FOR OFFICE USE ONLY Amendments 1 2 3 4 5 6
PROTOCOL # Date
APPROVAL DATE #
UNIVERSITY OF COLORADO DENVER
STANDARDIZED PROTOCOL FOR PRODUCTION OF TRANSGENIC MICE BY DNA OR STEM CELL
INJECTION INTO MOUSE EMBRYOS
Note: Complete this protocol form only if you are having transgenic mice produced for you by the UCDenverTransgenic/Knockout Microinjection Core
Laboratory (TMCL). Any breeding or manipulations you plan to perform on the transgenic mice produced under this protocol must be described on a
separate complete protocol form.
Please complete all the sections which are presented in green
SECTION A - ADMINISTRATIVE INFORMATION
1) PRINCIPAL INVESTIGATOR (PI) NAME:
b. CONTACT INFORMATION FOR PI: TELEPHONE: MAIL BOX: EMAIL:
c. PROTOCOL AFFILIATIONS: UCHSC Webb Waring Barbara Davis
Cancer Center Denver Health
2) CAMPUS/FACILITY WHERE RESEARCH IS CONDUCTED:
ANSCHUTZ MEDICAL CAMPUS 9 and COLORADO OTHER:
a. Location of Laboratory
P15 BUILDING 0384 ROOM NUMBER (if using Transgenic Core)
b. Location Of Area Where Animal Procedures Are Performed
P15 BUILDING 0384 ROOM NUMBER (if using Transgenic Core)
3) PROTOCOL TITLE: Production of Transgenic Mice by DNA or Stem Cell Injection into Mouse Embryos
TYPE OF PROTOCOL: NEW PILOT REPLACEMENT FOR #:
4) PROTOCOL ASSOCIATES
a. INVOLVED IN ANIMAL PROCEDURES : (if using Transgenic Core) Joe Anderson; Abby Knight, Ling Wang,
Saiphone Webb (PQ forms on file)
b. CONTACT PERSON:
TELEPHONE: MAIL BOX: EMAIL:
5) TOTAL NUMBER OF EACH SPECIES IN APPLICATION: Mouse
6) GRANT/PROJECT TITLE:
a. FUNDING AGENCY/SOURCE: (Projects funded with internal support require departmental chair approval letter)
NIH NON PROFIT AGENCY (include name) SBIR TTO-POC(Tech Transfer Office
FOR PROFIT COMPANY (include name) OTHER (include name) MOU Attached
b. GRANT NUMBER/ID (If known): OGC ROUTING NUMBER:
IN REVIEW, DATE: AWARDED, DATE:
7) INCLUDED IN THE APPENDIX (select all that apply):
DEPARTMENTAL APPROVAL LETTER
PERSONNEL QUALIFICATION FORMS(please complete and SIGN a PQF for PI)
IACUC Application Form – Production of Transgenic Mice Revised 11/07 Page1 of 12
SECTION A2 – INDIVIDUAL INVESTIGATOR SPECIFIC PROJECT INFORMATION
1. What specific transgenes or embryonic stem cells will be established in these mice?
2. How many constructs will be used? (if more than one please adjust the total number of animals requested in
section A5 and B 5b)
3. What is the expected effect(s) of this gene on the animals?
4. Are there any known deleterious health effects associated with this genetic manipulation?
If so, provide details of the health effect and how they will be monitored and/or alleviated.
5. What is the potential benefit, of establishing these transgenic animals, to human medicine or biology?
6. Will the resulting transgenic mice be housed and/or used at UCHSC?
No. Where (at what institution) will the transgenic mice be housed/used?
Yes. What is the number of the protocol that describes the use of the transgenic mice?
7. At what age will tail tissue be sampled for genotyping?
21 days Other – Justification must be provided for taking tail tissue samples later than 21 days.
