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Rev 11-2011

MaineHealth

Institutional Animal Care & Use Committee

Application of Intent to Use Vertebrate Animals in Research

Animal Facility Training Required Prior to Starting Any Project(Refer to the Animal Facility's SOPs located on the share drive)

Any Animals Arriving From Non-Approved Vendors Are Rederived(Refer to SOP: 99-001C Animal Procurement and SOP: 99-005C Quarantine)

Questions: MMCRI - Research Compliance Office: 207-396-8195

The Maine Medical Center Institutional Animal Care & Use Committee (IACUC) is required by law as stated in the Animal Welfare Act and the 1989 amendments to this Act to review and approve all activities or proposed activities involving the use of vertebrate animals.

Maine Medical Center provides assurance to all PHS agencies that the use of vertebrate animals in research or instruction has been approved by the Institutional Animal Care & Use Committee and that all requirements of the governing agencies have been met.

Unapproved or disapproved procedures may not be carried out under any circumstance. Failure to observe this rule could result in removal of all access to animals by the investigator's laboratory. Maine Medical Center's Public Health Service Assurance No. is: A3862-01.

REVISED FORM – 11/2011

Rev 11-2011Maine Medical Center Institutional Animal Care & Use Committee

IACUC New Submission/Triennial/Amendment Form IACUC #: Old # if

Triennial:Today’sDate: Species:

Principal Investigator: Email: Phone:

Work Address if not MMCRI:

Title of Protocol:

Research: Teaching/Educational: Student Project or Fellowship:

Grant & Funding Section:#1 Title of Grant:

Funding Agency: Grant or Contract #: Cost Center #:

#2 Title of Grant:


#3 Title of Grant:


By checking box to right, PI confirms grant matches PHS funded protocol.(Applies to new grant submissions and continuing reviews)

Initials Here:

Attach the grant page that contains grant number information andthe grant page that explains animal experiments. (NIH Requirement)

Protocol Submission Section:

Type of Submission New Protocol: Triennial: Amendment:

Complete entire form if you are submitting either a New Submission or Triennial

Amendment Section:Adding Additional Strains? List Each Strain Below Additional Animals/Staff:

How many more animals are added to the protocol because of this amendment?

#

Add Procedure(s) Below: Name(s) of Personnel being Added:

Name(s) of Personnel to Remove:

Notes:

SECTION: II INVESTIGATOR ASSURANCE

Rev 11-2011

The undersigned Principal Investigator for the research project described on the preceding pages document, hereby gives assurance that he/she will comply fully with Federal Law as set forth in the Animal Welfare Act.Further, if the protocol described is approved by the MMC IACUC, the investigator will:

A. Adhere strictly to the protocol as described herein;

B. Seek consent of the IACUC for any change(s) in the protocol before being implemented, and notify theIACUC of any change(s) in the location(s) where the animal s research is carried out;

C. Ensure all personnel listed are qualified and competent to carry out the procedures described and understand and accept the strict compliance with all laws pertaining to any type of animal

experimentation involved in this study;

D. Assure activities involving vertebrate animals in this application do not necessarily duplicate previous experiments;

E. Agree to emergency veterinary care by the appointed MMC veterinarian, if there is evidence of pain or illness;

F. Utilize the alternative to animal use information to prevent unnecessary animal use. Ensure that all animal program SOPs are followed Ensure protocols involving hazardous material or infectious agents are managed by trained

staffed and appropriate safeguards are in use Ensure appropriate measures are in place and are being given for minimum pain and distress,

and humane treatment of animals. Inform Animal Care Staff when you will not be available, (vacation, and conference).

Therefore appropriate animal care can be given. Check on animals at least once per week.

There have been no changes made to this protocol since my last approval.

Initials Here:

_____________________________________________________Signature of Principal Investigator Date

This Assurance must be signed at time of submission.

_____________________________________________________Signature of Department Chief Date

Rev 11-2011SECTION: III NON-TECHNICAL LAY SUMMARY OF PROJECT

1. PURPOSE/GOALS:

MANDATORY: Include publications with your triennial submission.

The lay summary MUST be written in clear, simple language which describes the purpose and potential benefits of the project, as well as the animal species to be used and their importance in the project. The summary should be an overview of the goals of the project, and should be in terms that non-scientists can understand. Please limit the length of this summary to one page.

** MUST BE IN CLEAR, SIMPLE LANGUAGE**

Do activities unnecessarily duplicate previous experiments or activities? Yes: No:If yes, describe and justify need for duplication here:

Please Note: SOP 10-031A Protocol Submission and Renewal Process, Sec. 3.4.4 allows the PI three weeks to respond to an IACUC request for changes. If no response is received, the submission will be closed.

Rev 11-2011

SECTION: IV IBC/IRB/ESCRO REVIEW - (NEW)

If your protocol requires review from the Institutional Biosafety Committee (IBC), the Institutional Review Board (IRB), or the Embryonic Stem Cell Research Oversight Committee (ESCRO), the IACUC will not issue approval until other committees have done so first. However, the other review(s) may take place concurrently.

