PROTOCOL FOR ANIMAL USE AND CARE

PROTOCOL FOR ANIMAL USE AND CARE Score 1: Score 2: Total: 1. Contacts Primary Investigator Alternate contact Name Sandra Weisker Name Email sweisker@ucdavis.edu Email Dept Animal Science Dept Telephone 530 752 3642 Telephone After hours After hours 2. Project Title: Familiar vs Unfamiliar Cages for Post operative Housing of Male Mice 3. Protocol Type: Research 4. Species Common Names Total number for study Source of animals Mice 32 Approved vendor 5. Procedures Male mice will undergo a sham vasectomy under anesthesia. Following the procedure they may or may not be given an analgesic. Half the mice will be housed in their home cage following the procedure and half will be housed in a new, clean cage following the procedure. They will be observed for 48 hours, using video recording under UV light, for changes in behavior. 6. Animal Location(s) Overnight Housing (Vivarium) Study Area/Laboratory Animals Maintained by Cole B N/A Vivarium 7. Special Husbandry Requirements Cages will be changed by the PI the day prior to surgery for mice undergoing treatment. Cages should not be changed for 48 hours post surgery unless flooded or otherwise uninhabitable. 8. Will hazardous materials be used in this study? No If yes, of what nature? Choose an item. 9. Special Procedures and/or Activities (check all that apply) None Survival Surgical Procedures Anesthetics or Tranquilizers Terminal Surgical Procedures If any of the following procedures or activities are checked, please provide justification Multiple Major Survival Surgical Procedures Prolonged Restraint

Monoclonal Antibody Productions by Ascites Neuromuscular Blocking Agents Use of Non Pharmaceutical Grade Drugs Laboratory Housing Death as an endpoint Justification: Use of Wire Bottom Cages for Rodents Food/Water Regulation Single Housed Rodents Deviation from Standard Environmental Cond. Deviation from Routine Sanitation Schedule Deviation from Normal Space Allocation 10. Funding and Funding Sources Animal Science Teaching 11. What veterinarian or veterinary service will provide care for your animals? Campus Vet Services (CVS) 12. Objectives and Significance Objectives In several recent studies, investigators have looked at the relationship between anesthesia, analgesia, and post operative behavior. One study observed behavior in 2 lines of inbred mice for 18 hours following anesthesia administration alone, anesthesia administration coupled with a surgical procedure, and anesthesia administration coupled with a surgical procedure plus the administration of an analgesic. They found that behaviors such as self grooming and locomotion were affected to varying degrees in each of the above groups when compared to a non treated control. Specifically, locomotion was reduced, particularly in the dark phase, and self grooming behavior was increased for the entire 18 hour period. Another article looked at similar groups to those in the above study for differences in post operative behavior between animals housed in a familiar versus an unfamiliar cage following treatment. This study looked only at females, and although they found difference between groups with and without surgery and with and without analgesia particularly in burrowing and self grooming behavior, there were no significant differences between mice house post operatively in a home cage versus a new cage. My hypothesis is that we would see a more profound difference in mice recovering in their home cage vs an unfamiliar cage if the mice tested were male. Male mice are more territorial than are females. They have been shown to rely more on odor cues for social stability. It is possible that in the male the stress of a novel environment would compound with the stress of a surgical procedure to produce changes in behavior not seen clearly in the female model. My objective is to observe C57BL/6 male mice in either a familiar or unfamiliar cage following surgery with and without analgesia to study whether the familiar cage will lessen signs of post operative distress and whether the use of an analgesic will negate these signs. I will use a ketamine + xylazine + acepromazine cocktail as my anesthesia, and Carprofen as my analgesic. The surgical procedure will be a sham vasectomy. I expect to see a decrease in self grooming in the mice in the familiar cages over those in the unfamiliar cages and an increase in locomotion in the mice in the familiar cages over those in the unfamiliar cages, both indicating decreased stress levels with the familiar cage. I may also see temporary changes in body

