TITLE: Reproductive Tissue Collection (Mammals): Post-mortem Sampling
AUTHORS: sanaz.arenivas, Barbara Durrant, comizzolip, mhouck, Jacquelyn Mountcastle, Budhan Pukazhenthi, phil.purdy

[DESCRIPTION]
Version date: April 2023

The following protocol illustrates how to collect and ship living reproductive tissue from a deceased wild or captive mammal for long-term cryopreservation. Collected tissues can either be immediately cryobanked at -196°C or processed for gamete collection and later cryobanked at -196°C. 

To print this protocol: Click the arrow to the right of the "MORE" button at the top of this page and select the option to print or download as PDF version. 

[BEFORE_START]
Tissue Handling
Collected specimens are stored in vials containing a nutrient-rich media to keep the cells alive and a mixture of antibiotics to prevent bacterial growth. All samples must be collected under aseptic conditions to avoid contamination. Using sterile tweezers, scalpels, and scissors, as well as cleaning the sampling site with alcohol or surgical scrub (e.g. chlorhexidine) will decrease the chances of contamination. Collect testes and/or ovaries in a sterile container large enough to hold the entire organ, such as a large 50ml centrifuge tube, a clean Tupperware, or ziplock bag.

Cold Storage
Avoiding temperature fluctuations is very important for preserving the tissue samples. Use ice packs for transport of testicular tissues only from the field and ensure refrigeration is available immediately after returning from the field site. Do not use wet ice or other commercial therapeutic packs. Chill ice packs in the freezer the night before collection. Frozen ice packs will remain cold in an insulated container for up to 24 hours. If the field site is more than 24 hours from refrigeration, plan to bring a portable electric refrigerator. Vials containing tissue should not have direct contact with the ice packs. Never freeze tissue biopsies before shipping to a biobanking facility.

Shipping
All testicular tissue samples must be shipped to a biobanking laboratory via overnight express. Shipment Monday through Wednesday will guarantee arrival at the receiving facility the next day. Do not ship samples on Friday. Immediate transport of ovaries to the receiving facility is highly advised. Overnight shipment of ovarian tissue is not recommend but may be done if it is the only option available. Consult with the receiving facility for any questions regarding shipping timelines before collecting or sending samples. 

[GUIDELINES]
The collection of samples should be opportunistic and follow all applicable regulations.  Reproductive tissues collected from deceased animals should be collected as close to the time of death as possible to avoid tissue decay. Temperature, body condition, and time are factors that accelerate or decelerate tissue decomposition, dictating the chances of collecting living cells. 

If the animal dies suddenly or if the animal must be euthanized, the carcass or any tissues must not be frozen. The carcass can be kept in a cool area and reproductive tissues must be dissected immediately and shipped the same day as collection to a biobanking facility. 

Testes maximum time after collection before arrival at facility: 24 hours, at 4-15°C
If testicular tissue is warm at time of collection, it should be kept at room temperature during the collection process. If the tissue is cold, it must be kept cold throughout the process to avoid rewarming and recooling. Testicular tissues must be kept at 4°C during shipment.

Ovaries maximum time after collection before arrival at facility: 4 hours, at 15-20°C 
Ovaries held at low temperatures (less than 15°C) for more than 24 hours will begin to rapidly degrade the oocytes inside. Ovarian tissue must be kept at 15-20°C at all times. Contact receiving facility to discuss transport of samples prior to collection.

For any animal carcass found in the wild, time, ambient temperature, and storage methods are critical factors that can impact how quickly a sample must be collected. If the time of death is unknown, tissues can be harvested as long as it has not been frozen, become necrotic, or started decomposing. In such cases, tissues should still be shipped immediately.