In order to determine if you need IBC, IRB, or ESCRO approval, please read below what each committee has authority over and whether or not this protocol should receive review by any of them.

IBC: o This standing committee has oversight of recombinant DNA (rDNA) research, and the use

of biohazardous materials for research. All activities proposed to involve the use of recombinant DNA and/or biohazardous materials must first seek approval from the Institutional Biosafety Committee.

IRB:o This standing committee is responsible for protecting the rights and welfare of people who are

subjects in research activities. All research projects involving human tissue or blood draws are required to be reviewed and approved by the MMC IRB prior to implementation.

ESCRO:o The Embryonic Stem Cell Oversight committee ensures all federal and state regulations

governing the conduct of human embryonic stem cell research are met and that all Human Embryonic Stem cell research is conducted in accordance with the general principles expressed in the NAS Guidelines for Human Embryonic Stem Cell Research (Guidelines), with the Policies and Procedures adopted by the Maine Medical Center hESCRO Committee, with other relevant Maine Medical Center research policies.

Please make a selection in the table below:

Select One From Below ⊠ This protocol is currently in reviewwith the following committee: ⊠

I do not require approval from any of the committees listed to the right: IBC

IRB

*I have already received approval from one of the committees listed to the right:

ESCRO

*If you have already received approval from one of the committees, please attach a copy of the approval letter to this protocol.

http://www.mmcri.org/home/webSubContent.php?subCatID=30&catID=2&headType=escro&catLevel=subCat

http://www.mmcri.org/home/webSubContent.php?subCatID=4&catID=2&headType=irb&catLevel=subCat

http://www.mmcri.org/home/webSubContent.php?subCatID=6&catID=2&headType=ibc&catLevel=subCat

http://www.nap.edu/openbook.php?isbn=0309096537

http://www.nap.edu/openbook.php?isbn=0309096537

Rev 11-2011SECTION: V ANIMAL INFORMATION

Please fill in #1 thru #5 below for each species. If you are using more than one species, you will have to copy and paste Section V for each successive species used. Begin your next species after #5 of this section.

NEW! " Alternative Search Training Module" and "Instructions for Alternatives Literature Searching"

1. JUSTIFICATION FOR THE SPECIES: 2.

Alternatives to the use of vertebrate animals must be considered. Have you considered alternatives? YES NO

Please provide an electronic copy of your literature search and the keywords that were used, per species. (Refer to SOP: 99-011A Alternatives to Animal Use Policy)

Attach your search results to this document.Send in MS Word Format or NotePad Formats Only (Website links are not accepted.)

Date search was conducted:Years Covered:(Ex. 1999-2010)Databases Used:Keywords used:

2. NUMBER OF ANIMALS NEEDED

Breeding Colony:1. Strain: # of Breeders:2. Strain: # of Breeders:3. Strain: # of Breeders:4. Strain: # of Breeders:5. Strain: # of Breeders:

Experiment Colony:1. Strain: Experiment 1:2. Strain: Experiment 2:3. Strain: Experiment 3:4. Strain: Experiment 4:5. Strain: Experiment 5:

Tri Breeding (Harem Breeding): The Guide has new regulations regarding tri breeding. You will need to be exempted by the IACUC in order to continue this procedure. Please explain why you will need to tri-breed:

http://www.mmcri.org/home/webSubContent.php?list=webcontentlive&id=45&catID=2&subCatID=5

Rev 11-20113. JUSTIFICATION FOR THE NUMBER OF ANIMALS:

To ensure all animals within MMCRI Animal Facility are covered under an approved protocol for proper care and use:

All animal species and strains to be used must be listed on the protocol Anyone producing animals with this intention in mind must first add these strains to their protocols, as

an amendment, and gain IACUC approval prior to any production of transgenic mice for this purpose. Exemptions will be given in the case if the external user/company directly requests transgenic mouse

production service to the Mouse Transgenic Core Facility. PI’s who wish to transfer a strain to another MMCRI investigator, refer to SOP: 99-004C, The

Transferring of Unused Animals to a Different Protocol. Each protocol utilizing animals has an IACUC approved animal allotment and justified amount. Upon reaching 80% usage of the allotted approved amount of animals, the PI will be notified by the

Animal Facility. The PI has the option to submit an amendment for IACUC approval to add additional animals to the protocol. If the PI has chosen not to amend the protocol, the existing breeders will be separated and no further breeding and animal orders will be allowed. (Pups are added to the census at the time of weaning.)

Reportable OLAW Incident: If the PI exceeds the number allotted without amending the protocol, and it is funded by an NIH grant, this is considered a reportable OLAW incident.

