Biomethodology Of The Mouse

1. Biomethodology Of The Mouse Office of Laboratory Animal Care University of Tennessee, Knoxville

2. General Behavior • Nocturnal • Non-aggressive towards humans • Social • Barbering is common • Males are more likely to fight if housed with non-littermates

3. Weight Gain Chart C57BL/6

4. Reproduction • Estrus Cycle 4–5 d • Gestation 19-21 d • Litter Size 10-12 pups • Eyes open 14 d • Weaning 21 d • Postpartum estrus • Reproductive life ~8 mo

5. Sexing • Newborns have a subtle difference in anogenital distance • At 9 days of age, nipples are evident in the female and absent in the male • Adults have a marked difference in anogenital distance

6. Housing • Adequate housing should provide the following: • Clean, dry and safe area with adequate ventilation, food and water • Visualization by personnel • Sufficient space to turn around and make normal postural movements

7. Enclosure Recommendations • Primary enclosure space recommendations per the Guide for the Care and Use of Laboratory Animals • UTK Mouse Cage Density Policy • http://www.vet.utk.edu/olac/pdf/Mouse_Cage_Density_Policy.pdf

8. Primary Enclosure • Cage Bottom • Easy visualization • Solid bottom flooring • Bedding and enrichment • Microisolator top • Reduces spread of pathogens • Wire-bar lid • Water bottle • Feed

9. Environmental Conditions • Temperature 68 – 79⁰F • Humidity 30 -70% • Ventilation 10 -15 air changes/hr. • Noise ≥85 db can cause • Stress • Metabolic changes • Reduced fertility Room Recommendations

10. Identification • Ear Tags • Ear Punches

11. Identification • Cage Card

12. Procedures • Institutional Animal Care and Use Committee (IACUC) protocol describes all procedures that can be performed • A procedure is defined as any activity carried out on a mouse such as: • Behavioral observation • Venipuncture • Surgery

13. Manual Restraint • Lift mouse by the base of the tail and place on wire bar lid • Push the mouse against the wire bar to prevent escape and advance the hand toward the head. • Grasp the scruff of the neck • Tuck tail between finger and palm

14. Manual Restraint • Click to Watch Video

15. Mechanical Restraint Decapicone Plastic Adjustable Restrainer

16. Blood Collection • Survival Procedures • Orbital sinus • Tail vein prick • Facial vein • Non-Survival Procedures • Cardiac puncture • Cranial vena cava puncture • Axillary cut down

17. Blood Collection • Blood Collection Guidelines • Single blood draw • ≤1.0 ml per 100 grams of body weight • Multiple blood draws • Maximum of 1.5 ml per 100 grams of body weight within a 2 week period • Note: Average mouse body weight is ~20 grams but can be highly variable between stock/strain

18. Blood Collection • Orbital sinus • For collecting up to 0.2 ml of blood • Anesthetize mouse • Hold the head steady • Insert pipette in the medial canthus of the eye • Rotate the tube between thumb and finger • Keeping eyelids closed, apply direct pressure using gauze for hemostasis

19. Blood Collection • Tail Nick • For collecting up to 0.2 ml of blood • Warm tail to dilate blood vessel • Use proper restraint • Prep with 70% alcohol • Use #11 scalpel blade or needle to nick the lateral tail vein • Apply direct pressure for hemostasis

20. Blood Collection • Facial Vein • Hold the mouse securely • Locate the puncture site slightly caudal to the freckle • Apply petroleum based lubricant to the site • Use a lancet or 18 gauge needle to puncture the skin • Collect blood • Apply direct pressure for hemostasis

21. Blood Collection • Cardiac • For collecting up to 1.0 ml of blood • Anesthetize mouse • Insert ≤25 gauge needle under sternum at a 20⁰ angle • Aspirate slowly • Euthanize mouse

22. Blood Collection • Click to Watch Video

23. Tissue Collection • Tail Biopsy • Limited to a maximum of 2 times • Maximum of 5 mm • Analgesia/Anesthesia is required for mice 21 days of age and older

24. Compound Administration • 25 to 27 gauge needle Maximum Administration Volumes (in ml/kg)

25. Compound Administration • Subcutaneous (SC or SQ) • The sides of the mouse serve as good site for SQ injections • Insert needle underneath skin • Aspirate negative pressure • Inject compound and watch for SQ bleb • To reduce leakage from the injection site, pause before retracting the needle