Note: The TMCL cannot accept DNA constructs or transgenic stem cells for injection if any resulting
animals would require housing in a facility at more secure than ABSL2 containment level. If
hazardous material use is planned please contact the IACUC Office for further advice prior to
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SECTION B - PROJECT DESCRIPTION
1. PROJECT GOALS IN NON-SCIENTIFIC (i.e. “LAY”) LANGUAGE
The Goal of this study is to produce mice which carry artificially induced genetic material (genes or gene
fragments) into the mouse genome. The incorporated DNA becomes part of the normal mouse genome and
allows the study and manipulation of specific genes in a living organism. (Individual Investigators must
provide specific information about their DNA construct in section A2)
2. NARRATIVE DESCRIPTION OF ANIMAL PROCEDURES
Part 1: Pronuclear DNA Injection. Fifteen 21-28 day old weanling female mice are superovulated (SO) for
each day of injections (max. four days or 60 mice per week). The embryo donors are injected with 2.5 to 5 IU
pregnant mares' serum gonadotropin (PMSG) each. 44 hours later, 2.5 to 5 IU chorionic gonadotropin (hCG)
is injected per donor and mated with males from the Transgenic Lab's standing colony of fertile FVB males.
Pseudopregnancy (PsPg) is stimulated by mating 15 mature female mice for each day of anticipated embryo
transfers by mating them with males from the Transgenic Laboratory's standing vasectomized male colony.
Those females with a vaginal plug the next morning will be the recipients for the embryo transfers. (This
usually produces 5-8 plugged females). The non-plugged females, along with those not used for embryo
transfer are placed back in the Transgenic/Knockout Laboratory core colony.)
The donors are sacrificed by C02 narcosis followed by cervical dislocation. The Transgenic laboratory
(0410B) is equipped with a C02 flow meter and cage top for euthanasia. Embryos are collected from the
oviducts by flushing with appropriate media and sorted.
Following the transgenic injections, the embryos are transferred to the oviducts of the PsPg recipient
females as described in appendix A. The transferred females will be housed in a VAF (viral antibody free)
colony until birth and weaning of the putative transgenic founder mice.
Tissue samples will be taken from the pups for analysis indicating successful integration of the transgene in
the mouse genome. One or more of the following procedures may be used:
Tail Biopsies for DNA Analysis:
Standard procedure for mice between 3-4 weeks of age, one tail sample is allowed without anesthesia or
ligature. (A waiver may be granted for tail snips for genotyping in mice between 21 and 28 days old if
no more than 1.5mm tissue is taken and if the line requires the extra days after the normal weaning
age for development. The need for this should be detailed by the individual investigator, in
consultation with the Core, when completing the application as it must have specific IACUC approval).
Very little bleeding occurs as the tail tip is cartilage and is not ossified. Older mice are anesthetized and the tail
is ligated with #1 silk thread about 1.0-1.5 mm from the tip. The tail is clipped with scissors immediately distal
to the ligature and the mouse placed back into its home cage to recover. Alternatively, the tail is cauterized
with a cautery pen.
Ear Biopsies for DNA Analysis: The mouse is restrained by hand and an ear punch biopsy is taken or a small
snip (2.0mm) of pinna periphery is excised with scissors. No analgesia is necessary for ear biopsies. There is
no bleeding and pain is momentary similar to ear tagging (or ear punching) for identification.
Blood samples: Blood samples are taken from Avertin-anaesthetized mice by one of two methods. For
amounts of 0.25 ml, retro orbital blood sampling is used. For lesser amounts (0.1 ml), the lateral tail vein will
be nicked and the blood collected.
The foster mother and non-transgenic pups will be sacrificed by CO2 narcosis followed by cervical
dislocation, and the positive pups transferred to the investigator's appropriate IACUC protocol for
breeding and experimental use.
Part 3: Blastocyst Injection. Ten female 21-28 day old mice are superovulated as in the previous section.
The embryo donors are sacrificed as in the earlier section at 84 hours post coitus and the uteri dissected out.
Embryos are collected from the uteri by flushing with the appropriate medium. Following injection of the stem
cells into 3.5 day blastocysts, embryos are transferred to the uteri of 2.5-day PsPg recipients. Postoperative
care and recovery is monitored as described in the previous section for oviductal transfers.
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Tissue samples are generally not required to assess integration of the transgene in this procedure. Usually a
"marker" such as coat color is used to indicate integration of the transgenic embryonic stem cell into the host
blastocyst and the resulting mouse will be a chimera with two different hair colors. Directed mating is primarily
used to assess germ line transmission of the transgene.
In both matings with the donor and VX males, the males are transferred back to their colony for repeat
utilization. At the end of their useful mating life span (ca. one year), the males are euthanized by CO2
Following weaning of the putative transgenic animals, they are transferred the investigator's IACUC protocol.