*Verify the Number of animals needed per procedure and number of animals for maintenance of colony. (Include USDA Procedure Category B, C, D, E)

Please retain the statistical tests, strategy, or method for evaluating data for your study.*Verification: (Cell will expand)

FLOW CHARTS ARE MANDATORY FOR ALL EXPERIMENT DESIGNS

To View an Experimental Design Samples, Click Here4. EXPERIMENT DESIGN:

Experiment Design # 1:

Strain & Age n =

DAY PROCEDURE CATEGORY

1

Day #/End Point Euthanize

Rev 11-2011



Strain & Age n =


1




Strain & Age n =


1




Strain & Age n =


1


Rev 11-20115. STRAIN/LINES:

#1 Fill in all informationSpecies:Strain/line:Phenotype/special care:Supplier:If to be created @ MMCRI, IBC #:Name of construct:Function of the gene product:

#2 Species:Strain/line:Phenotype/special care:Supplier:If to be created @ MMCRI, IBC #:Name of construct:Function of the gene product:

#3Species:Strain/line:Phenotype/special care:Supplier:If to be created @ MMCRI, IBC #:Name of construct:Function of the gene product:



Rev 11-2011

SECTION: VI USDA STUDY PAIN CATEGORIES

The definition of pain states, "Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain and distress in other animals." In defining animal stress, we must properly distinguish between "stress" and "distress." Distress is defined as "an aversive state in which an animal is unable to adapt completely to stressors and the resulting stress and shows maladaptive behaviors."

Category C: Studies on Live, Vertebrate Animals Causing No More Than Minimal Pain, Distress, or Discomfort

Involving No Pain or Use of Pain Relieving Drugs

Examples include: o Routine examinationso Blood samplingo MRI

o Short periods (up to 24 hours) of withholding food and water

o Injection of non-toxic materials

○ Approved methods of euthanasia that induce rapid unconsciousness

Acceptable levels of "minimal pain, distress, or discomfort" in this category would be those procedures that are normally done on animals given routine physical examinations at veterinary clinics. (How would a human undergoing a similar procedure be treated? Would a sedative or analgesic be provided?) Animals that are euthanized and then have tissues/organs removed would be included in this category.

Category D: Studies on Live Animals Involving Pain, Stress, or Distress Which Is Alleviated or Controlled ByAnesthesia or Analgesia.

Examples include studies on: Anaesthetized animals (that may or may not regain consciousness), such as survival

surgical procedures that may result in even minor post surgical discomfort. Also included are studies using noxious stimuli from which escape is possible; some

tumor or device implants; and the use of Freund's complete adjuvant. Other examples include major surgery under general anesthesia that results in:

Significant post-operative discomfort Prolonged periods (several hours or more) of uncooperative physical restraint Deprivation of the animals' environmental necessities, such as maternal

deprivation; aggression; and predator-prey interactions Also included are studies in which diseases or toxicities are induced and the animals are

expected to become sick or abnormal. Animals in Type 2 studies may experience pain, but the necessary treatments to alleviate

the symptoms are available and provided, or the animals are euthanized.

Involvement of trained technicians, scientists, and veterinarians is critical if this pain is to be minimized or avoided. Adherence to acceptable veterinary practices is mandatory and will vary depending on the project, i.e. post-op analgesia, fluid therapy or intensive nursing care. The standard is to provide analgesics in all instances in which a human in a similar situation would receive them.

Category E: Projects Involving Significant Pain or Distress without the Benefit of Pain-Relieving Drugs. (Death as an End Point )

Examples include: Application of noxious stimuli from which escape is impossible. The use of muscle relaxants in surgery without concurrent use of anesthetics, induction of

aggressive behavior leading to self-mutilation or fighting where death is the end-point. Also included are studies in which death is the end-point, i.e. diseases are induced and

infected animals are permitted to succumb rather than be treated.

Comment: Animals are expected to show clinical symptoms of pain or distress as a result of the research objectives, but these symptoms cannot be treated or otherwise alleviated with the use of drugs or intensive care because doing so would interfere with the research objectives. Type 3 studies place an explicit responsibility on investigators to explore alternative designs to ensure that these methods have to be used. Scientific justification for using these procedures and withholding analgesia or treatment must be explained in a statement by the Principle Investigator. This statement is required by federal law. The IACUC submits these statements in the annual reports to the government.

Rev 11-2011 Procedures and Staffing

Procedures that may cause more than momentary or slight pain must, in their planning, involve consultation with a veterinarian.

Mouse tail and toe biopsy are standard procedures, therefore, procedure requests are not required as long as SOP 00-021C Mouse Procedures is being followed. Attach all certifications and training documentation relevant to the personnel and procedures listed below.

Please attach any comments and recommendations made by the veterinarian.

If Category D is chosen fill out “Category D” section. If Category E is chosen fill out “Category E” section. Species: Category:

Procedure #1:

Personnel Names # of Years Performing the Procedure on Species Above

Where did or will you receive the training for this specific procedure?

If Category D is chosen fill out “Category D” section. If Category E is chosen fill out “Category E” section.

Species: Category:Procedure #1:



If Category D is chosen fill out “Category D” section. If Category E is chosen fill out “Category E” section.

