Drexel University and Drexel University College of Medicine

Institutional Animal Care and Use Committee

Application to Use Animals in Research and Teaching

(Form must be typed for submission)

Please use hyperlinks for help or additional information.

1. Title of Your IACUC Study / Collaborative Research: Baseline Survey of the Lower Xingu River Rapids, Brazil: a Highly Diverse, Globally Unique, and Immediately Imperiled Ecosystem
2. Financial Sponsorof the Study / NSF, DEB: Biodiversity Discovery and Analysis Program
3. Principal Investigator (must be faculty, postdoctoral fellow, medical, dental resident or visiting scientist)
3a. Name / John G. Lundberg
3b. Academic Rank / Professor and ANS Curator
3c. Department / BEES and Academy of Natural Sciences
3d. Mail Stop or Address / Dept Ichthyology, Academy of Natural Sciences, 1900 Ben Franklin Pkwy, Phila, PA 19103
3e. Work Phone / 215 405-5069
3f. Home Phone / 215 880-2175
3g. Beeper or Cell Phone Number / 215 880-2175
3h. Fax Number / NA
3i. E-mail Address /
4. Faculty Sponsor for postdoctoral fellow, medical/dental resident or visiting scientist
4a. Name of Faculty Sponsor
4b. Academic Rank
4c. Department
4d. Mail Stop or Address
4e. Work Phone Number
5. Responsible Persons authorized to make research decisions in the absence of the principal investigator (if none, enter none in the Name column)
Name / E-mail / Work Phone # / Home Phone #
Mark Sabaj Pérez / / 215 299-1026
6. Review Status of Protocol(please check a, b, or c)
6a. Completely new protocol /

Yes

/

YES

/ No
6b. Re-submission due to three completed years of the study (Mark Yes or No)? /
Yes
/

No

If yes, enter your current protocol number. /
Protocol Number
6c. This protocol is exactly same as the protocol # shown, except for a change of sponsor and/or title / Protocol Number
(For any other minor changes, you must submit an explanatory memo to the IACUC chairperson) / Change of Sponsor
Change of Title
For question 6c only, complete the boxes to the right
If you have answered 6c, you need not complete the rest of this form / Your Signature
Faculty Sponsor Signature
Date
7. Approval Period: Check the number of years from the approval
date that you want this protocol active / One year / Two years / Three Years
The approval date will be assigned as the date of the IACUC meeting the protocol was approved / 3 years
  1. Non Scientific Summary: In language understandable to a high school senior. Briefly describe the goals and significance of this study

8a. Goals
The NSFBiologicalDiscovery and Analysisprogramsolicits proposals todiscover and document biological species diversity of all forms of life on Earth,with special emphasis ongeographic regionsthat are poorlysurveyed.NSF BDA is recommending for funding ourproposalto inventory and analyze the fish fauna of the lower Xingu River, ParáState, Brazil. Preliminarydata suggeststhat thefishfauna ofthelowerXinguRiverishighlyspeciesrich, withahighdegree ofendemism.In2011,constructionbeganonalargecomplexofdams that will eventually eliminateflowtovastrapids complexwherediversity andendemismisthought tobe particularlyhigh.Todate, thefewsurveys that have beenconducted inthis regionhave consistently discoveredfishspecies that are new-to-science.Wewillconductthree collecting expeditions in2013-2014,withthegoalofthoroughly documenting and characterizingfishfaunal diversity intheareato beimpacted bythedamsnowunder construction. The rationale for site and species selection are based on the urgent need to get baseline knowledge of this fauna before riverine habitats are disrupted.
8b. Significance
This project will provide the most thorough inventory of the fish and invertebrate fauna of the soon-to-altered lower Xingu River, Brazil, through documentation with research specimens deposited in natural history collections, habitat and taxonomic data and analyses, scientific publications and electronic databases. This project is collaborative with collaborators at Texas A&M University and with Brazilian scientists and their institutions, and we have secured all necessary permits from the Brazilian government to proceed with the field work and collaborative study of the fauna.
8c. Provide a simple chronological outline or what will happen to the animals in this study. Do not give details use a flow chart if necessary (e.g. animals arrive, so many days later surgery takes place, so many days after surgery they are euthanized).
Three expeditions will be conducted from 2013 to 2014.30 to 40 study sites, representingtherangeofhabitattypesavailableinthelowerXinguRiver,willbesampled duringeachexpedition.Ateachsite,fisheswillbecapturedfromthewildusingseinesand gillnets.Following capture,specimens willbeeuthanizedbyimmersion ina concentrated solution ofMS-222(tricaine methane sulfonate, 200-500mg/Ldependingonfishsizeand observed responseand bufferedtosaturationwithsodium bicarbonate[AVMA,2007])for atleast10minaftersignsofgillventilation havestopped. Allpersonnel willwearprotective gloves, respirator and eye goggleswhen handlingMS222andformalin.
8d. If this protocol is a renewal, provide a brief summary of your progress in addressing the goals of the protocol since it was last
approved. Indicate how the experiments described in the renewal will extend the previous work.
9. Animal model:
  1. Why is it necessary to use laboratory animals rather than in vitro methods, human studies, computer models or other methods?