weight, body condition, and hydration between the groups. If body weight, condition, and hydration levels drop more in the unfamiliar cage than the familiar cage, it would be a further indication of decreased stress levels in a familiar cage post operatively. If these effects are seen only in the mice without analgesics on board; it may render the advantage of a familiar cage mute in terms of the welfare of the male mice. Significance The significance of this study is to determine whether the current method of post operative housing is in the best interest of male mice. We currently house them in clean cages with clean bedding to prevent the possibility of infection. Studies with females have shown that the risk of infection is limited; however, it failed to show a clear difference in welfare. Male mice may have a more profound response to the use of a familiar cage following surgery. This knowledge could improve post operative care for male mice. 13. The 3 R s Refinement, Replacement, and Reduction: a) Database Search for Alternatives (minimum 2 databases): Database name Years covered Keywords/Search Strategy Date(s) Biosis All Lab animals, mice, male mice, surgery, home Sept 2016 cage, novel environment, and behavior PubMed All Lab animals, mice, male mice, surgery, home Sept 2016 cage, novel environment, and behavior CAB All Lab animals, mice, male mice, surgery, home cage, novel environment, and behavior Sept 2016 13 b. Refinement (Considered Alternatives): We are looking at a complex set of interactions; environment, social cues, pain, stress, and drug interactions and their effect on behavior. There are no alternatives in the literature to create these interactions in vitro. I am choosing an injectable anesthesia over the inhalant anesthesia used in the previous studies because it is the anesthesia used in my facility and the results will be more significant to research being carried out in my facility. The use of analgesics following rodent surgery has been documented to decrease signs of pain and distress; however, mice, being prey animals, tend to hide signs of stress, and the use of analgesics may increase this tendency. It is therefore necessary to compare animals with and without analgesics to see whether the familiar cage is actually affecting the stress levels of the animal. Pain from a procedure of this nature is expected to be mild. Analgesics may be administered post operatively if signs of moderate to severe pain are observed. 13 c. Has this study been previously conducted? No 13 d. Replacement (Species Rationale): The goal of the study is to improve the post surgical treatment of mice. Mice are the best model to achieve this goal. There are no non animal models able to look at the complex actions between pain, perception, and

behavior. 13 e. Reduction (Animal Numbers Justification): The total number animals required for this study is N = 32 The number of groups required is 4: 2 groups comparing the effects of familiar vs non familiar post operative cages on signs of pain. This is my major variable to be tested. 2 groups comparing the effects of an analgesic on post operative signs of pain. This variable is needed because analgesia may mask the signs of stress making the differences harder to see. The number of animals per group required is n = 8: Similar studies, including the one on which I based my experimental design, have used 8 mice per group. Eight mice are expected to give a statistically significant result. N = 8 mice/group x 4 groups = 32 total mice 13 f. Study Groups and Numbers Table: (You may insert additional rows as needed. Let me know if you need assistance.) Group Species Number of Animals Procedures/Treatments 1 U/A Mice 8 Unfamiliar post op cage Anesthesia + surgery + pre op analgesia 2 U/O Mice 8 Unfamiliar post op cage Anesthesia + surgery (no pre op analgesia) 3 F/A Mice 8 Familiar post op cage Anesthesia + surgery + pre op analgesia 4 F/O Mice 8 Familiar post op cage Anesthesia + surgery (no pre op analgesia) Click Click here Click here here Click Click here Click here here Click Click here Click here here Click here Click here Click here 14 a. Procedure Details Describe the use of animals in your project from receipt of animals to study endpoint. Thirty two male C57Bl/6 mice, age 3 weeks, will be purchased from an approved vendor. Upon arrival mice will be housed 2 per cage and each cage randomly to an experimental group. Mice will be allowed a minimum of 3 weeks to acclimate and reach sexual maturity. Home cages will always be standard shoebox cages with a minimum of 60 square inches of floor space. Cages will be bedded with a mixture of Tekfresh, Paperchip, and Enviro dri. Cages will be changed weekly as per colony protocol; however, the PI will change cages the day before treatment so that bedding is relatively clean on the day of surgery.