Species: Category:Procedure #1:



Mandatory – Certificate Info for All listed on this Protocol:(Must be renewed every 3 years – expired training will not be accepted)

Enter date taken or N/A if not applicable. Missing information will be requested. (See MMCRI Website)

Personnel Names“Working with the IACUC”

Date:

“Post-Procedure Care of Mice and Rats”

Date:

“Working with Mice in Research Settings”

Date:

“Working with Swine in Research Settings”

Date:


Rev 11-2011

Euthanasia: A second method of euthanasia must be used**.

SpeciesAgent/Method ⊠

Agent/Method ⊠ Other ⊠ Route of

administration

FollowSOP: 99-

016C* (Yes/No)

Mouse 1st Method Isoflurane CO2

Mouse 2nd

Method**Cervical

DislocationDecapitation

Swine 1st Method Fatal Plus

Swine 2nd

Method** Open Chest

**After any animal has been declared dead, the animal has to be observed an additional 10 minutes to confirm death. At this time a second method of euthanasia must be used. Refer to SOP 99-016C Pain and Distress Policy.

* For any method of euthanasia which is not IACUC approved (refer to SOP: 99-016C Pain and Distress Policy), provide scientific justification for using the method in this project.

Is the time of spontaneous death of any animal subject an endpoint in any of the studies in your project? Yes: No:

If yes, provide scientific justification why euthanizing the animal, when it exhibits distress or becomes moribund, will bias or compromise the experimental result: (Below)

Yes: No:

Justification:

Continue . . .

Category D

NOTE:The Principal Investigator must provide a written narrative of the sources consulted to determine whether or not alternatives exist to procedures which may cause pain or distress. From the USDA Animal Care Resource Guide -- Policy #12 Consideration of alternatives to each procedure which may cause pain or distress must state sources consulted, such as Biological Abstracts, Index Medicus, Medline, the Current Research Information, Services (CRIS) and the Animal Welfare Information Center (AWIC).


Attach your search results to this document.Send in MS Word Format or NotePad Formats Only (Website links are not accepted.)

Date search was conducted:

Years covered: (Ex. 1999-2010)Databases Used:

Keywords:

Alternatives were found?Place “X” next to choice:

*YES NO

*If alternatives were found, explain below why they cannot be used.Explanation:

Continue . . .


Category E

NOTE:The Principal Investigator must provide a written narrative of the sources consulted to determine whether or not alternatives exist to procedures, which may cause pain or distress. From the USDA Animal Care Resource Guide -- Policy #12 : Consideration of alternatives to each procedure which may cause pain or distress must state sources consulted, such as Biological Abstracts, Index Medicus, Medline, the Current Research Information, Services (CRIS) and the Animal Welfare Information Center (AWIC).


Attach your search results to this documentSend in MS Word Format or NotePad Formats Only (Website links are not accepted.)

Date search was conducted:Years covered: (Es. 1999-2010)Databases Used:

Keywords:

Alternatives were found?Place “X” next to choice:

*YES NO

*If alternatives were found, explain below why they cannot be used.Explanation:

Justify No Pain ReliefProvide a scientific justification why drugs which might alleviate pain will be withheld. Document the rationale for this decision and provide references, if possible. (Use the box below.) This information is required in our annual USDA report and may be quoted directly from this protocol form.

(Cell will expand)

Expected Clinical Signs of Pain/DistressPlease describe clinical signs to be expected. Indicate the severity and duration of each clinical sign, the frequency the animals will be monitored and when the pain will be eliminated or managed (euthanasia, drugs, or withdrawal of painful stimulus). The Committee must understand that the pain is the minimum needed forthe shortest time possible, consistent with the experimental goals. (Use the box below.)

(Cell will expand)


SECTION: VII QUESTIONSAnswer “Yes” or “No” to All Questions Below

#1 Will you be bringing mice up to your lab? YES NOIf yes, complete attached form Bring Live Mice to Your Lab

#2 Will imaging equipment and/or irradiator be used on live mice? YES NOIf yes, complete attached form Equipment

#3 Will non-survival* surgery be performed on the animals? (*animal are euthanized at the end of the procedure w/o recovery) YES NO

If yes, complete attached form Acute Surgery

#4 Will survival* surgery be performed on the animals? (*Survival is defined as recovery from anesthesia.) YES NO

If yes, complete attached forms Survival Surgery and Anesthesia

#5 Does this study require anesthesia, analgesia, sedation or tranquilization of animals? YES NO

If yes, complete attached form Anesthesia, analgesia, sedation or tranquilization

#6 Will you be using animals to produce either monoclonal or polyclonal antibodies? YES NO

If yes, complete attached form Antibody Production

#7 Will you be collecting body fluids from animals, other than fluid collection following euthanasia? YES NO

If yes, complete attached form Ante-mortem Fluid Collection

#8Will exogenous substances or tissue (e.g., drugs, infectious agents, carcinogens, toxin, etc.) be administered to animals other than for anesthesia or production of antibodies?