The foster mothers are subjected to embryo transfer surgery one time and euthanized by CO 2 narcosis
following weaning of their transgenic pups
3. STUDY END-POINT FOR ANIMALS
Animals will be euthanized:
after completion of the experimental protocol.
4. ANIMAL IDENTIFICATION:
a. Species: Mouse
b. Strain (for rodents): any
c. Sex: male/female
d. Age and/or weight range: 3-4 weeks; 6 weeks – 4 months
5. EXPERIMENTAL GROUPS AND NUMBERS:
Experimental/Control Group Number of Animals per Number of Repetitions Total number used
Names Group (per group) in group (sum per row)
15 4 60
10 4 40
PsPg females 6 4 24
b. Number of animals required for the entire study: 124 per construct. Please indicate the
number of constructs to be used and adjust the total number of animals accordingly .
Please also enter the total number of animals required in section A 5
c. How was the number of animals in each group determined?
Experience has shown that up to 20 donors are required to produce usable numbers fertile embryos
(150-250) for injections. 50%60% of the embryos generally survive the construct injection, resulting in
75 to 150 viable embryos. Five recipients are needed to receive 23-30 embryos each. Therefore, the
number of animals used represents the minimum number of animals required to generate an adequate
and usable number of transgenic founder mice. Historically, 0.5-1 % of embryos collected will result in
production of transgenic animals.
The number of transgenic lines produced by each injection series (injection-day) is widely variable,
being dependent on the number of positive transgenic animals born. Each transgenic animal will
become the founder animal of his own line. The number of positive animal produced per injectionday
can run from zero to an average of 20% of the animals born
Estimated number of requests for transgenic mouse production originated by investigators in the previous period
d. How was the number of repetitions for each group determined?
The numbers/group represent the number of animals used per injection-day. The number of
IACUC Application Form – Production of Transgenic Mice Revised 11/07 Page4 of 12
repetitions is the anticipated maximum number of injection-days per year.
6. EXPERIMENTAL AGENTS:
Frequency of Duration of Treatment
Experimental Agents Dose Route) Volume
PMSG (Embryo donors)
2.5-5 IU IP 0.1 ml Single Dose 48 hours
HCG Embryo donors
2.5-5 IU IP 0.1 ml Single Dose 12 hours
7. NEUROMUSCULAR BLOCKING AGENTS (I.E., PARALYTICS): N/A
8. HAZARDOUS MATERIAL USE: N/A
SECTION C - ANESTHESIA AND ANALGESIA
1. PRE-ANESTHETIC/ANALGESIC OR SEDATIVE DRUGS: N/A
2. ANESTHETIC DRUGS :
Drug Initial Dose Supplemental Route For Which Expected
Dose Procedure? Time Under
5% 3-3.5% Inhalation
Isoflurane Embryo Transfer 20 minutes
Xylazine/Ketamine 100mg/Kg 10 mg/Kg IP
Cocktail Ketamine/ Ketamine/
Laparotomies 20 minutes
5-10mg/Kg 0.5 mg/Kg
3. ANESTHESIA MONITORING (CHECK ALL THAT APPLY):
b. Response to toe/skin pinch.
4. FREQUENCY OF MONITORING:
b. Every 2-3 min.
5. POST PROCEDURAL ANALGESIC OR TRANQUILIZING DRUGS:
DRUG DOSE ROUTE FREQUENCY DURATION WHICH ANIMALS?
OF DOSING OF
Buprenorphine 0.05 - 1.0 IP or SQ Once every 12 48 hours Recipient females
mg/kg hours following embryo transfer
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SECTION D - POTENTIAL HEALTH CHANGES
1. POTENTIAL HEALTH CHANGES
a. Decreased food and water intake. j. Coma.
b. Abscesses. k. Labored breathing.
c. Dehydration. l. Hypothermia.
d. Infection m. Hyperthermia.
e. Malnutrition. n. Skin abnormalities.
f. General weakness. o. Paralysis.
g. Diarrhea. p. Ataxia.
h. Constipation or ileus. q. Urinary incontinence.
i. Seizures. r. Excessive urination.