On the day of treatment, mice are weighed within the first hour of lights on. Mice are transported to the surgical room in their home cage fitted with a microisolator top. Mice are removed from their home cage and placed in a clean induction cage. Each mouse will receive an SQ injection of saline (groups 0) or saline plus Carprofen (groups A). Each mouse will receive an IP injection of anesthesia and be returned to the induction cage. When mice have reached a surgical plane of anesthesia, a sham vasectomy will be performed as described below. Mice will be placed in a recovery cage and monitored until they are fully awake. Details of the sham vasectomy Each mouse will be anesthetized with a cocktail of ketamine + xylazine + acepromazine, IP. Surgical plane will be evaluated using the toe pinch reflex. Supplemental anesthesia will be given as needed until the mouse reaches a surgical plane. Eyes will be coated in a sterile ophthalmic lubricant to prevent dehydration and mice will be kept on a microwave gel pack to prevent hypothermia. SQ saline for dehydration is administered with or without Carprofen as described above. Campus policy for aseptic sterile technique will be followed. After scrubbing and draping the surgical site, a small incision (~1 cm) will be made through the skin, and then through the peritoneum, on the midline of the lower abdomen The testicles will be brought out through the incision and then returned intact to the abdomen using blunt forceps. The peritoneum will be closed with simple interrupted sutures using absorbable suture material. The skin is closed using a wound clip. Mice are allowed to recover from anesthesia in a cage containing paper toweling. The cage will have a temperature gradient achieved by placing one half of the cage on a microwave gel pack. Mice will be monitored every 15 minutes until sternally recumbent and clearly awake. Groups A: Analgesia Each mouse will receive 10 20 ml/kg of Carprofen in sterile saline, SQ Carprofen dose is 5 10 mg/kg Carprofen concentration is 50 mg/ml Carprofen is diluted 1:100 in sterile saline Groups 0: No Analgesia Each mouse will receive 10 20 ml/kg of sterile saline SQ Volume will be dose matched to the dose of Carprofen in groups A Groups U: Unfamiliar cage Upon recovery from anesthesia, each pair of mice is placed into a standard shoebox cage with clean bedding. A microisolator top is placed on the cage and the mice are transported back to their home room for monitoring. Groups F : Familiar cage Upon recovery from anesthesia, each pair of mice is placed back into their original cage so that the cage and bedding have familiar odors and pheromonal cues but no smells from induction or recovery of anesthesia. A microisolator top is placed on the cage and the mice are transported back to their home room for monitoring.

