?
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no
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Where should you never pick up a mouse?
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middle or tip of the tail
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room temp for mice should be around
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22 – 25 C
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mice are highly susceptible to
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hypothermia and hyperthermia
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how do mice lose heat?
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via tail and ears
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what mice might need higher than normal room temp? (2 examples)
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nude mouse breeding colonies
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room humidity that is ideal for adult mouse is…
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50-70% humidity
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Low humidity might cause what in a mouse?
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ring tail dehydration (in young mice)
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high humidity might cause what ….
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increased bacteria and ammonia levels in cages
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how many air changes should there be per hour for conventially housed mice?
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15-20 air changes per hour
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open top shoe box advantages and disadvantages
open top shoe box advantages and disadvantages
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Advantage: Good ventilation at cage level Disadvantages: Spread of disease and allergens Material can fall into cage from other cages
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micro-isolator with filter top (static cage with filter top) advantages and disadvantages
micro-isolator with filter top (static cage with filter top) advantages and disadvantages
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Advantages: Less spilling of food & bedding out into the environment Reduction in disease transmission due to filter top Disadvantages: Rise in temperature within the cage Reduced ventilation inside the cage requires: Increased air flow at room level More frequent cage changing
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Individually Ventilated Cages (IVC) advantages and disadvantages
Individually Ventilated Cages (IVC) advantages and disadvantages
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Advantages: Cage level ventilation No allergens or disease organisms in the room air Automated water supply-sipper tubes available Less frequent cage changing Disadvantages: Critical to check water system daily- sipper tubes Check airflow
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When would you not use ear tags for identification?
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If you want to use the ear for blod collection
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common colors of mice coats
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black white agouti
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hole punching the left ear of the mice is….
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whole punch 10, 20, 30 half punch 40, 50, 60 double half punch 70, 80, 90
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hole punching in the right ear of mice is….
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whole punch 1, 2, 3 half punch 4, 5, 6 double half punch 7, 8, 9
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3 steps to assess appearance and behaviour
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1. observe from outside of cage 2. observe with lid and food hopper removed 3. physical exam of each mouse
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ABNORMAL appearance and behaviour of mice
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Weight loss Dehydration Sunken eyes Change in urine and fecal output (including consistency) Hunched posture Lethargy, depression and withdrawal from the group Change in gait and/or movement Ocular and nasal discharge Attitude: depression, difficult to arouse Activity level: hypo activity (hunched, huddled, lethargic), hyperactivity Discharge, bleeding, evidence of trauma Change in behaviour (vocalizing or biting) Excessive scratching, biting at painful areas, self-mutilation
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MGS is….
MGS is....
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Mouse grimace scale. based on facial expression assists in animal pain management
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Mouse BCS
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similar to that of cats and dogs. BC 3 = vertebrae and dorsal pelvis not prominent; palpable with slight pressure
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Sendai Virus
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RNA virus aorosol transmission respiratory secretion transmission Signs: weight loss ruffled hair coat hunched posture anorexia dyspnea chattering sudden death secondary infections
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Mouse Hepatitis Virus (MHV)
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caused by coronavirus May cause encephalitis/hepatitis (in mice) May cause wasting disease in immuno suppressed Signs: asymptomatic dehydration weight loss diarrhea sudden death
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Tuzzer’s Disease
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Caused by Clostridium piliformis bacteria commonly affects immuno suppresed commonly affects those w/poor housing conditions ingestion of spores transmission ingestion of feces transmission signs: dehydration weight loss diarrhea sudden death ISOLATE animals
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Epizootic Diarrhea of Infant Mice (EDIM)
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cause by rotavirus (affects suckling mice) fecal oral route transmission signs: soft yellow feces fecal straining abdominal distention up to 2wks old ISOLATE affect animals DECONTAMINATE rooms
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Mice MITES 2 examples… transmitted via….
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Myobia musculi Mycoptes musculinus transmission: direct contact contaminated beeding contaminated objects
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Mice MITES clinical signs… Treatment….
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Alopecia Pruritus Dermatitis Ivermectin
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Pinworms (Syphacia obvelata) found primarily….. Diagnostics….
Pinworms (Syphacia obvelata) found primarily..... Diagnostics....
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in the cecum. Shape: banana (asymmetrical) Tape test Fecal flotation PCR done on feces
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Pinworms (Aspiculuris tetraptera) found primarily….
Pinworms (Aspiculuris tetraptera) found primarily....
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in the cecum shape: long oval (symmetrical) Fecal flotation necropsy (adults primarily in colon) PCR test done on feces
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Clinical signs of Pinworms….
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Few hair loss weight loss Animals not suitable for research (pinworms may influence experimental results)
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explain Ulcerative Dermatitis
explain Ulcerative Dermatitis
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mild to severe extremely itchy (pruritic) some more prone to ulcerative dermatitis (strain C57B1/6) Signs: moist itchy lesions around neck and scapular area
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concerns with ulcerative dermatitis
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secondary infection
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Murine norovirus
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neonatal death ill health in immune suppressed mice
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Helicobacter spp.