s. Weight loss, specify as a % of total body weight:
t. Hyper/hypo- glycemia. Explain:
u. High incidence of carcinogenesis. Explain:
v. Behavioral changes. Explain: due to pregnancy and lactation
w. Other. Specify:
2. MONITORING FOR ANTICIPATED HEALTH CHANGES (Check all that apply):
Observed/Assessed Parameter Frequency of Assessment
a. Food/water consumption / (# / day, week, or month)
b. Body weight / (# / day, week, or month)
c. Pain or discomfort 1/day (# / day, week, or month)
d. Signs of infection 1/day (# / day, week, or month)
(redness, swelling, discharge or dehiscence)
e. Behavior, activity, or posture. 1/day (# / day, week, or month)
f. Blood glucose / (# / day, week, or month)
g. Tumor growth / (# / day, week, or month)
h. Other: / (# / day, week, or month)
i. Not applicable.
3. CRITERIA FOR PREMATURE REMOVAL FROM STUDY (Check all that apply):
a. Inability to eat or drink adequately.
b. Weight loss more than 15% of body weight.
c. Excessive generalized or localized pain and/or discomfort.
d. Uncontrollable infection, sepsis.
e. Markedly reduced response to stimuli or inability to ambulate properly.
f. Chronic hyper/hypo-glycemia; specify criteria:
g. Excessive tumor burden (>2cm length or >10% of total body weight)
Specify and justify alternative criteria:
h. Veterinarian’s discretion based upon humane issues. (required)
i. Other. Specify:
4. ANIMALS REMOVED FROM THE STUDY WILL BE:
SECTION E - RESTRAINT, DEPRIVATION, AND EUTHANASIA
2. SPECIAL HOUSING, CONDITIONING, DIET OR OTHER CONDITIONS :
a. None Apply.
b. Yes. The following apply (Check all that apply):
(1) Prolonged exposure to high or low temperatures.
(2) Prolonged exposure to non-standard humidity.
(3) Prolonged exposure to non-standard atmosphere.
(4) Non-standard housing.
(5) Prolonged exposure to non-standard light cycle.
(6) Water restriction for longer than 12 hours.
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(7) Food restriction for longer than 24 hours (simple stomach animals) or longer
than 48 hours (ruminants).
(8) Specialized or purified diet (include diet formulation datasheet in the appendix).
(9) Other. Specify:
c. If yes, justify the special conditions: Light cycle may be adjusted to 14 hours light :10 hours
dark to optimize breeding and reproductive success
3. METHOD OF EUTHANASIA (check all that apply):
f. Carbon dioxide exposure (followed by cervical dislocation).
SECTION F - ANIMAL ORDERING AND HOUSING INFORMATION
1. SOURCE (check one):
a. Purchased from an approved vendor.
2. ANIMAL HOUSING (Check all that apply):
b. UCHSC Center for Comparative Medicine
3. WILL ANIMALS WILL BE REMOVED FROM THE ANIMAL HOUSING FACILITY?
The IACUC strongly encourages researchers to utilize the animal procedural areas in the approved housing facilities. If this is not possible,
time animals spend out of an approved facility should be minimized and cannot be longer than 12 hrs.
SECTION G - RATIONALE FOR USE OF ANIMALS
AND APPROPRIATENESS OF THE SPECIES AND NUMBERS USED
1. LIVING ANIMALS ARE REQUIRED FOR THIS STUDY BECAUSE:
a. The complexity of the processes being studied cannot be duplicated or modeled in simpler
b. There is not enough information known about the processes being studied to design
c. Preclinical studies in living animals are necessary prior to human testing.
d. Other: there is no other way to obtain living embryos for transgenic integration
2. THIS SPECIES HAS BEEN SELECTED BECAUSE:
a. A large database exists, allowing comparisons with previous data.
b. The anatomy or physiology is uniquely suited to the study because: rapid generation time;
desirable embryo grafts
c. This is, phylogentically, the lowest species that provides adequate size, tissue, or anatomy
for the proposed study.
d. It provides a particularly good model for duplicating the human condition.
e. Previous studies using this species formed the background of this project.
f. It has the following unique features that make it the best available choice:
SECTION H - PAIN OR DISTRESS
1. ANIMALS IN THIS STUDY HAVE THE FOLLOWING MANIPULATIONS (Select the appropriate option):
b) Survival procedures, treatments or studies that could potentially cause pain or distress will be conducted.