Post surgical monitoring and data collection: Once mice have returned to their home room, behavior is recorded using an infrared sensitive camera. Each cage has a camera and infrared light source mounted 1.5 meters above the cage. Video recording will continue for 48 hours post surgery. Videos will be analyzed for appearance and duration of each of the following behaviors: o Resting: sitting or lying flat; sleeping o Locomotion: Walking, running, jumping, or climbing o Nest building: manipulating bedding material into a nest o Self grooming: licking or wiping their own fur o Eating o Drinking In addition, every 12 hours, in the hour before lights out and the hour after lights on, each animal will be weighed and observed for surgical complications. Animals will be scored for clinical signs of pain such as body condition, coat condition, and wound healing. Animals indicating moderate pain will be given Carprofen analgesic, 5 10 mg/kg SQ, every 12 hours as needed. At the end of the 48 hour observation period, animals will either be euthanized. If animals can be transferred to another approved protocol, they will continue to be monitored daily until 7 to 10 days when wound clips are removed. 14 b. All Drugs and Compounds to be Administered to the Animals (except for euthanasia). Include anesthetics, analgesics, neuromuscular blocking agents, antibiotics, and/or experimental compounds. (You may insert additional rows as needed) Species Drug Dose Route When and how often will it be given? Mouse Ketamine 50 100 mg/kg IP One procedure, to effect Mouse Xylazine 5 15 mg/kg IP One procedure, to effect Mouse Acepromazine 0.5 2.0 mg/kg IP One procedure, to effect Mouse Carprofen 5 10 mg/kg SQ One dose prior to procedure in groups A Up to every 12 hours as needed in all groups post op. Click here. Click here. Click here. Click here. 14 c. Anesthesia monitoring (during the procedure) Depth of anesthesia will be determined using the pedal withdrawal reflex. If the initial dose fails to eliminate this reflex, supplemental doses of ketamine at ¼ to ½ of the initial dose will be administered to effect. During anesthesia, depth is monitored by observations of breathing patterns, jaw tone, and pedal withdrawal reflex. If breathing becomes shallow, jaw tone returns, or purposeful movement is observed, the animal will be tested for a pedal withdrawal reflex and additional anesthesia will be administered if necessary. 14 d. Post Anesthesia Monitoring (during the anesthesia recovery period)

Animals are observed every 15 minutes until sternal and clearly awake. 14 e. Surgery i) Location Building Room Surgeon (s) Cole B 124 Sandra Weisker ii) Post Surgical Monitoring (from after recovery of anesthesia until healed, typically 7 to 14 days) a) Please identify the parameters monitored, and the interval(s) and for what duration of monitoring Mice are monitored every 12 hours for 48 hours, and daily thereafter until wound clips are removed at 7 to 10 days post surgery. Mice are monitored for weight loss and/or body conformation, dehydration, changes in activity, wound healing, and signs of infection. b) When will analgesics be administered and at what interval(s)? Analgesics will be given to groups A prior to surgery. Analgesics will be given post surgically to any animals if signs of moderate pain are observed; i.e., listlessness, dehydration, signs of infection. c) If post operative analgesics cannot be given, please provide scientific justification. Analgesics are one of the variables being studied; therefore not all animals can receive them preoperatively. However, analgesics will be given post operatively if indicated. 15. Adverse Effects a) Describe all significant adverse effects that may be encountered during the study. Potential adverse effects include a stress from handling or injections, negative reaction to anesthesia or analgesia, pain or infection resulting from the sham vasectomy. b) Describe frequency for monitoring the well being of animals on the study and criteria for terminating/modifying the procedure(s) if adverse effects are observed. Animals will be monitored every 15 minutes during post anesthetic recovery and every 12 hours for 2 days post surgery. Animals exhibiting signs of moderate pain; i.e., listlessness, dehydration, weight loss of 10% or more, body condition of 2 or less, licking or scratching at the incision site. If signs of pain are consistently severe in groups O, we will drop groups 2 and 4 and administer Carprofen pre operatively to all treatment groups. c) How will the signs listed above be ameliorated or alleviated? All animals will be handled gently by trained personnel. Stress from handling and injection is expected to be momentary and mild. In groups A, all mice will receive 1 dose of Carprofen prior to surgery to alleviate pain. Groups 2 will not receive Carprofen prior to surgery as this is the variable for the group. However, animals that exhibit signs of moderate pain as described above, will receive Carprofen postsurgically. d) List the criteria to be used to determine when euthanasia is to be performed or the animal will be removed from the study. An animal with moderate to severe pain that does not respond to analgesics, animals with a weight loss of 20% or BC of 1 or less, or an animal with a post surgical infection will be euthanized. 16. Methods of Euthanasia Species Method Drug Dose Route Justification if physical method is being used. Mouse Asphyxiation CO2 To Effect inhalation Click here. Click here. Click here. Click here. Click

here. Click here. Click here. Click here. Click here. Click here. Click here. Click here. Click here. Click here. Click here. 17. Disposition of Animals