The goal of this study is todiscover and document biological speciesof fishes in their natural habitats of the lower Xingu River.
b. Why is the animal species you have chosen most appropriate for your study?
See 9a.
10. Number of Animals Requested: Please list below the total number of each species to be used in your
study for a three year period.
Species Name (e.g. rat) / Approximately 300 species will be encountered. A list species known to the sampling areais available upon request but an indeterminate number of species will be added as new records or discoveries.
Strain/Stock (e.g. Tac: N(SD) fBR) / NA
Number to be purchased / NA
Number resulting from in house births (including fetuses > Embryonic day 16 ) / NA
Number being transferred from another study / NA
Total Number of animals / Approximately 1 to 300 specimens per species.
Age or weight / Indeterminate
Housing site (Center City, Queen Lane, Calhoun) / NA
Usage Site (Building & Room #) / NA
  1. Justification of Animal Numbers: Justify why you need the number of animals listed in question 10.
Show the experimental groups to be used in your study and the number of animals in each group. In your
justification, use power analyses, previous publications of similar work, or other means.
The Amazon Basin isknown tohostmore than 5000fishspecies,more than any other river system onEarth.TheXinguRiver,whereoursurveys arefocused,likelyhosts morethan10% ofthisdiversity, orgreaterthan 500species.Inorder tofulfillthestated goalsofNSF’sBiodiversity Discovery and Analysis program,wewillstrivetocollect,identify and catalog asmuch ofthisdiversity aspossible prior to completion of dam construction and natural habitat disruption.Similar previousexpeditions run bythePIs and our Brazilian colleagues haveyieldedupwards of 300speciesand upto10,000specimens.Weconsider theseto bereasonablegoalsfortheexpeditions wewill direct, especially because the natural rapids habitatsofthe Xingu River are soon-to-be destroyed. Given the prevalenceof mimicry among tropical fishes, the commonly cryptic natureofbiodiversityinthetropics,andtheneedtothoroughlycharacterizebiodiversityof this region prior to completion of dam construction,all specimens captured will be euthanized.Euthanizedspecimenswillbepreserved informalinsothatgutcontentscanbe analyzed and theidentificationsofthespecimensmaybeconfirmed inthelaboratory.All specimenswillbecataloged and permanentlyarchived and curatedattheAcademyofNaturalSciences inPhiladelphiaand our primary collaborating Brazilian institution Instituto Nacional de Pesquisas da Amazonia (INPA) in Manaus. Taxonomic research and knowledge at the species level requires large sample sizes in order to document phenotypic and genotypic variation within and among species.
  1. Maximum Number of Cages: What is the maximum number of cages you anticipate using for each
species at any one time.
Species / Maximum Number of Cages
13. Special Care for Animals: Will your research or the genetic composition of the animal lead to a condition that requires special care, such as a barrier housing, special diet, food or water restriction (Mark yes or no). / Yes /

No

/

NO

  1. Who will provide this care?

  1. In the space below, describe the care required. In addition, if deprivation and/or restrictions of ad libitum food or water is required, describe the duration, extent, and the reason for the reduction, as well as the monitoring methods used to insure the well being of the animal.

  1. Does your protocol preclude the use of environmental enrichment?
/ Yes /

No

/

NO

NOTE: According to the Guide for the Care and Use of Laboratory Animals (NRC 2011) animals should be provided with environmental enrichment that promotes the expression of species-typical behaviors and psychological well-being through physical exercise, manipulative activities and cognitive challenges. For further information, see the IACUC Policy for Animal Enrichment

Justification:

  1. Will any portion of the protocol require that animals be singly housed?
/ Yes /

No

If yes, provide a rationale as to why this is required. NOTE: This does not apply to temporary single housing following surgery during the immediate post-operative period. (According to the Guide for the Care and Use of Laboratory Animals (NRC 2011) single housing of social species should be the exception and justified based on experimental requirements.)