For urgent questions regarding protocol steps or collection guidelines, please contact the Revive & Restore Biobanking Team at informedbiobanking@reviverestore.org 

[STEPS]
SECTION: Preparation
1. Pre-chill icepacks in the freezer the day before planning to collect and ship samples (testicular tissues only).
SECTION: Preparation
2. Record all information about the species and individual, including a picture of the animal for identification and GPS location where the animal was found. 
SECTION: Preparation
3. Proper protective equipment must be worn (gloves, etc). Sterility must be maintained as much as possible.
SECTION: Tissue Collection
4. Use a sterile container large enough to hold the entire organ, such as a large 50ml centrifuge tube, a clean Tupperware with a tight-sealing lid, or a durable ziplock bag. Gather containers and have them readily available. 
SECTION: Tissue Collection
5. Wet the entire sampling area of the carcass in rubbing alcohol and blot with sterile gauze. 
SECTION: Tissue Collection
6. With sterile scissors or scalpel, dissect open the pelvic cavity. The reproductive tract will lie under the large intestine/rectum. 
SECTION: Tissue Collection
7. Collect the entire reproductive tract, including both ovaries, the uterine body, and oviduct. For larger animals, collect only the ovaries. 
SECTION: Tissue Preservation
8. Thoroughly soak gauze in phosphate buffered saline (PBS) and antibiotics and gently wrap around the tissue. Ensure that the gauze fully covers the tissue, keeping it moist. Place in sample container and close the cap tightly. If the specimen is too large for the container, you may use a clean ziplock bag.
SECTION: Tissue Preservation
9. Using an alcohol-resistant marker, label the container with an identifier that matches exactly the recorded metadata. Check to make sure that each container is easy to identify with the information provided.
Required information to record: 
Scientific name of animal
Sex of individual denoted as♀(female)
Date of tissue collection
Tissue type
Any other identification number of individual
SECTION: Tissue Preservation
10. Ovarian tissue must be kept at 15-20°C. Do not refrigerate or freeze samples.  

**Notify the receiving facility immediately.**
SECTION: Transport
12. Enclose all paperwork and sample/animal information in the insulated shipping container. 
SECTION: Transport
13. Contact the receiving facility to arrange immediate pick up or shipment. 
SECTION: Tissue Collection
5. Before dissecting from the body, clean scrotum thoroughly with rubbing alcohol.
SECTION: Tissue Collection
6. Cut away the testicles from the base of the body using a sterile scalpel or scissors. Use gauze to absorb blood from the cut portion of the testis. Discard used gauze. 
SECTION: Tissue Preservation
7. Thoroughly soak gauze in phosphate buffered saline (PBS) and antibiotics and gently wrap around the tissue. Ensure that the gauze fully covers the tissue, keeping it moist. Place in sample container and close the cap tightly. 
SECTION: Tissue Preservation
8. Using an alcohol-resistant marker, label the container with an identifier that matches exactly the recorded metadata. Check to make sure that each container is easy to identify with the information provided
Required information to record: 
Scientific name of animal
Sex of individual denoted as ♂ (male)
Date of tissue collection
Tissue type
Any other identification number of individual
SECTION: Tissue Preservation
9. Place samples on cold ice packs in an insulated cooler for transport back to your facility. Use about 3 inches of newspaper or bubble wrap to ensure that the tubes are not in direct contact with the ice packs. Do not freeze samples.  

**Immediately ship overnight express. Do not wait to ship testicular tissues. For samples collected on Thursday-Friday, contact receiving facility to arrange off-hour delivery**

 
SECTION: Shipping
10. When you are ready to ship the samples, make sure the ice packs are frozen ahead of time. 
Below is a detailed video example of how to pack your box:
 

SECTION: Shipping
11. Open the shipping box and place one frozen ice pack at the bottom of the styrofoam container inside.
SECTION: Shipping
12. If you do not have the insulated Tupperware container like the one demonstrated in the video, wrap each vial in bubble wrap or newspaper and secure with tape. Add additional 3 inches of bubble wrap or newspaper between the samples and the icepacks to ensure that they do not have direct contact. 
SECTION: Shipping
13. Placed wrapped samples on top of the first ice pack.  
SECTION: Shipping
14. Place the second frozen icepack on top of the wrapped samples. 
SECTION: Shipping
15. Close the lid to the styrofoam container. Enclose all paperwork and sample/animal information. Close the shipping box and place the prepaid shipping label to the top. 
SECTION: Shipping
16. Contact the receiving facility when you have shipped your sample and provide the tracking number so that personnel know when to expect your shipment. 
SECTION: Transport
11. Place the containers with samples in an insulated shipping box. Add additional 3 inches of bubble wrap or newspaper between the samples to ensure that the samples do not move or become damaged during transport.