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SECTION I - ALTERNATIVES TO POTENTIALLY PAINFUL OR DISTRESSING PROCEDURES
1. Please detail the specific procedures in this protocol which have the potential to cause pain or distress
(assume that any procedure which causes pain in a human has the potential to cause pain/distress in an animal)
Survival surgery to implant embryos in pseudopregnant mice.
2. METHODS USED TO DETERMINE THAT ALTERNATIVES TO POTENTIALLY PAINFUL OR
DISTRESSING PROCEDURES WERE NOT AVAILABLE:
a. Data base or sources consulted (check one or more of the following):
JOHNS HOPKINS CENTER FOR ALTERNATIVES TO ANIMAL TESTING
MEDLINE BIOSIS TOXLINE Index Medicus AGRICOLA Biol. Abstracts
Animal Welfare Information Center
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Office Use Only, Protocol #
Current Research Information Service
Other Search Engine (describe):
1) Date of data base search: 10/30/07
2) Years covered by search: 1966-2007
3) Key words (2-3) used in search: transgenic; mice; blastocyst
b. Scientific Meeting (name):
2) Discussion relevant to alternatives:
c. Scientific Journal (name):
2) Discussion relevant to alternatives:
d. Consultation with Expert (name):
1) Qualifications of expert:
2) Date of consultation:
3) Discussion relevant to alternatives:
e. Provide a narrative of the methods or logic used in determining that alternatives to actually or potentially
distressful or painful procedures are not available. Describe any alternative to painful/distressful
procedures found in the literature. If a bona fide alternative was identified but not used, the
narrative should explain why:
http://altweb.jhsph.edu/searchalt.htm; http://www.aphis.usda.gov/ac/policy/policy12.pdf ;
There are no means of producing transgene or gene knockout/knockin mice without performing embryo transfer using
surgical procedures. A living recipient is necessary to carry the embryos to term. No alternative methods were
discovered via Medline search.
3. EXPLAIN WHY DISTRESS OR PAIN CANNOT BE RELIEVED BY DRUGS
(skip this section unless Section H1c or H1d has been checked)
Buprenorphine 0.05 - 1.0 mg/kg is administered every 12 hours
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Office Use Only, Protocol #
SECTION J - REGULATORY ASSURANCES
1. ASSURANCE FROM THE PI OR COURSE DIRECTOR:
A. ASSURANCE FROM THE PI OR COURSE DIRECTOR
(check all that apply)
A. ALTERNATIVES TO PAINFUL PROCEDURES ASSURANCE: I certify that I have
considered alternatives to potentially painful/distressful procedures, as indicated in section H 2.
(check only if sectionH2 was required)
B. NON-DUPLICATIVE ASSURANCE: In planning this experiment, I have reviewed the relevant
literature (e.g., by a computer database literature search, use of comprehensive review articles,
or consultation with the Animal Welfare Information Center, etc.). Based on the available
literature, I certify that the activities involving animals described in this protocol do not
unnecessarily duplicate previous research. (required for research protocols)
C. RESEARCH STUDIES: I certify that the above statements are true. If this protocol is
associated with a grant application, I certify that this protocol is essentially the same as the
study found in grant application or program/project (please state funding agency, project title & award ID and
date of award) . The IACUC will be notified of any changes in the proposed project, or
personnel, relative to this application, prior to proceeding with any animal experimentation.
I will not proceed with animal experimentation until approval by the IACUC is granted. (required for
D. TEACHING EXERCISES: I certify that the information in this application is essentially the
same as contained in the course outline. The IACUC will be notified of any changes in the
proposed teaching exercises, or personnel, relative to this application, prior to proceeding with
any animal manipulation. I will not proceed with any animal manipulation until approval by the
IACUC is granted. (required for teaching protocols)
Principal Investigator Date
2. VETERINARY REVIEW AND ASSURANCE
UCHSC Veterinarian Date
3. IACUC CHAIR REVIEW AND ASSURANCE
UCHSC Chair Date
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Office Use Only, Protocol #
APPENDIX A - SURVIVAL SURGICAL PROCEDURES
PROTOCOL TITLE: Production of Transgenic Mice by DNA or Stem Cell Injection into Mouse Embryos
Are animals expected to survive surgery and regain consciousness?