Justification:

  1. Non-ULAR Facilities and Animal Transportation:

  1. Will any animals be kept out of the ULAR facility for more than 12 hours
(Mark yes or no)? If yes, provide scientific justification for that need in the space below. / Yes /

No

/

NO

  1. Will you be transporting live animals between buildings for any reason?
If yes, ULAR must be notified prior to transport and approve the conditions under which the animals will be transported. / Yes /

No

/

NO

Justification:

15. Second Use of Animals: Have any of the animals to be used in your study been used for any other research or teaching protocol (other than breeding) within or outside of this university (Mark yes or no)? If yes, in the space below please explain and justify why they will be used again. / Yes /

No

/

NO

16. Animal Restraint: Will any animal be kept awake and restrained for more than 60 minutes (Mark yes or no)? If yes describe and justify composition of the animal lead to a condition that requires special care, such as a barrier housing, special diet, food or water restriction (Mark yes or No) /

Yes

/

No

/

NO

  1. Reason

  1. Length

  1. Conditioning procedure:

17. Pain Relief: Will you be using any anesthetic, analgesic, tranquilizer or neuromuscular blocking drugs for any procedure? If yes, complete the table below.
Species
Experimental group
Drug Name (generic)
Induction Dose (mg/kg, %gas)
Maintenance Dose (mg/kg, %gas)
Frequency of use (e.g. every 4 hours)
Route of Administration
Volume
If the volume is a dilution, please enter the dilution factor and the diluent.
a. Will you be using a volatile anesthetic agent (Mark yes or no)? If yes, in the space below please describe the precautions you will take to scavenge waste anesthetic gases. / Yes /

No

b. Indicate the method you will use to ensure that the animals are properly anesthetized
Toe Pinch / Palpebral Reflex / Reaction to Incision
Blood Pressure / Other / Please specify other
  1. Will you be using neuromuscular blocking drugs (Mark yes or
no)? If yes, in the space below please justify their use and explain how you will ensure that the animals are adequately anesthetized and not just paralyzed. / Yes /

No

  1. Pain or Distress: Would any surgical or nonsurgical procedures described in this protocol, if performed without anesthesia, analgesia, or tranquilization, cause more than momentary or slight pain or distress(Mark yes or no)? Whether yes or no, please complete the table below.
/ Yes /

No

Category A
No Pain or Distress / Category B
Alleviated
Pain or Distress will be alleviated by drugs or other means / Category C
Pain or Distress will not be alleviated
Species
Number of animals
Duration of relief required after the procedure /
Not Applicable
a. If there will be intentionally unalleviated pain or distress, please provide, in the space below,
scientific justification why this is necessary for your research.
b. If you indicated that you will be alleviating pain or distress, when will this occur
When first noticed / Immediately after surgery / Before surgery ends
Other (please specify)
c. If applicable, please describe your protocol for monitoring the pain, distress, health, and well-being
ofyour animals. Your response must include (1) monitoring methods, (2) frequency, (3) animal removal criteria you will use to remove an animal from the study, (4) disposition of animals removed, and (5) responsible individuals.
19.Animal Use and Pain and Distress: If you have indicated that animals in your study will experience pain or distress, even if it will be fully alleviated, please mark the appropriate checkbox(s) below and fill in the requested information for each item marked.
I have considered alternatives to the use of animals in my study. In addition, I have also considered alternatives to procedures that might cause more than momentary pain or distress, and I have not found such alternatives. I have used the following methods and sources to search for alternatives:
AGRICOLA Data Base (National Agricultural Library). Date of search, years covered by search, and keywords used:
MEDLINE Data Base. Date of search, years covered by search, and keywords used:
CAB Abstracts Data Base. Date of search, years covered by search, and keywords used:
TOXLINE Data Base. Date of search, years covered by search, and keywords used:
BIOSIS Data Base. Date of search, years covered by search, and keywords used:
ATLA (Alternatives to Laboratory Animals, Journal). Issues Used:
Animal Welfare Information Center (Tel. 301-504-6212). State information requested from AWIC.
Direct contact with colleagues or other sources (Must document the source and substance of the information).
20.Other Agents used during your research (excluding antigens for antibody production, anesthetics, analgesics, tranquilizers, and neuromuscular blocking drugs):
  1. List any drug, cell line or vehicle used in this study (do not include antigens for antibody
production, anesthetics, analgesics, tranquilizers or neuromuscular blocking drugs)
Species/Strain
Experimental Group
Drug, cell line or vehicle name (generic)
Vehicle (for research agents only)
Dose (ml/Kg or mg/kg)
Frequency of use (e.g. 2x per day for 4 days)
Route of Administration
Volume used (if appropriate)
Procedure (e.g. surgery, label)
Use spaces below, if more than four agents are to be used
Species/Strain
Experimental Group
Drug, cell line or vehicle name (generic)
Vehicle (for research agents only)
Dose (ml/Kg or mg/kg)
Frequency of use (e.g. 2x per day for 4 days)
Route of Administration
Volume used (if appropriate)
Procedure (e.g. surgery, label)
b.Any known adverse effects of the agents listed at the dose level and frequency used in this study (Mark yes or no)? If yes, in the space below describe the adverse effects and include possible treatment or why a treatment cannot be used. / Yes
c. Will non-pharmaceutical grade medication such as antibiotics, anesthetics, analgesics or experimental compounds be used in protocol activity? If yes, indicate the type of medication, justify usage and describe how the medication will be prepared, sterilized, labeled with expiration date, and stored. For further information, see the IACUC Policy for use of Non-Pharmaceutical Grade Drugs – Cocktails. / Yes
21. Non-Surgical Procedures:
a.Will any animal undergo a nonsurgical procedure (Mark yes or no)? If yes, in the space below describe your nonsurgical procedures in a stepwise fashion. / Yes / Yes /

