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Amur leopard Necropsy

The Complete Amur Leopard Necropsy Protocol

Notification and Reporting

Time critical gamete and genome collection- Prior to or immediately upon death, contact a reproductive physiology program to discuss gamete collection and preservation. The Omaha Zoo Center for Conservation and Research (CCR), (contact below) can do Amur leopard gamete preservation post mortem. Semen preservation is most successful although oocyte may be preserved as well. Ovaries or testicles need to be collected within minutes if at all feasible. A protocol for initial testicle or ovary collection is provided at the end of this protocol if you are unable to make contact with a reproductive program immediately. However recommended procedures change so it would be useful to talk to the person who will be doing the work to determine their current recommendations.

Programs preserving genome cell lines are also in place although less common as of this writing. Preservation of this material also requires quick action. Contact the San Diego Zoo Institute for Conservation Research (contact below) for the most current instructions for this procedure.

Routine reporting- Any time an Amur leopard dies, please notify the SSP Program Leader and the North American Amur leopard Studbook Keeper by email within 48 hours of the event with the following information:

Institution and local contact


Studbook number

Local identification number such as ISIS number and/or house name

Any unique physical identifiers such as tattoos or microchips

Date of Death


Once the death has been recorded in ZIMS or other relevant record-keeping software, a specimen report should be sent to both the AZA studbook keeper and the International Studbook keeper so the studbooks can be updated.

A final pathology report, including the gross necropsy results, histopathology and results of any ancillary diagnostic testing such as cultures, electron microscopy, special stains, virus isolation and other diagnostic procedures, should be submitted with all the identifying information listed above to the Amur leopard Species Survival Plan Veterinary Advisor. Again, Please include the above identifying information in the final report so all records are well correlated.


Contact List

Current June, 2015

AZA Amur leopard Species Survival Plan Program Leader and

Amur leopard Studbook Keeper – North America

Cynthia Kreider

Zoo Director

Erie Zoo

P.O. Box 3268

423 West 38th Street

Erie, PA 16508

Phone: 814-864-4091 Ext 226

Cell: 814-434-2632

Fax: 814-864-1140



AZA Amur leopard Species Survival Plan Vice-Chair

Beth Jo Schoeberl

Curator of Primates

Denver Zoological Society

2300 Steele St.

Denver, CO 80205

Phone: 720-337-1505

Fax: 720-337-1501



AZA Amur leopard Species Survival Plan Secretary

Tammy Schmidt

Assistant Curator of Mammals

Zoo Atlanta

800 Cherokee Ave. SE

Atlanta, GA 30315

Phone: 314-322-2665



Amur leopard International EEP Studbook Keeper

Jo Cook

ALTA Coordinator

Zoological Society of London

Regent’s Park

Phone: 020 7449 6564 and 020 7449 6566



Veterinary Advisor- Amur leopard SSP- also interim contact for post mortem gamete preservation

Julie Napier, D.V.M

Senior Veterinarian

Center for Conservation and Research

Omaha’s Henry Doorly Zoo and Aquarium

3701 S. 10th St.

Omaha, NE 68107

Office: 402-738-2045

Hospital: 402-738-2080

Cell: 402-676-6453



Pathology Advisor – Amur leopard SSP

D McAloose, VMD, Dipl ACVP

Wildlife Conservation Society

Zoological Health Program

Head of Pathology

2300 Southern Blvd

Bronx, NY  10460

Phone: 718-220-7105

Fax: 718-220-7126



Necropsy Protocol


This protocol is written as a “complete” necropsy protocol. However the author fully recognizes resources and time constraints do not always allow for as thorough a necropsy and tissue collection as might be preferred. At a minimum we need everyone to do a “basic” necropsy and tissue collection. Please complete as much of the more thorough “complete” protocol as possible.


There are some population disease concerns that require additional special tissue collections, processing or submission. At this time these include renal disease, reproductive tract disease, and cardiac abnormalities. Prior to or during necropsy if possible, please contact the following people to get the most current requests for tissue collection or handling.


Reproductive Tract Disease:

Complete tracts from all Amur leopards needed, male and female, whether diseased or not, implanted or not. Entire tract in formalin.