NO (If NO, delete Appendix A from your application as it is not applicable)
YES (If YES, complete the remainder of Appendix A)
1. Specie: Mouse
2. Surgeon's name: Joe Anderson; Peter Koch
3. Anesthetist's name: Joe Anderson Peter Koch
4. Location where surgery will be done: P18 0410B
5. Describe the pre-op preparation of the animals.
a. Food restricted for hours
b. Water restricted for hours
c. Pre-op medications given:
Please describe other preparations in detail.
6. Minimal sterile techniques will include (check all that will be used):
a. Sterile instruments
b. Sterile gloves for surgeon
c. Scrubs or laboratory coat (rodents only)
d. Sterile surgical gown (not required for rodents or aquatics)
e. Surgical mask and surgical cap
f. Sterile operating area - table, drapes
g. Clipping of hair
h. Skin preparation with a sterilant such as betadine
i. Practices to maintain sterility of instruments during surgery
7. Describe the following surgical procedures:
a. Incision size and location: On flank ~ 1 cm caudal to rib cage and ~1.5 cm lateral to spine
b. Method of skin closure: Wound clip
c. General surgical details: The pseudopregnant (PsPg) female is anesthetized with isoflurane (5%
induction with 3-3.5 % maintenance using a veterinary vaporizer which is recirculation type which
actively scavenges minimizing potential exposure of personnel to isoflurane gas. Upon full
anesthesia (lack of toe/foot pinch response – 3-5 minutes), the mouse is placed on her right side
under a dissection microscope. The incision site is located approximately 1 cm caudal to the rib
cage and 1.5 cm lateral to the spine. The skin of the prepared surgical site is grasped with small
forceps and a small incision (0.5 cm) is made in the skin. The incision is moved around over the
underlying body wall until the ovarian fat pad beneath can be seen and a small incision is made at
that point, taking care to avoid cutting the vascular and nerve bundles in the muscle. The ovarian
fat pad is grasped with forceps and gently pulled through the incision, bringing with it the ovary,
oviducts and proximal portion of the uterus. Exterioization of the reproductive tract is kept to the
minimum necessary. A small bulldog clamp is attached to the fat pad if necessary to keep the
oviduct exteriorized. A small opening is blunt dissected in the bursal membrane, allowing access
to the ostium of the oviduct.
Fifteen to twenty washed embryos are loaded into a transfer pipet in sterile media. The pipet is
introduced into the ostium of the oviduct and the embryos gently expelled into the ampullary end of
Following successful transfer, the mouse's reproductive organs are gently placed back into the
body cavity. A single 5.0 vicryl suture is used to close the opening in the body wall if necessary.
The edges of the skin at the incision are opposed and closed with a wound clip. The wound clip
will be removed in <14 days.
The mouse is placed back into her cage to recover. Buprenorphine (0.05-1.0mg/kg) is
administered. If the animal is exhibiting sign of pain (hunching, stiffness) another dose will be
administered 12 hours from the initial injection. No further analgesia is typically required. Gentle
heat is applied by placing one end of the cage on a heating pad (aquamat) at low setting.
Following recovery of the female in about 30-60 minutes, she is transferred to the mouse room.
8. Provisions for Post Surgical Care of the Animals:
a. Who will be responsible for post operative care? Joe Andeerson; Peter Koch
b. Post-operative analgesics be given:
(1) Routinely to all animals
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Office Use Only, Protocol #
(2) As needed, determined by:
c. Where and how will animals be recovered from anesthesia? Room 0410C; animal will be
placed on a warming blanket (aquamat) to facilitate recovery.
d. How frequently will animals be monitored during anesthesia recovery? Every 15 minutes
e. What long term post-surgical care will be provided?
(1) Wound monitoring and/or care
(2) Provision of analgesics
(3) Fluid supplementation
(4) Special diet provisions
(5) Antibiotics (specify type, dose and route)
Please describe this care in detail.
9. Will any animal have more than one major survival surgical procedure?
YES. If yes, then:
a. Time interval between surgeries:
b. Scientific necessity or rationale for performing more than one procedure in the same
animal instead of using more animals:
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