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may affect immune system
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Murine parvovirus
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affects neonatal mice causes diarrhea wasting
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Injection for mice sometimes….
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have to be diluted with sterile saline or sterile water
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0.1mL = _____ uL
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100 uL
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Common mice injections
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SC IM (caudal thigh, point caudally, 45 angle) IP (lower right quadrant) IV (tail vein) Oral gavage
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needle size for SC =
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23G
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needle size for IP =
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25G
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Needle size for IV =
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25G
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Needle size for IM =
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25G
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____ – _____% total blood volume can be withdrawn every ____ to _____ _____
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10-15% 2-3 weeks
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Blood collection technique for NON-terminal (mouse will wake afterwards)
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Submandibular (cheek bleed) Jugular Saphenous vein Lateral tail vein Retroorbital sinus (not recommended by CCAC)
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Blood collection technique for TERMINAL (mouse will NOT wake afterwards)
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cardiac puncture
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what is balanced anesthesia?
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using several drug in combination
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criteria for selecting anesthetic regimes
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1. you have to be able to collect valid research results 2. need to provide post-operative analgesia 3. appropriate for species and strain of animal 4. appropriate for age and size of animal 5. environment dependent (fish vs rodent) 6. Logistic effect (1 rat vs 100 rats) 7. equipment available in lab for support 8. availability of anesthetic (not all available in Canada)
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Rodents do not……
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vomit so no need to withold food and water prior to anesthesia.
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What happens if food or water is witheld from rodents?
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they become hypoglycemic
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What happens if food is witheld from rabbits or guinnea pigs?
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can cause digestive disturbances
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Rodent HR normally between…..
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350-600 bpm
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What cannot be assessed during surgery?
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CRT
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Where can you assess color of a rodent?
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footpads nose tail ears should be PINK
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In rodents don’t forget to give ___ ____. Rodents do not have …..
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eye lube. palpebral eye reflex
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Rabbit RR
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30-60 bpm
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Rodents RR
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50-100 bpm
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what can sometimes be seen just before death?
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agonal breathing
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Reflexes
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Pedal withdrawal reflex (hind leg – middle toe) Tail pinch reflex
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IP Advantages Disadvantages
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Advantages Commonly used in rodent anesthesia and dosages are well documented in literature easily learned larger volumes can be given Disadvantages Possible tissue damage Inaccurate placement of agent slow induction & long recover Rabbit: IP route not recommended
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IM Rodents (locations) Rabbits (locations)
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Rodents: (limit as can be painful) quadriceps gluteals Rabbits quadriceps epaxial
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IV Note about rodents Note about rabbits
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rodents: difficult to gain access Rabbits: good ear veins
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Suitable pre-anesthetic agents for rabbits
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acepromazine diazepam midazolam hypnorm meditomidine Xylazine
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rabbits metabolize atropine with…..
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atropine esterase enzyme (found in their livers)
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Acepromazine excellent for….
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sedation in rabbits (useful for performing radiographs)
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If used alone, what does NOT effectively immobalize rodents?
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Cyclohexamine agents (Ketaset, Vetalar)
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Concentrated forms of what are used for euthanasia
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Barbiturates like pentobarbital Should be administered IV
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induction generally achieved within…..
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2-3 minutes
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brief procedures last
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<30 seconds
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Advantages and disadvantages of inhalation anesthesia
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Advantages fast recovery & fast induction cleared by the respiratory tract Disadvantages need for scavenging of gases because of health and safety concerns expensive require highly training staff
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Explain Open Drop-bell jar delivery system
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cotton ball soaked in inhalent anesthetic requires fume hood / proper ventilation difficult to adjust levels of anesthetic Health/safety issue for user may be used for wildlife
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How do rodents receive their anesthetic gas?
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nasal catheter or intubated
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What should be done until animals are ambulatory (post-op)
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flip from one side to the other every 15-20 minutes To avoid/prevent congestion in lungs
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What needs to be done immediatlyu after surgery for rodents?
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provide nutrition because of high metabolic rate example: Clear H2O diet gels softened rodent pellet (mush)
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what may observed when pain is present (in the coat appearance)
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piloerection
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explain discharge around eyes in rodents (Chromodacryorrhea)
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secreted from Harderian glands called Porphyrin secretions black/reddish brown non-specific response to illness and/or pain and/or stress
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Rabbits may do what when they experience abdominal pain?
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grind their teeth
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many analgesic agesnts are used ________ for rodents
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off label
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What is important to note about analgesia with rodents because of their high metabolism?
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analgesia may not last as long as in cats and dogs
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Duration of buprenorphine in rodents
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6-12 hrs sustained release buprenorphine – 48-72 hrs
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Duration of NSAIDS in rodents
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12-24 hrs
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What is the ethical goal of biomedical research?
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– For animals to experience the least amount of pain – Pain experienced for the fewest number of animals possible – Pain experienced for the shortest possible time