Dalen Agnew,

Michigan State University
Michigan State University
Diagnostic Center for Population and Animal Health
Lansing, Michigan 48910-8104

Phone: 517-432-5806

Fax: 517-353-5096


Complete Procedure: The necropsy should be thorough, two sets of formalin tissue should be collected and a liberal set of frozen tissue should be collected. Please ask the lab(s) that process the tissues for histology to keep the wet tissues, blocks, and slides if at all possible or to return them for archiving at the zoo that sent in the tissues. Many labs will keep these materials for a period of time and then discard them at a later date. Gross photos of everything possible should be collected and stored where they can be retrieved. Lesions should be measured, and if possible, some tissues should be weighed, such as heart. Remember the common sense part: for instance, if the cat has CNS disease, then a serious effort to get the whole brain and cord out would be a good thing. If it has heart disease, measure the walls, the luminal diameter, photo the valves and so forth. Holding the carcass until histology has been completed is also a good thing if possible.


Record the following information

Institution: _________________________________________________________________

Address: ___________________________________________________________________





Species: ____________________________________________________________________

Studbook No: _______________________ In house or ISIS number: ____________________

In-house identity (microchip, tattoo, name): ____________________________________________________________________________

Sex: ____________________________________

Date of birth: _____________________________                     Weight: __________________

Date of death: _____________________________               

Date of necropsy: __________________________

Necropsy number: _________________________



Please hold samples at your institution for future toxicological or nutritional analysis if necessary.

____ Liver

____ Kidney

____ Heart




Preserve the following tissues in 10 % neutral buffered formalin at a ratio of 1 part tissue to 10 parts formalin. Tissues should be no thicker than 1 cm (with the exception of the eye and brain).  INCLUDE SECTIONS OF ALL LESIONS AND SAMPLES OF ALL TISSUES ON THE TISSUE LIST.  Photograph and measure lesions.  Weigh organs that are larger or smaller than expected. 


SSP SURVEILLANCE TISSUES and recommended tissue sampling procedures:

       ____Liver - sections from 3 lobes, including gall bladder

       ____Spleen - Cross section including capsule.

       ____GI Tract - 3 cm long sections of:


       ____Stomach - multiple sections from cardia, fundus (body), and antrum of pylorus

       ____Small intestines - duodenum, jejunum, ileum

       ____Large intestines - cecum, colon

       ____Omentum - ~3 cm square

       ____Pancreas - representative sections from two areas including central ducts

       ____Adrenal - entire gland with transverse incision.

       ____Kidney -cortex and medulla from each kidney

       ____Urinary bladder, ureters, urethra - cross section of bladder and 2 cm sections of ureter & urethra.

____Reproductive tract - Entire uterus and ovaries with longitudinal cuts into lumens of  uterine horns. Both testes (transversely cut) with epididymis.  Entire prostate, transversely cut.

       ____Salivary gland

____Oral/pharyngeal mucosa and

       ____Tongue - cross section near tip including both mucosal surfaces. 

       ____Lung - sections from several lobes including a major bronchus


       ____Thyroid/parathyroids - leave intact.

       ____Lymph nodes - cervical, mediastinal, bronchial, mesenteric and lumbar. Cut transversely.


       ____Heart - longitudinal sections including atrium, ventricle and valves from right and left sides.

       ____Eye - both eyes intact.  Remove extraocular muscles and periorbital tissues.    

       ____Brain - cut longitudinally along midline. Submit entire brain and pituitary gland.

       ____Spinal cord (if neurologic disease) - sections from cervical, thoracic and lumbar cord.

       ____Diaphragm and Skeletal muscle - cross section of thigh muscles

       ____Opened rib or longitudinally sectioned ½ femur - marrow must be exposed for 

               proper fixation

       ____Skin - full thickness of abdominal skin, lip and ear pinna.

       ____Neonates: umbilical stump - include surrounding tissues.




The following checklist is also provided as an aid in being thorough and keeping track of tissues, photos, etc.


Tissue examination and collection checklist






-20, -80

Ancillary Dx








Oral cavity & teeth














Skin and nails














Skeletal muscle







Peripheral nerves







Mammary gland





















Bone marrow (femur)































































Thoracic cavity







Abdominal cavity



































Trachea & Lungs







Heart/Pericardial sac







Aorta & other vessels







Thymus/lymph nodes














 Small intestines            
 Large intestines            
 Liver & gall bladder            
 Lymph nodes            
 Aorta & vessels            
 Urinary bladder            
 Adrenal glands            
 Access sex gland            
 Ears & bullae            
 Skull/nasal cavity            
 Pituitary gland            
 Vertebral column            
 Spinal cord            