YES NO

If yes, complete attached form Administration of Exogenous Substances or Tissues

#9 Does this project involve the production or study of tumors in animals? YES NOIf yes, complete attached form Tumor Studies

#10 Does the protocol require behavioral conditioning of animals? YES NO

If yes, complete attached form Behavioral Conditioning

#11 Does the research protocol require deviation from standard diet or other than normal environmental conditions? YES NO

If yes, complete attached form Diet Manipulation and Environmental Modifications

SECTION: VIII BRING LIVE MICE TO THE LAB

Refer to SOP 02-020A The Use of Animals within the Principal Investigator’s Lab

Any animal brought up to the lab is not allowed back into the barrier animal facility, but if needed, can go to quarantine. Transgenic animals shall be cared for under Animal Biohazard Level 1 condition, unless a higher degree of biohazard conditions are needed to ensure proper care.

Answer all items below that are applicable.Lab location:

Does your lab area have the following?Place “X” Below for All that Apply:

Restricted access to the roomBiohazard sign posted at all entries to the area and work areaAn appropriate see-through container for euthanasia to prevent any unnecessary suffering Live traps within the roomHand washing sink

Doors to Lab Rooms:Sweeps

Will the animals be staying in the lab over a 12-hour period? *YES: NO:If “YES”, who will be providing care/observation of the animal(s)?

Length of stay:Reason:

Continue …

SECTION: IX EQUIPMENT TO BE USED ON LIVE MICE

Answer “Yes” or “No” for All Listed Below (Additional info for any “Yes” answers)

#1 MRI YES: NO: Will be following: SOP 05-024B Internal MRI Use & SOP 07-029C MRI & SOP 07-030C MRI tail vein

a. How often will this procedure be performed per animal?b. What would be the procedure schedule?

#2 X-Ray YES: NO: Will be following: SOP 07-032C Faxitron X-ray analysisa. How often will this procedure be performed per animal?b. What would be the procedure schedule?c. Amount of time in unit?

#3 Maestro Imaging

YES: NO: Refer to manual for use

a. How often will this procedure be performed per animal?

b. What would be the procedure schedule?c. Amount of time in unit?

#4 Mouse ECG YES: NO: Will be following : SOP 09-038C

a. How often will this procedure be performed per animal?b. What would be the procedure schedule?c. Amount of time in unit?

#5 Irradiator YES: NO: Will be following : SOP 07-028C Irradiation of Mice


b. What would be the procedure schedule?c. Amount of time in unit?

#6 PIXIMUS ™ II Mouse Densitometer YES: NO:

Will be following : SOP 07-033C PIXImus densitometer analysis


b. What would be the procedure schedule?c. Amount of time in unit? (typically, this takes 2-5 minutes)

#7 vivaCT 40 YES: NO: Will be following : SOP 08-035Ca. How often will this procedure be performed per animal?b. What would be the procedure schedule?c. Amount of time in unit?

#8 Blood Pressure Monitor YES: NO: Will be following: SOP 09-037C

a. How often will this procedure be performed per animal?b. What would be the procedure schedule?c. Amount of time in unit?

#9 Vevo2100 High-resolution Ultrasound YES: NO: Will be following: SOP 08-036C


b. What would be the procedure schedule?

c. Amount of time in unit?

SECTION: X ACUTE SURGERY

The animal will be euthanized at the end of the procedure, without recovery from anesthesia.Describe the surgical procedure(s):

SECTION: XI SURVIVAL SURGERY Refer to SOP: 00-022C *Major Survival Surgery on Non-rodent Mammals Guidelines,

SOP:99-013C Rodent Survival Surgery*Major surgery is defined as opening a body cavity.

*Please attach any comments and recommendations made by the veterinarian. 1. Major or minor surgery:2. Number of survival surgery procedures per animal: *If more than one procedure, fill out question 8, in this statement.

3. Describe in detail the surgical procedure(s) per species (refer to SOP: 00-023C Rodent Carotid Artery Ligation & Denudation, if needed). Include how long the animal will be maintained following surgery.

4. Pre-operative care:

5. Post-operative Analgesics:

Species Analgesics agent Dosage Route Schedule

mouse major Buprenorphine 0.05-0.1mg/kg SC BID for two days after surgerymouse minor Buprenorphine 0.05-0.1mg/kg SC BID for one day after surgery

6. Post-operative care:

Species Frequency of Observation

Will Fluids Be Administered? Dosage Route Schedule

7. Antibiotics: *YES: NO: *If yes, complete the table below.

Species Will Antibiotics Be Used? Yes/No)

Antibiotic Agent Dosage Route Schedule

8. Provide written scientific justification for multiple surgical procedures on the same animal. Refer to SOP:99-012A Multiple Survival Surgical Procedures

SECTION: XII ANESTHESIA, ANALGESIA, SEDATION, OR TRANQUILIZATION

1. Please place an “X” next to those that apply below:

SpeciesMark “X”

