UNIVERSITY OF COLORADO DENVER
STANDARD PROTOCOL FOR REDERIVING MOUSE COLONIES USING IN VITRO FERTILIZATION (IVF)
Please complete all the sections which are presented in green
SECTION A - ADMINISTRATIVE INFORMATION
1) PRINCIPAL INVESTIGATOR (PI) NAME:
a. DEPARTMENT:
b. CONTACT INFORMATION FOR PI: TELEPHONE:
c.
PROTOCOL AFFILIATIONS:
MAIL BOX:
UC Denver
Barbara Davis
Denver Health
Other
EMAIL:
Cancer Center
2) CAMPUS/FACILITY WHERE RESEARCH IS CONDUCTED:
ANSCHUTZ MEDICAL CAMPUS
OTHER:
a. Location of Laboratory
P15BUILDING 0384 ROOM NUMBER (if using Transgenic Core)
b. Location Of Area Where Animal Procedures Are Performed
P18 BUILDING 0410B ROOM NUMBER (if using Transgenic Core)
3) PROTOCOL TITLE: REDERIVING MOUSE COLONIES USING IN VITRO FERTILIZATION (IVF)
TYPE OF PROTOCOL:
NEW
PILOT
REPLACEMENT FOR #:
4) PROTOCOL ASSOCIATES
a. INVOLVED IN ANIMAL PROCEDURES : (if using Transgenic Core) Abby Zamora, Saiphone Webb, Peter Koch
(PQ forms on file)
b. CONTACT PERSON:
TELEPHONE:
MAIL BOX:
EMAIL:
5) TOTAL NUMBER OF EACH SPECIES IN APPLICATION: Mouse (See Section B.5. for Experimental Groups
and Numbers Tables) =
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)
b. GRANT NUMBER/ID (If known):
IN REVIEW, DATE:
c.
(Requires departmental chair approval letter)
MOU Attached
OGC ROUTING NUMBER:
AWARDED, DATE:
SPEEDTYPE NUMBER:
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 – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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SECTION A2 – INDIVIDUAL INVESTIGATOR SPECIFIC PROJECT INFORMATION
1. What specific strain(s) and/or transgenic line(s) will be expanded or rederived?
2. Why is the line(s) being expanded or rederived?
3. Describe any special features/characteristics of the line/strain, especially those that pertain to the animal’s
health, longevity and abnormal phenotype.
4. POTENTIAL CONTRIBUTION(S) TO BIOLOGY AND/OR HUMAN MEDICINE:
These procedures may help produce more rapid and accurate accumulations of experimental results
which may benefit development of new medicines and may contribute to elucidation of the biochemical
processes of living organisms. IVF can decrease the number of animals required to perform a successful
rederivation.
(Please add a statement of the potential contribution to biology and/or human medicine for your particular line)
5. Source of sperm to be used in the rederivation?
Cryopreserved (please provide collection/cryopreservation number)
Fresh (please provide protocol number)
6. Will the mice produced by the colony expansion/rederivation procedures be housed and/or used at UC Denver?
Yes. If yes, what is the number of the protocol that describes the use of the mice?
No. If no, where (at what institution) will the mice be housed/used?
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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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 rederive mouse strains (breeds) using either in vitro fertilization (IVF) from fresh or
cryopreserved sperm to generate embryos for surgical implantation (rederivation) for one of the following
reasons:
a) Produce pathogen-free animals from a compromised mouse line or colony.
b) Rapidly produce larger numbers of animals more quickly than normal mating. This process is termed
speed expansion or colony expansion.
c) Colony rescue
Mouse pathogens can give rise to a variety of problems in the experimental colony. Immunologic studies may be
influenced by circulating antibodies. Animal fertility may be adversely affected threatening loss of valuable
transgenic and other research colonies. Rederivation is an effective technique to ’clean up’ or remove the
majority of mouse pathogens and ectoparasites from a mouse colony. Since most pathogens are spread via
fecal or oral contact, surgical transfer of fertile embryos produced by natural mating or through in vitro fertilization
(IVF) into SPF foster mothers can eliminate the majority of pathogens.
Colony Speed Expansion (CSE) is a valuable tool as the name implies, to expand the size of an existing colony
within a shorter time that could be achieved through normal mating. This is of particular importance when
experimental groups of age-matched animal must be obtained and used for a specific experimental protocol.
Similarly, a colony is not reproducing effectively (diminished fertility) and is at risk of being lost, can be ‘rescued’
using the techniques outlined above.