Gross = Gross appearance: N=normal/no gross lesions; AB=abnormal; NE=not examined; NF=not found; NP=not present

FF = Tissue fixed in formalin: + = yes                 

Histo = Tissue submitted for histology: + = yes


-20/-80 + = Frozen tissue temperature: list storage temp as                                                                                      –20, -80 or other

FP = Filter paper sample: + = yes                            

Ancillary Dx: + = yes; please include list and results with report



Collection and Processing of Testicles or Ovaries

for Shipment




As soon as possible after death, remove testicles enclosed within the scrotal sac and place them into a clean plastic bag or specimen container with some saline-soaked gauze at room temperature and seal tightly.  Place ice packs or ice sealed in a plastic bag on the bottom of a small styrofoam box or other insulated container.  DO NOT USE DRY ICE.  Cover the ice with several layers of newspaper or paper towels, then place the testicles on top and seal the box.  Note:  it is very important that the testicles cool very slowly to 4-7oC (refrigeration temperature); therefore, be sure to not place them in direct contact with the ice (or the sperm will die of cold shock).


Testicles may be sent by overnight priority delivery service, but you should expect a reduction in sperm survival – particularly from aged or chronically ill animals.  Ideally – and for very valuable animals – counter-to-counter airfreight service is preferable (e.g., American Airlines or United Airlines offer such services).  Current technology in assisted reproduction has demonstrated the potential of producing live offspring from embryos derived from in vitro fertilization as well as sperm injection of either motile or non-motile sperm collected from testicles.  Therefore, sperm samples should be salvaged and cryobanked as part of a conservation program for any species.




For unexpected deaths:  To avoid any loss in egg survival, ovaries must be received within 8 hours of death; therefore, they must be shipped by counter-to-counter airfreight service.  Before removing ovaries, pre-warm physiological saline and several warm packs (to physiological temperature).  Using sterile technique, cut the end of the oviducts (Fallopian tubes) at the utero-tubal junction, and remove the ovaries.  Place them directly into a sterile specimen container or sealable, leak-proof plastic bag containing the physiological saline pre-warmed to body temperature.  Pack the specimen cup in a thick styrofoam box containing several warm packs.  Use cloth towels to ensure that the ovaries are properly insulated.


For anticipated deaths or euthanasia:  Ideally, oocytes should be recovered from the ovaries as soon as possible after the death of the animal.  If a minimum of 24 hours is anticipated before the death of the animal – call the Center for Conservation and Research (CCR) at Omaha’s Henry Doorly Zoo & Aquarium (see below) and request a kit for recovering and maturing the ovarian oocytes.  You will be sent media (Hepes-TL Solution containing heparin and antibiotics) for processing the ovaries and recovering the oocytes.  The solution should be warmed to physiological temperature before beginning.  Small ovaries should be sliced in approximately 5 mm sections using a sterile scalpel blade, then the follicles punctured using 25-20 gauge needles.  The oocytes can then be collected (using an embryo handing device provided in the kit or, alternatively, any type of micropipette with sterile tips) and placed directly into tubes of media containing oocyte maturation medium (TCM 199 containing hormones, serum, energy substrates and antibiotics).  These tubes should be placed into the transportable incubator (provided) which should be then sent by overnight courier to Omaha’s Henry Doorly Zoo & Aquarium CCR.  Once liberated from the ovarian follicles, the oocytes will resume meiosis and mature to metaphase of meiosis 2 in approximately 24 hours at which time they will be prepared for in vitro fertilization by staff at the CCR.




                        Reproductive Physiology

                        Center for Conservation and Research

                        Omaha’s Henry Doorly Zoo & Aquarium

                        3701 South 10th Street

                        Omaha, NE  68107-2200

                        Phone: 402-733-8401, ext. 5055

                        Or Zoo Hospital

Phone: 402-738-2080



Participation in this is voluntary, but highly encouraged for the benefit of the species.  The long-term storage of the gametes and the processing costs will be the responsibility of Omaha’s Henry Doorly Zoo & Aquarium.  We ask that institutions budget for overnight courier (testicles) or counter-to-counter (ovaries) shipping costs.  If you are unable to fund these costs, please contact us in advance to discuss alternatives.


*The SSP will authorize the use of any rescued gamete material, according to value, genetics, and banked quantity.  In the event of successful propagation, institutions will be contacted and breeding loan agreements and documentation will be drafted at that time.




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