Below

Reason(i.e. Sedation, Anesthesia)

Agent Dose Frequency Route Duration Anesthesia

Mouse anesthesiaAvertin

(tribromoethanol)SOP 10-040C

0.2ml of 1.2%solution/10g body wt or 240 mg/10g body wt

once IP ~ 1 hr

If you are using Avertin, explain why you are not using a pharmaceutical grade drug:Is there another drug you could use instead of Avertin:

Mouse anesthesia Ketamine + Xylazine

K:90-120mg/kg + X:10 mg/kg once IP ~ 0.5 hr

Mouse anesthesia Isoflurane To effect on going inhalation on going

Swine sedation Ketamine & atropine

23mg/kg & 1cc/22 kg 1 IM 15-20

minutes

Swine anesthesia Isoflurane 2% on going inhalation on going

Swine Anesthesiapre-intubation

TelazolXylazineAtropine

Telazol 4.4 mg/kgXylazine 2.2

mg/kgAtropine 0.05

mg/kg

1 IM

2. How will depth of anesthesia be monitored? (Refer to SOP: 99-015C Monitoring Animal(s) while under anesthesia)

3. Who will perform the anesthesia? 4. If controlled substances are being used, who will be responsible for this substance? (Refer to SOP: 99-017C Drug Storage and Control) 5. Paralytic Agent Use

SpeciesParalytic Agent

Being Used? Yes/No

Agent Dose Frequency Route Duration

6. What is the purpose of using a paralytic agent?

7. Describe the methods used to monitor the level of anesthesia in the paralyzed animal :

SECTION: XIII ANTIBODY PRODUCTION*

Refer to SOP 99-006A Monoclonal Antibody Production in Vivo (ascites production)SOP 99-008A Guidelines for Use of Adjuvants in Animals

AB1 AB2 AB3 AB4Name Species:

Polyclonal/Monoclonal:

Antigen:Route of Admin:Site of Injection:

Vol./Site:Frequency:

*Anesthesia:Restraint Method:

Adjuvant #1/Primer:Route of Admin:Site of Injection:

Vol./Site:Frequency:Anesthesia

Restraint Method:

Adjuvant #2/Booster:When Given?

Route of Admin:Site of Injection:

Vol./Site :Total Volume:

Frequency:Anesthesia:

Restraint Method:

Abdomen Primed?If yes then: Agent:

Volume:Number/Schedule:Total Volume of

Antibody Needed :

*Ante-mortem Fluid Collection needs to be filled out.

SECTION: XIV ANTE-MORTEM FLUID COLLECTION*

Collection 1 Collection 2 Collection 3 Collection 4 Collection 5Species

Fluid

Volume/ Collection

Site

Method of collection

# Collections

Frequency of CollectionsMethod of Restraint

*Anesthesia(Yes/No)

*If YES, then fill out attach form Anesthesia, Analgesia, Sedation, and Tranquilization of Animals

Refer to following SOP’s:

1. 99-005A Use of Physical Restraint 2. 99-012C Mouse Blood Collection procedures 3. 00-020C Rat Procedures

PI Last Name & IACUC #: __________________________________

SECTION: XV ADMINISTRATION OF EXOGENOUS SUBSTANCES OR TISSUES

Item listed below will be sent to the Safety Committee or the Institutional Biosafety Committee (IBC) to obtain approval.

Attach product information: MSDS, Cell line: MAP or biological contaminant test One PRODUCT per page

Product Name:Product #: CAS #:

Manufacturer:Insert “X” Next to Item Below:

Chemical: Biological agent(cell lines):

Carcinogen: Radioactive materials:

Infectious agents: Recombinant DNA or genetically altered materials:

Other: \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ Refer to: SOP: 99-013A Use of Biohazards & Radioactive Material in Animals SOP: 07-031C Guidelines for Cell Line and other Biological Material

Name Species:Dose Per Animal:

Route of Delivery: Frequency:

Total # of Treatments:

Length of Time Treated Animals

Maintained:Route of

Excretion:Excretion and

Rate:Will physical or physiological impairment

result from the product?Will the animals be anesthetized or sedated

during the procedure?

To Be Filled Out by Safety or the Institutional Biosafety Committee (IBC)NFPA rating:

Health:Fire:Reactivity:

Biohazard level: IBC Approval #:Level of physical and biological containment required:Personnel safety requirements to be imposed:Limitations imposed by the facility:Comments:Signature:

Date

SECTION: XVI TUMOR STUDIES

1. Before any cell line can be introduced, mandatory MAP testing or acceptable alternative must be conducted. Refer to SOP: 99-007C Animal Disease Surveillance and Diagnostic Policy

2. Attach MAP testing results refer to SOP 07-031C Cell Line Policy.

SOP 99-007A - General Tumor Guidelines

1.1 The tumor(s) should not be allowed to become ulcerated or necrotic.1.2 The tumor(s) should not significantly interfere with the movements of the animal, especially its ability to

obtain food and water and its ability to bear its own weight. Additionally, the tumor should not prevent the animal’s ability to regain normal posture if placed on its back.

