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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
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.
Current June, 2015
AZA Amur leopard Species Survival Plan Program Leader and
Amur leopard Studbook Keeper – North America
P.O. Box 3268
423 West 38th Street
Erie, PA 16508
Phone: 814-864-4091 Ext 226
AZA Amur leopard Species Survival Plan Vice-Chair
Beth Jo Schoeberl
Curator of Primates
Denver Zoological Society
2300 Steele St.
Denver, CO 80205
AZA Amur leopard Species Survival Plan Secretary
Assistant Curator of Mammals
800 Cherokee Ave. SE
Atlanta, GA 30315
Amur leopard International EEP Studbook Keeper
Zoological Society of London
Veterinary Advisor- Amur leopard SSP- also interim contact for post mortem gamete preservation
Julie Napier, D.V.M
Center for Conservation and Research
Omaha’s Henry Doorly Zoo and Aquarium
3701 S. 10th St.
Omaha, NE 68107
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
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.
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
Studbook No: _______________________ In house or ISIS number: ____________________
In-house identity (microchip, tattoo, name): ____________________________________________________________________________
Date of birth: _____________________________ Weight: __________________
Date of death: _____________________________
Date of necropsy: __________________________
Necropsy number: _________________________
STANDARD FROZEN (-70oC IF POSSIBLE) TISSUE CHECK LIST:
Please hold samples at your institution for future toxicological or nutritional analysis if necessary.
STANDARD FIXED TISSUE CHECK LIST:
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.
____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
____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
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
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.
FOR ANY SHIPMENT, PLEASE NOTIFY SOMEONE AT THE REPRODUCTIVE BIOLOGY LABORATORY AT OMAHA’S HENRY DOORLY ZOO & AQUARIUM CCR AS SOON AS POSSIBLE AS TO WHAT IS BEING SHIPPED AS WELL AS ANY PERTINENT TRACKING INFORMATION AT TELEPHONE: 402-733-8401, EXT. 5055; FAX: 402-733-0490; E-MAIL: REPRO@OMAHAZOO.COM . SEND TISSUES IN CARE OF:
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
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.
Leigh Clayton, Director of Animal Health at the National Aquarium, speaks about being an aquarium veterinarian. Listen to the PODCAST.