(Individual Investigators must provide specific information about their strain or line in section A2)
2. NARRATIVE DESCRIPTION OF ANIMAL PROCEDURES:
A. In vitro fertilization (IVF) using freshly collected sperm or using thawed cryopreserved sperm.
a. Oocyte production and collections.
i. Ten 3-4 week old female mice (oocyte donors) will be ordered for the investigator from the
appropriate vendor. The background strain is the same as that of the sperm donors
provided by the investigator. Upon arrival, the oocyte donor females are housed in the
Transgenic Core (TGTC ) and superovulated as follows: Following an hCG injection
(human chorionic gonadotropins; 2.5-5 IU; 0.1 ml; IP) at 12 noon on the third day, the
donors are placed back into their cage until oocyte harvest the next morning (day 4). The
donors are not mated.
ii. The next morning, (day 4) the unfertilized oocytes are collected from the unmated donors.
The donors are euthanized by CO2 exposure followed by cervical dislocation and the
oviducts removed. The oocytes are harvested from the oviducts and the adhering cumulus
cell removed. The cleaned oocytes are washed through three changes of embryo media
and transferred to embryo culture media. They are kept in the incubator until use.
b. Sperm Collection:
i. Fresh sperm - The investigator provides two young breeder males as sperm donors.
These males are transferred to the Transgenic Lab where the males are euthanized by
CO2 exposure followed by cervical dislocation and the testes and epididymi are quickly
removed. The organs are transferred through three to five washes of culture media or
sterile saline and placed into fertilization medium where the sperm are harvested. The
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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sperm is removed by cutting the epididymus and testis into small pieces. Sperm will swim
out of the organ pieces where they will be collected, washed and transferred to fertilization
medium.
ii. Cryopreserved sperm - may be used if desired and available. An aliquot of the
cryopreserved sperm is thawed and transferred into fertilization medium
c. In vitro Fertilization:
i. The sperm obtained from either source is used to fertilize non-fertilized mouse oocytes.
The oocytes are added to small drops to the sperm fertilization media where fertilization
takes place over 2.5-3 hr. The fertilized oocytes are collected and transferred to culture
medium overnight. The next morning, successfully fertilized oocytes are now 2-cell
embryos and can be surgically transferred into pseudopregnant females. (Surgical details
provided in Appendix A.)
Embryo Collection:
To generate a transgenic strain of mouse, the TGTC will produce mice by surgical embryo transfer into
pseudopregnant (PsPg) recipient females.
A. Pseudopregnancy - (PsPg) is stimulated by mating 10 mature female mice, for each day of anticipated
embryo transfers, 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 4-6 plugged females. The non-plugged females along with those not used for embryo transfer are
placed back in the TGTC's colony.)
B. Embryo Transfer - In both cases (IVF using fresh sperm or from cryopreserved sperm) the embryos
produced by IVF 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 mice.
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
(~0.2 cm) is allowed without anesthesia or ligature. Very little bleeding occurs as the tail tip is cartilage and
is not ossified. Older mice (> 21 days) are anesthetized and the tail is ligated with #1 silk thread about 1.01.5 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. The tail
biopsy samples are delivered to the investigator for screening.
After screening, the investigator will provide results to the TGTC. At this time, the foster mother and
non-transgenic pups will be sacrificed by CO 2 narcosis (followed by cervical dislocation) and the
positive pups transferred to the investigator's appropriate use IACUC protocol.
3.
STUDY END-POINT FOR ANIMALS (Select all that apply)
a. Animals will not be euthanized at study completion. Animals (unused PsPg females)
b. Animals will be euthanized: oocyte donors/sperm donors
c. without significant treatments or experimental procedures. Breeder males. If used
d. after defined period of time: oocytes donors 3-4 weeks; recipients 21 days post partum; and recipient
post weaning
4.
ANIMAL IDENTIFICATION:
a.
Species: Mouse
b.
Strain (for rodents): as in section A 2.1
c.
Sex: male/female
d.
Age and/or weight range: 3 weeks – 1 year
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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5.
EXPERIMENTAL GROUPS AND NUMBERS: (Choose one table) Stress Category D
a. Tables:
A1 Freshly Collected Sperm for IVF ONLY
Experimental/Control Group Number of Animals per
Names
Group
Number of Repetitions
(per group)
Total number used
in group (sum per row)
Oocyte donor females
10
2
20
Sperm donor males
2
2
4
Recipient females
5
2
10
Number of animals required for the entire study: 34 x # lines = Total number________
A2 Cryopreserved Sperm for IVF only
Experimental/Control Group
Number of
Names
Group
b.
Number of Repetitions
(per group)
Total number used
in group (sum per row)
Oocyte donor females
Up to 10
2
20
Recipient females
5
2
10
Number of animals required for the entire study: 30 x # lines = Total number________
c.
