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PROTOCOL FOR ANIMAL USE AND CARE
1. Contacts
Primary Investigator
Name
Email
Dept
Telephone
After hours
2.
Title:
3.
Protocol Type:
Sandra Weisker
sweisker@ucdavis.edu
Animal Science
530-752-3642
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Alternate contact
Name
Email
Dept
Telephone
After hours
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Familiar vs Unfamiliar Cages for Post-operative Housing of Male Mice
Research
4. Species
Common Names
Mice
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Total number for study
32
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Source of animals
Approved vendor
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5. Procedures
The mice on this study will be used to look at the effect of a familiar vs unfamiliar cage for post–
operative housing. Mice will be pair housed.
All mice will undergo a sham vasectomy under injectable anesthesia.
Mice in groups A (analgesic) will receive a pre-operative analgesic; mice in groups 0 (no analgesic) will
not. Mice in either group will be evaluated for the need of a post-operative analgesic.
Mice in groups U (unfamiliar) will be housed in a new cage post-surgically. Mice in groups F (familiar)
will be housed in a familiar cage post-surgically.
Indication of pain or surgical complications, i.e., listlessness, dehydration, infection, should be reported
to the investigator immediately.
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 ☐
☒Anesthetics or Tranquilizers
☐Prolonged Restraint
☒Survival Surgical Procedures
☐Food/Water Regulation
☐Terminal Surgical Procedures
☐Death as an Endpoint
☐Neuromuscular Blocking Agents
☐Single housing of social species
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. Literature search for alternatives and unnecessary duplication:
a) Databases (minimum 2 databases):
Database name
Years covered Keywords/Search Strategy
Biosis
All
mice AND surgery; surgery AND “home cage
behavior” AND “male mice”; “male mice” AND
novel environment” AND stress ; surgery AND
alternatives AND “lab animals”
PubMed
All
mice AND surgery; surgery AND “home cage
behavior” AND “male mice”; “male mice” AND
novel environment” AND stress ; surgery AND
alternatives AND “lab animals”
CAB
All
mice AND surgery; surgery AND “home cage
behavior” AND “male mice”; “male mice” AND
novel environment” AND stress ; surgery AND
alternatives AND “lab animals”
Date(s)
Sept 2015
Sept 2015
Sept 2015
13 b. Results of the Literature Search:
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. 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.
13 e. 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: (if you need additional rows, let me know)
Group
Species
Number of
Procedures/Treatments
Animals
U-A
Mice
8
Unfamiliar post-op cage
Anesthesia + surgery + analgesia
U-0
Mice
8
Unfamiliar post-op cage
Anesthesia + surgery (no pre-op analgesia)
F-A
Mice
8
Familiar post-op cage
Anesthesia + surgery + analgesia
F-0
Mice
8
Familiar post-op cage
Anesthesia + surgery (no pre-op analgesia)
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14 a. Summary of Procedure
Describe the use of animals in your project from receipt of animals to final desposition.
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.
 At the end of the 48 hour observation period, animals will either be euthanized or will continue
to be monitored daily until 7 to 10 days when wound clips are removed.
14 b. Drugs to be used (except for euthanasia). Include anesthetics, analgesics, neuromuscular blocking
agents, antibiotics, and/or experimental compounds. (If you need additional rows, let me know)
When and how often will it be given?
Species
Drug
Dose
Route
Mouse
ketamine
50-100
IP
One procedure, to effect
mg/kg
Mouse
xylazine
5-15
IP
One procedure, to effect
mg/kg
Mouse
acepromazine
0.5-2
IP
One procedure, to effect
mg/kg
Mouse
Carprofen
5-10
SQ
One dose prior to the
mg/kg
procedure, as needed post-op
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14 c. Anesthesia monitoring
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
Animals are observed every 15 minutes until sternal and clearly awake.
14 e. Surgery
i) Location
Building
Cole B
ii) Post-Surgical Monitoring
a)
Room
124
Surgeon (s)
Sandra Weisker
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., dehydration, listlessness, 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, will be evaluated for
the administration of analgesics or euthanasia.
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 (If you need additional rows, let me know)
Species
Method
Drug
Dose
Route
Mouse
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Asphyxiation
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CO2
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To effect
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Justification if physical
method is being used.
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17. Disposition of Animals
Mice may go to another approved protocol or be euthanized at the end of the study.