1.3 If the tumor(s) causes the animal to become comatose, moribund, or in apparent distress the animal should be given the proper veterinary care and, if necessary, sacrificed.

1.4 In general, clinical problems occur in animals with masses located on the dorsum and flank, which exceed 10% of the normal weight of the animal. A significantly lower tumor burden is acceptable with the tumor masses on the ventral aspect of the animal because these are subjected to more physical trauma.

1.5 The tumor burden should not be so significant as to impair the normal daily activity of the animal1.6 The tumor(s) should not be allowed to cause the animals any undue pain or stress.1.7 Any deviation from these guidelines must be approved by IACUC prior to starting the project.

Will the project deviate from the guidelines above? *If yes provide written scientific justification below: *YES NO

(Cell will expand):

Use “Example” column below as a template for your Model. (If you need more columns, copy table)FOR

EXAMPLE: Model #1 Model #2 Model #3

Tumor Model:Renal

Carcinoma

Tumor Source: Human Kidney

Host Species: Mouse

Host Strain: Tg

Method on Induction:Implant in

kidney

Route of Inoculation:Implant in

kidney

Tumor Site: Kidney

Genetically Altered Animals: (Yes/No)

Yes

SECTION: XVII BEHAVIORAL CONDITIONING

NameSpecies

Reinforcement is Used? If yes, what? Duration Frequency By Whom?

1. What is the purpose of the conditioning?

2. What criteria will be used to monitor the long-term condition of the animals during the training and experimental periods? (Refer to: SOP 00-018C Animal Euthanasia Due to Poor Health)

Continue …

SECTION: XVIII DIET MANIPULATION AND ENVIRONMENTAL MODIFICATION

Attach a Copy of the Nutritional Information

1. How does this diet different from the approved vendor diet feed? (Cell will expand):

2. Diets

Name Species

Frequency of Feeding Supplier of Diet? Duration

of Diet

Physical Impairment (*Yes/No)

PhysiologicalImpairment (*Yes/No)

*If “YES”, describe below the impairment in detail, including criteria for termination of the experiment. (Refer to: SOP 00-018C Animal Euthanasia Due to Poor Health)

*(Impairment details):

3. Describe how dietary manipulations or restrictions will be accomplished:

4. Storage requirements of the feed:

5. Environment

Name Species

Environmental Change Duration

Physical Impairment (*Yes/No)

Physiological Impairment (*Yes/No)

*If “YES”, describe below the impairment in detail, including criteria for termination of the experiment. (Refer to: SOP 00-018C Animal Euthanasia Due to Poor Health)

*(Impairment details):

Thank You!

PLEASE DELETE ALL PAGES BEYOND THIS POINT

EXAMPLES: Simple Flow Chart/Experimental Designs(To go back: Click Here)

FLOW CHARTS ARE MANDATORY FOR ALLEXPERIMENT DESIGNS

EXPERIMENT DESIGN: Experiment Design # 1: Tumor Growth

Strain: C57BL/6 and C57BL/6.Col11 (SGA/PGE). n = 20DAY PROCEDURE CATEGORY

Day-1

Day-7

Day 14

Day 21

Day 21

Subcutaneous injection of 0.5 x 106 B16F10 melanoma cells in 10 each of the 2 strains of C57BL/6 mice (8 weeks old).

Caliper measurement of tumors within all mice



All mice sacrificed and tumors dissected for analysis.

D.

EXAMPLE: Simple Flow Chart/Experimental Design

Experiment Design # 2: Experimental Metastasis

Strain: C57BL/6 and C57BL/6.Col11 (SGA/PGE) mice. n = 20DAY PROCEDURE CATEGORY

Day-1

Day 21

Intravenous injection of 0.5 x 106 B16F10 melanoma cells in 10 each of the 2 strains of C57BL/6 mice (8 weeks old).

All mice sacrificed and lung tissues dissected for analysis.

D.

Continue ….

EXAMPLES – Experimental Flow Designs

#4 EXPERIMENT DESIGN: Comprehensive developmental skeletal analysis of B6.C3H-10-7 female mice at 8 &16 weeks of age (Courtesy of Dr. Rosen)

Total animals: n=60Wean at 3 weeks

Negative control groupC57BL/6J/CJR female n=30

Experimental groupsB6.C3H-10-7 female n=30

Developmental skeletal analysis at 2 different time points

8 weeks old:Group A: C57BL/6J/CJR female, n=15Group B: B6.C3H-10-7 female n=15

16 weeks old:Group C: C57BL/6J/CJR female, n=15Group D: B6.C3H-10-7 female n=15

Group 1: (n=3 from each Group A-D: total 12 animals)1. PIXImus & FACITRON bone mineral density2. MRI marrow and body fat composition3. Terminal bleed to measure levels: Osteocalcin, Trap5b, P1NP,

IGF-14. Tissue harvesting: femurs for pQCT, uCT, histomorphometry*;

liver, peripheral fat pads for RNA

Group 2: (n=12 from each Group A-D: total 48 animals) 1. PIXImus & FACITRON bone mineral density2. Terminal bleed to measure levels: Osteocalcin, Trap5b, P1NP,



*All animals will be given interperitoneal injections of reagents for bone histomorphometry at two time points prior to sacrifice: 1 week prior (Calcein) & 2 days (Demeclocycline) prior to sacrifice.