How was the number of animals in each group determined?
Each group represents the number of animals required to produce sufficient oocytes necessary for
successful regeneration. See following notes:
i. Oocyte donors will be ordered from vendors. The exact source of the donors depends upon the
requirements of the Investigator's transgenic mouse line and strain.
ii. The estimated number of recipients represents the minimal number of animals necessary to support
successful regeneration.
d.
How was the number of repetitions for each group determined?
Occasionally the donor females don’t produce sufficient numbers of oocytes or the sperm donors provide
inadequate numbers of quality of sperm. In either case, the procedure might have to be repeated.
6. EXPERIMENTAL AGENTS:
Experimental Agents
Frequency of Duration of Treatment
Dosing
Effect
Dose
Route)
Volume
PMSG
5 IU
IP
0.1 ml
Single Dose
48 hours
HCG
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: PMSG and HCG
SECTION C - ANESTHESIA AND ANALGESIA
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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1.
PRE-ANESTHETIC/ANALGESIC OR SEDATIVE DRUGS:
Drug
Banamine
2.
Dose
Route)
Volume
2.5 mg/Kg
SC
0.05 cc
Initial dose
Maintenance
dose
Route
Isoflurane
5%
3-3.5%
Inhalation via
Vaporizer
Procedure
Time Under
Anesthesia
Mouse
Laparotomies
3-5 min
Response to toe/skin pinch.
FREQUENCY OF MONITORING:
b.
5.
12 hours
ANESTHESIA MONITORING (CHECK ALL THAT APPLY):
b.
4.
Single Dose
ANESTHETIC DRUGS :
Primary
anesthetic
3.
Frequency of Duration of Treatment
Dosing
Effect
Every 2-3 min.
POST PROCEDURAL ANALGESIC OR TRANQUILIZING DRUGS:
DRUG
Banamine Analgesic
DOSE
ROUTE
2.5 mg/Kg
SC
FREQUENCY
OF DOSING
once every 12
hours post
surgery
DURATION
OF
TREATMENT
48 - 72 hrs
WHICH ANIMALS?
Embryo Transfer
surgery
SECTION D - POTENTIAL HEALTH CHANGES
1. POTENTIAL HEALTH CHANGES
a. Decreased food and water intake.
b. Abscesses.
c. Dehydration.
d. Infection
e. Malnutrition.
f.
General weakness.
g. Diarrhea.
h. Constipation or ileus.
i.
Seizures.
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:
w. Other. Specify:
x. None
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
j.
k.
l.
m.
n.
o.
p.
q.
r.
Coma.
Labored breathing.
Hypothermia.
Hyperthermia.
Skin abnormalities.
Paralysis.
Ataxia.
Urinary incontinence.
Excessive urination.
(Revised11/10)
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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
(# / 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:
a.
Euthanized.
SECTION E - RESTRAINT, DEPRIVATION, AND EUTHANASIA
1. RESTRAINT:
a.
Routine.
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.
(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:
3. METHOD OF EUTHANASIA (check all that apply):
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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f.
Carbon dioxide exposure (followed by cervical dislocation).
SECTION F - ANIMAL ORDERING AND HOUSING INFORMATION
1. SOURCE (check all that apply):
a.
Purchased from an approved vendor.
f.
Other. Specific details on strains and protocol source in section A2
2. ANIMAL HOUSING (Check all that apply):
UC Denver Center for Comparative Medicine
3.
a.
b.
WILL ANIMALS BE REMOVED FROM THE ANIMAL HOUSING FACILITY?
Yes
No.
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 systems.
b. There is not enough information known about the processes being studied to design
nonliving models.
c. Preclinical studies in living animals are necessary prior to human testing.
d. Other:
This is the only method available to perform IVF to produce 2-cell embryo for surgical
implantation and birth of offspring.
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:
3.
POTENTIAL CONTRIBUTION(S) TO BIOLOGY AND/OR HUMAN MEDICINE:
These procedures may help produce more rapid and accurate accumulations of experimental results which
may benefit development of new medicines and may contribute to elucidation of the biochemical processes of
living organisms. IVF can decrease the number of animals required to perform a successful rederivation.
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.