No

Three ichthyological collecting expeditions will be conducted inthelowerXinguRiver from 2012 to 2013.30 to 40 study sites, representingtherangeofhabitattypesavailableintheXinguRiver,willbesampled duringeachexpedition.Ateachsite,fisheswillbecapturedfromthewildusingseinesand gillnets.At once following capture,specimens willbeeuthanizedbyimmersion inconcentrated solution ofMS-222(tricaine methane sulfonate, 200-500mg/Ldependingonfishsizeand observed responseand bufferedtosaturationwithsodium bicarbonate[AVMA,2007])for atleast10minaftersignsofgillventilation havestopped.Tissue samples for genetic work will be harvested from epaxial musculature after MS-222 euthanasia and before formalin fixation. Euthanized specimens are then placed in 10% buffered formalin, and taken to the museum lab for identification and study. Allpersonnel willwear appropriate respirators, and protective eye goggles and gloveswhen handlingMS222andformalin.Our procedures for fish capture, handling and euthanasia are consistent with the latest (2003)published “GuidelinesforThe Use of Fishes in Research” by the the American Fisheries Society, American Institute of Fisheries Research Biologists and American Society of Ichthyologists and Herpetologists.
b. Breeding colony maintenance. Describe the reason for maintaining a breeding colony. Discuss the breeding
scheme, weaning age, duration for which animal will be used for breeding, etc. Describe the genetic monitoring program. Indicate how samples are collected for genotype determination. Indicate method of individual animal identification. The Guide for the Care and Use of Laboratory Animals (NRC2011) space recommendations allow for one male and one female with a litter to be maintained in a standard shoebox mouse cage. If you plan to utilize trio breeding, provide justification for this increased density including management techniques which will allow pups to develop to weaning without any detrimental effects for the mother or litters.
c.Blood sampling: Will you be obtaining blood samples (other than during terminal procedures (Mark yes or no)? If yes, complete the table below. / Yes /

No

Experimental Group / Amount of Blood Removed (ml) / Sampling Frequency / Sampling Site / Method (specify)
e.g. I.V. or postorbital bleeding
22. Survival Surgery:
a.Survival Surgery Animal Numbers and Locations: Will survival surgery be conducted on any animals (Mark yes or no)? If yes, complete the table below, and answer all other questions that pertain to survival surgery. / Yes /

No

Species/Strain
/ # Of animals having only one survival surgery / # Of animals having more than one survival surgery / Building / Room / Group Identification
In the space below provide scientific justification for requiring more than one survival surgical procedure on the same animal
b.Describe the aseptic technique you will follow:
How will the animal be prepared for aseptic surgery?
What procedures will the surgeon use to prepare himself/herself for aseptic surgery?
How will the instruments be prepared for aseptic surgery? (Sterile instruments must be used for each animal).
c.Survival surgery description of surgical procedure.
Describe the major steps in your survival surgical procedure. Indicate group will undergo survival
surgery. Remember to indicate the site of the incision, major intraoperative steps and how the
incision will be closed.
How will the animal’s body temperature be maintained during surgery?
23.Postoperative and Postanesthesia Care. Answer this question only if an animal will undergo survival surgery or general anesthesia for any survival purpose. (NOTE: In addition to completing this form, the IACUC requires that you complete Surgical and Postoperative Care forms for all non-rodent survival surgery. These forms are available in every Drexel aseptic surgical suite and in the University Laboratory Animal Resources office.)
a. Where will the animal be kept (room/building) until it recovers from anesthesia? (NOTE: Recovery implies that the animal is awake enough to maintain sternal recumbency.)
b. How often will the animal be observed until it recovers?
c. Who will observe the animal until it recovers? [Supply name(s)]
d. How will postoperative/post-anesthesia body temperature be maintained?
e. If the animal will not be kept in its own cage until fully recovered, explain why.
  1. If skin sutures or clips are used, when will they be removed? (Note: Do not use absorbable sutures for skin closure unless they are subcutaneous).

24. Nonsurvival Surgery:
a.Will any animals undergo nonsurvival surgery (Mark yes or no)? If yes, please complete the table below. / Yes /

No