5.EXPERIMENT DESIGN: Comprehensive developmental skeletal analysis of B6.C3H-10-10 female mice at 8 &16 weeks of age







Group 1: (n=3 from each Group A-D: total 12 animals)5. PIXImus & FACITRON bone mineral density6. MRI marrow and body fat composition7. Terminal bleed to measure levels: Osteocalcin, Trap5b, P1NP,






*All animals will be given interperitoneal injections of reagents for bone histomorphometry at two time points prior to sacrifice: 1 week prior (Calcein) & 2 days (Demeclocycline) prior to sacrifice.

6. EXPERIMENT DESIGN: Comprehensive developmental skeletal analysis of B6.C3H-10-12 female mice at 8 &16 weeks of age







Group 1: (n=3 from each Group A-D: total 12 animals)9. PIXImus & FACITRON bone mineral density10.MRI marrow and body fat composition11.Terminal bleed to measure levels: Osteocalcin, Trap5b, P1NP,

IGF-112.Tissue harvesting: femurs for pQCT, uCT, histomorphometry*;





*All animals will be given intraperitoneal injections of reagents for bone histomorphometry at two time points prior to sacrifice: 1 week prior (Calcein) & 2 days (Demeclocycline) prior to sacrifice.

MAINE MEDICAL CENTER RESEARCH INSTITUTE

PROCEDURE #

10-040C

DATE ADOPTED 1/22/2010

REVISION DATES:PREPARED BY

Toni St. Peter, Laboratory Animal Manager

REVIEWED BY

IACUC

Last review 5/11 TSP

PROCEDURE TITLE: The Use of Tribromoethanol (Avertin)

1.0 Purpose: To ensure appropriate medications are used in research animals.

2.0 Responsibility: It is the responsibility of the principal investigator and all staff performing the task to follow the procedures established in this document.

3.0 Rationale:3.1. Tribromoethanol is appropriate for short term procedures in mice, especially surgical

procedures. It is best used in situations where it will be only given on a single occasion. However, adverse reports about the efficacy and safety of Tribromoethanol (TBE), combined with the availability of effective pharmaceutical grade alternatives, have made the continued routine use of TBE for rodent anesthesia controversial.

3.2. Advantages:3.2.1. Tribromoethanol induces anesthesia rapidly and provides good surgical

analgesia for approximately one hour. 3.2.2. If it is given by injection, one is spared the occupational health risks and

technical difficulties associated with volatile anesthetics. 3.2.3. It is not a controlled drug and is, therefore, readily available.3.2.4. If used appropriately, TBE has a good margin of safety.

3.3. Disadvantages:3.3.1. TBE is an irritant, especially at high doses, high concentrations, or with

repeated use. Adhesions are sometimes seen in the abdominal cavity after IP injections.

3.3.2. TBE degrades in the presence of heat or light to produce toxic byproducts. Degraded solutions can be both nephrotoxic and hepatotoxic.

Administration of degraded TBE solutions has been associated with death, often 24 hours after surgery.

3.3.3. TBE can cause intestinal ileus (stopping of the gut motility and subsequent death of the animal) several weeks after injection. This is more common with

TBE stored in the presence of light or heat, stored at higher than recommended doses, or given in higher than recommended concentrations.

3.3.4. The effects of TBE are also somewhat unpredictable in mice younger than 16 days or in animals with altered carbohydrate metabolism, such as various mouse strains used for diabetes or obesity models.

4.0 Policy: Investigators may use non-pharmaceutical grade TBE, if approved by the IACUC in an animal protocol. TBE must be stored and disposed of as described below.

5.0 Instructions: 5.1. 100% stock Tribromoethanol (Avertin): Dissolve 5g Tribromoethanol (Sigma T48402)

in 5ml Tert-Amyl alcohol. Make sure all the Tribromoethanol goes into solution. This may require lots of shaking.

5.2. Mix 2.5ml of stock solution in 100ml isotonic saline. Warm the saline first in a 40°C bath. Shake, wrap in foil or amber bottle, and store at 40°C.

5.3. Storage: Refrigerate the properly labeled aliquots and protect them from light. The material degrades rapidly in the presence of heat or light. The stock solution must be dated and stored in an amber bottle or wrapped in foil at 4°C. Even refrigerated and wrapped in foil, the material is stable for only two months. If the material degrades it becomes toxic.

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