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)
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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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 http://caat.jhsph.edu/
MEDLINE
BIOSIS
TOXLINE
Index Medicus
AGRICOLA
Biol. Abstracts
Animal Welfare Information Center
http://awic.nal.usda.gov/nal_display/index.php?info_center=3&tax_level=1&tax_subject=183
Current Research Information Service
Other Search Engine (describe): PubMed
1) Date of data base search: 08/10/10
2) Years covered by search: 1985-2010
3) Key words (2-3) used in search: rederivation, IVF, mouse embryo
b. Scientific Meeting (name):
1) Date:
2) Discussion relevant to alternatives:
c. Scientific Journal (name):
1) Reference:
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 ;
http://www.nc3rs.org.uk/
Embryo transfer is a very successful method for generation of mice efficiently where typical breeding
schemes have been unsuccessful for a variety of causes: low libido, advanced age of the animals, genetic
background, or strain. Live animals must be used: 1) to obtain viable embryos 2) as recipients of those embryos
following collection to produce viable offspring. This method saves time, per diem costs and the total number of
animals used to achieve the same results from regular breeding.
3.
EXPLAIN WHY DISTRESS OR PAIN CANNOT BE RELIEVED BY DRUGS
Anesthetics and analgesics are used to relieve pain or distress.
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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SECTION J - REGULATORY ASSURANCES
Signature Page
PI:
PROTOCOL TITLE: STANDARD PROTOCOL FOR REDERIVING MOUSE COLONIES USING IN VITRO
FERTILIZATION (IVF)
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 (see Section A for funding information). 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
research protocols)
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
UC Denver Veterinarian
Date
3. IACUC CHAIR REVIEW AND ASSURANCE
UC Denver Chair
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
Date
(Revised11/10)
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APPENDIX A - SURVIVAL SURGICAL PROCEDURES
PI:
PROTOCOL TITLE: REDERIVATION OF MOUSE COLONIES PRODUCED BY IN VITRO FERTILIZATION OF
OOCYTES
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.
2.
3.
4.
5.
Species: Mouse
Surgeon's name: Abby Zamora; Saiphone Webb
Anesthetist's name: Abby Zamora; Saiphone Webb
Location where surgery will be done: P15 0384
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
7. Surgical site preparation
g.
h.
i.
8.
Hair removal
Skin preparation with a sterilant such as betadine
Practices to maintain sterility of instruments during surgery
Describe the following surgical procedures:
a. Incision size and location: ~0.5 cm incision. On flank ~ 1 cm caudal to rib cage and ~1.5 cm
lateral to spine
b. Method of closure: body wall: 5.0 absorbable suture (Vicryl or equivalent), skin: wound clip
c. General surgical details: The pseudopregnant (PsPg) female is anesthetized with isoflurane gas
anesthetic using a veterinary vaprorizer for delivery and Banamine analgesic administered sub
cutaneously (SC). 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 skin is moved around over the underlying body wall until the ovarian fat pad beneath
can be seen through the opening and a small incision is made at that point, taking care to avoid
cutting the vascular and nerve bundles in the fascia and muscle. The ovarian fat pad is grasped
with iris forceps and gently pulled through the incision, bringing with it the ovary, oviducts and
proximal portion of the uterus. Exteriorization 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. Alternately, a small puncture is made in the oviduct just distal to the
ampulla.
Twenty five to thirty washed embryos are loaded into a transfer pipet in sterile media. The pipet is
introduced through the hole in the bursa and threaded into the ostium of the oviduct or the
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
(Revised11/10)
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puncture in the oviduct. The embryos gently expelled into the oviduct through the opening at either
site.
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. 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 if necessary (the mouse generally removes the clip by herself).
Following surgery the mouse is placed back into her cage to recover. Gentle heat may applied by
placing one end of the cage on a heating pad (aquamat) at low setting. Following recovery of the
female in about 3-5 minutes, she is transferred to the mouse room. Another dose of Banamine
will be administered 12 hours from the initial injection and again at 24, 36 and 48 hours
c. Provisions for Post Surgical Care of the Animals:
a. Who will be responsible for post operative care? Abby Zamora; Saiphone Webb
b. Post-operative analgesics be given:
(1) Routinely to all animals: immediately prior to surgery and ~12, 24, 36 and 48 hr later.
(2) As needed, determined by:
c. Where and how will animals be recovered from anesthesia? Room P15 0384; animal will
be placed back into her cage. A warming pad (Aquamat) is available if necessary.
d. How frequently will animals be monitored during anesthesia recovery? Every 3-5
minutes
e. What long term post-surgical care will be provided? Observation only
(1) Wound monitoring and/or care daily observation
(2) Provision of analgesics
(3) Fluid supplementation
(4) Special diet provisions
(5) Antibiotics (specify type, dose and route)
Please describe this care in detail.
d. Will any animal have more than one major survival surgical procedure?
NO
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:
IACUC Application Form – Rederiving Mouse Colonies Using In Vitro Fertilization (IVF)
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