BIOMETHODOLOGY OF THE RAT Office of Laboratory Animal Care University of Tennessee, Knoxville General Behavior Nocturnal Photoperiod 12:12 or 14:10 for breeding colonies Non-aggressive, inquisitive, trainable Coprophagic Accept single housing Males unlikely to fight when housed together Body Weight Gain Chart CD Rat Reproduction Estrus Cycle 4–5 d Gestation 21–23 d Litter size 3-18 pups Haircoat 8-9 d Eyes open 10-14 d Weaning 21 d Postpartum estrus Sexing Anogenital distance is longer in the male and shorter in the female Male Female Genital Opening Anus Housing Adequate housing should provide the following: Behavior and physiological needs Social interaction Clean, dry and safe area with adequate ventilation, food and water Visualization by personnel Sufficient space to turn around and make normal postural movements Housing Recommendations Primary enclosure space recommendations per the Guide for the Care and Use of Laboratory Animals Rat Weight (g) Floor Area (in2) Height (in) <100 17 7 Up to 200 23 7 Up to 300 29 7 Up to 400 40 7 Up to 500 60 7 >500 >70 7 Primary Enclosure Enclosure Locking wire bar lid Easy visualization Solid bottom flooring Bedding and enrichment Water bottle Feed Microisolator top Reduces spread of pathogens Environmental Conditions Room Recommendations Temperature 68 – 79⁰F Humidity 30 -70% Ventilation 10 -15 air changes/hr. Light levels 130 -325 lux Noises sustaining ≥85 db can cause Stress Metabolic changes Reduced fertility Identification Temporary Markers and Dyes Hair Clipping Ear Tags Microchip Permanent Ear Punches Tattoos Identification Cage Card Manual Restraint Use gloves Grasp the tail 1-2 cm from the base and place rat on wire bar lid Manual Restraint Palm the rat over the back Push the rat against the wire bar Advance hand toward rat’s head Manual Restraint Place index and middle finger around the neck Keep the head between the middle and index fingers Manual Restraint The thumb and second finger should be placed under opposite axilla The tail can be held by the opposite hand Manual Restraint Click to Watch Video Mechanical Restraint Plastic adjustable restrainers Decapicones Blood Collection Survival Procedures Tail vein Orbital plexus Non-Survival Procedures Cardiac puncture Axillary cut down Cranial vena cava puncture 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 Blood Collection Tail Vein Use for collecting 0.1–2.0 ml of blood Heat source to dilate blood vessel Proper restraint ≤ 21 gauge needle Blood Collection Click to Watch Video Blood Collection Orbital plexus Use for collecting up to 4 ml of blood Anesthetize rat 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 Blood Collection Click to Watch Video Blood Collection Cardiac Use for collecting up to 15 ml of blood Anesthetize rat ≤21 gauge needle Insert needle under sternum at a 20⁰ angle Aspirate slowly Euthanize rat Blood Collection Click to Watch Video Tissue Collection Tail Biopsy Limited to a maximum of 2 times Maximum biopsy size of 5 mm Analgesia/Anesthesia is required for rats 21 days of age and older and in rats weighing 50 grams or more Compound Administration 21 to 25 gauge needle Maximum Administration Volumes (in ml/kg) IM 0.1–0.2 IP 10–20 IV PO (bolus) (gastric gavage) 5 10–40 SC 2-10 Compound Administration Subcutaneous (SC or SQ) Locate site of injection Insert needle underneath skin Aspirate negative pressure Inject compound and watch for SQ bleb Pause before retracting needle Compound Administration Intraperitoneal (IP) Assistance is helpful Locate lower right quadrant for injection site Aspirate If an unintended subcutaneous bleb occurs, reposition the needle Compound Administration Intravenous (IV) Lateral tail vein Proper restraint Heat source to dilate blood vessel After removing needle, apply direct pressure for hemostasis 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 rat for signs of distress 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 rat and procedure. 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 Aseptic Technique Place a sterile drape over the rat Anything that touches the surgical site must be sterile Non-absorbable sutures/clips should be removed in 7-14 days Surgical Monitoring Prevent pain, hypoxia, and hypothermia Monitor withdrawal reflex Provide a source of external heat Provide appropriate analgesics for postoperative pain management Surgical Record Euthanasia Inhalant Anesthetic Overdose Carbon Dioxide Isoflurane End the procedure with a thoracotomy or cervical dislocation Place rat in the chamber Turn on CO2 flow into the chamber Once the rat has stopped breathing, wait at least 1 minute before removing the rat from the chamber End the procedure with a thoracotomy or cervical dislocation Substantially prolonged in neonates Cervical Dislocation Performed on anesthetized rats weighing <200 grams Must be performed by skilled personnel Prevention of Infectious Disease Colony Health Surveillance Sentinel rats are tested on a routine basis Gross necropsy Serological assays Tests for parasites Rat Antibody Production Test (RAP Test) and PCR A test for cell lines and tumors for rodent viruses Included in every animal use protocol Occupational Health and Safety PPE (Personal Protective Equipment) Protects handler and rat May include: gloves, shoe covers, gowns, lab coats, masks, and head covers Zoonoses prevention Proper handling Health surveillance programs Routine sanitation Proper use of PPE Health Surveillance Rat Serology (RADIL) Rat coronavirus (RCV) Generic parvovirus (NS1) Rat parvovirus (RPV) Rat minute virus (RMV) Kilham rat virus (KRV) Toolan's H-1 (H-1) Rat theilovirus (RTV) Sendai virus Pneumonia virus of mice (PVM) Mycoplasma pulmonis Reovirus 3 Lymphocytic choriomeningitis virus (LCMV) Cilia associated respiratory bacillus (CARB) Hantaan virus Tyzzer’s Mouse adenovirus (MAD1) Procurement of Rats APPROVED vendors include Charles River, Jackson Labs, Harlan, NCI-Frederick, and Taconic Rats that are shipped from a non-approved vendor source must be approved by OLAC before the rats are procured. An animal requisition form must be submitted to the facility manager: http://www.vet.utk.edu/olac/pdf/animal_acquisition_form.pdf Quarantine Quarantine is required if receiving rats 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 Health Concerns Whenever possible, pain and distress should be eliminated General appearance Lethargy Aggressiveness Hunched posture Health Concerns Eyes Porphyrin staining = Stress Teeth Overgrowth Tooth breakage Malocclusion Health Concerns Body Condition Swellings or Tumors Poor Haircoat Ectoparasites Stress Cold Poor Nutrition Body Condition Scoring Hickman DL, Swan M 2010 Reporting Signs of Pain or Distress For Rats 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 animal’s condition and treatments will be recorded on the “Sick Animal” card References Guide for Care and Use of Laboratory Animals. National Academy Press, 2011. Hickman, DL, Melissa Swan. 2010. Use of Body Condition Score Technique to Assess Health Status in a Rat Model of Polycystic Kidney Disease. J Am Assoc Lab Anim Sci 49:155-159. Lawson, PT. Assistant Laboratory Animal Technician (ALAT) Training Manual. The American Association for Laboratory Animal Science, Memphis, TN. 2005. Sharp, Patrick and Marie LaRegina. The Laboratory Rat. CRC Press, Boca Raton, FL. 1988. Presentation: Chris Carter, OLAC UTK