26. Compound Administration • Click to Watch Video

27. Compound Administration • Intraperitoneal (IP) • Locate lower right quadrant for injection site • Aspirate • If an unintended subcutaneous bleb occurs, reposition the needle

28. Compound Administration • Click to Watch Video

29. Compound Administration • Intravenous (IV) • Lateral tail vein • Proper restraint • Heat source to dilate vessel • Apply direct pressure after injection

30. Compound Administration • Gastric Gavage (PO) • Use a bulb-tipped gastric gavage needle • Measure length of needle from mouth to last rib • DO NOT FORCE the needle down the esophagus • Inject solution • Observe mouse for signs of distress

31. Compound Administration • Click to Watch Video

32. Anesthesia • When planning any procedure involving anesthesia and/or surgery, please consult one of the laboratory animal veterinarians in the Office of Laboratory Animal Care (OLAC) at 974-5634. • The veterinarian can provide guidance and detailed information in selecting the most appropriate anesthetic and analgesic protocol for your mice and procedure.

33. Aseptic Technique • Surgical Prep • After induction of anesthesia, clip hair from the surgical site • Prep skin with povidone iodine, chlorhexidine or other appropriate skin antiseptic • Scrub in a circular pattern, beginning in the center and spiraling outward • Follow povidone iodine scrub with a 70% alcohol prep • Repeat Twice • End procedure with a light coat of povidone iodine solution (not scrub) to the surgical site

34. Aseptic Technique • Place a sterile drape over the mouse • Anything that touches the surgical site must be sterile • Non-absorbable sutures/clips should be removed in 7-14 days

35. Surgical Monitoring • Prevent pain, hypoxia, and hypothermia • Monitor withdrawal reflex • Provide a source of external heat • Provide appropriate analgesics for post-operative pain management

36. Record of Medical Care

37. Record of Medical Care

38. Euthanasia • Inhalant Anesthetic Overdose • Isoflurane • End the procedure with a thoracotomy or cervical dislocation • Carbon Dioxide • Place mouse in the chamber • Turn on CO2 flow into the chamber • Once the mouse has stopped breathing, wait at least 1 minute before removing the mouse from the chamber • End the procedure with a thoracotomy or cervical dislocation • Substantially prolonged in neonates • Cervical Dislocation • Performed on anesthetized mice • Must be performed by skilled personnel

39. Occupational Health and Safety • PPE (Personal Protective Equipment) • Protects handler and mouse • May include: gloves, gowns, lab coats, shoe covers, hair bonnets, face masks

40. Prevention of Infectious Disease • Colony Health Surveillance • Sentinel mice detect for presence of infectious pathogens and parasites in the research colony • Mouse Antibody Production Test (MAP Test) and PCR • A test for cell lines and tumors for murine viruses • Included in every animal use protocol

41. Procurement of Mice • APPROVED vendors include Charles River, Jackson Labs, Harlan, NCI-Frederick, and Taconic • Mice that are shipped from a non-approved vendor source must be approved by OLAC before the mice are procured. • An animal requisition form must be submitted to the facility manager: http://www.vet.utk.edu/olac/pdf/animal_acquisition_form.pdf

42. Quarantine • Quarantine is required if receiving mice from an unapproved vendor • The minimum quarantine period is six (6) weeks • No experimental manipulations or breeding can be initiated during the quarantine period unless approval has been granted by an OLAC veterinarian

43. Health Concerns • General appearance • Lethargy • Aggressiveness • Hunched posture • Coat • Piloerection • Hair loss • Unkempt appearance

44. Health Concerns Abnormal masses Skin lesions

45. Health Concerns • Teeth Normal Malocclusion

46. Body Condition Scoring Ullman-Cullere, et al. 1999

47. Tumor Production • Four criteria for euthanasia: • Tumor size of 1.5 cm in diameter or tumor ulceration • Body condition score of 1 • Tumor interferes with function of vital organs • Tumor significantly interferes with locomotion

48. Reporting Signs of Pain or Distress • For Mice That Require Veterinary Care • Complete the red “Sick Animal” cage card • Attach card to cage • Notify facility manager or • Print Clinical Case Request Form http://www.vet.utk.edu/olac/pdf/CLINICAL_CASE_REQUEST.pdf • Fax form to the OLAC office 974-5649 • Assessment of the mouse’s condition and treatments will be recorded on the “Sick Animal” card

50. 10 Question Quiz