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

[DESCRIPTION]
Version date: January 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. 

References: 
Brahmasani, S.R., Sontakke, S., Ghosh, S., Mallapur, G., Yadav, S., Vasudevan, K. (2021). GRB Manual- An Introduction to Genetic Resource Banks for Wildlife Conservation. 
Gaur, A., Umapathy, G., Vasudevan, K., Sontakke, S., Rao, S., Goel, S., Kumar, D., Gupta, B., Singh, D.N., Kumar, A. (2017). Manual for Biological Sample Collection and Preservation for Genetic, Reproductive and Disease Analyses. 
Trans Ova Genetics. (2022). Trans Ova Livestock Cloning. https://transova.com/service/cloning-services/
ViaGen Pets & Equine. (2021). Emergency Pets Protocol – U.S. Clients. https://www.viagenpets.com/emergency-pets/
White, C. L., & Dusek, R. J. (2015). Wildlife specimen collection, preservation, and shipment. In J. C. Franson, M. Friend, S. E. J. Gibbs, & M. A. Wild (Eds.), Techniques and Methods. US Geological Survey. https://doi.org/10.3133/tm15c4

[BEFORE_START]
Tissue Handling
Biopsied tissues 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 will decrease the chances of contamination. Collect as many samples as possible.

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 samples must be shipped to a biobanking laboratory via overnight express. You must use an insulated container. Ice packs must be used to ship testicular tissues only to maintain a temperature between 4-8°C. Considering that samples may be in queue at the receiving facility for a period of time before they are processed, ship your samples as soon as possible. Shipment Monday through Wednesday will guarantee arrival at the receiving facility before the weekend. Do not ship samples on Friday. Most facilities will not be available to receive shipments on weekends. If samples are collected Thursday through Sunday, samples must be stored in the fridge until Monday. Consult with the receiving biobanking facility for any questions regarding shipping timelines before 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 during shipment as well.

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.

[STEPS]
SECTION: Preparation
3. Proper protective equipment must be worn (gloves, etc). Sterility must be maintained as much as possible.
SECTION: Tissue Collection
4. Remove the biopsy container from the kit 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, only collection the ovary pair is needed.
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. 
SECTION: Tissue Preservation
9. Using an alcohol-resistant marker, label the biopsy vial with an identifier that matches exactly what is indicated on the biopsy form. Check to make sure that each vial is easy to identify with the information provided on the form. 

Required information to include on the form: 
Scientific name of animal
Sex of individual denoted as ♂ (male) or ♀(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 freeze samples.  

**Immediately ship overnight express. Do not wait to ship ovarian tissues. For samples collected on Thursday-Friday, contact receiving facility to arrange off-hour delivery**
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 biopsy vial with an identifier that matches exactly what is indicated on the biopsy form. Check to make sure that each vial is easy to identify with the information provided on the form. 

Required information to include on the form: 
Scientific name of animal
Sex of individual denoted as ♂ (male) or ♀(female)
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 the insulated Tupperware to transport your vials. If you do not have the Tupperware on hand, 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: Preparation
2. Record all information indicated in the biopsy form, including a picture of the animal for identification and GPS location where the animal was found. 
SECTION: Shipping
11. Place the biopsy vials inside the Tupperware container included in your kit. 

  


SECTION: Shipping
12. Close the lid and place the Tupperware container into the vinyl lunch bag provided in your kit. 

   



SECTION: Shipping
13. Place the vinyl lunch bag into the styrofoam container.

  

SECTION: Shipping
14. Close the lid to the styrofoam container. Enclose all paperwork to the top, including the Biopsy Form. Close the shipping box and place the prepaid shipping label to the top. 

  



SECTION: Shipping
10. When you are ready to ship the samples, make sure the ice packs are frozen ahead of time. 
Detailed video instructions on 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.
Photo credit for the following photos: ViaGen Pets & Equine, 2021
 
  

SECTION: Shipping
12. Place the biopsy vials inside the insulated Tupperware container included in your kit. If you do not have the insulated Tupperware container, 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. Close the lid and place the Tupperware container into the vinyl lunch bag provided in your kit. 

   



SECTION: Shipping
14. Place the vinyl lunch bag into the styrofoam container, on top of the first ice pack. 

  

SECTION: Shipping
15. Place the second frozen icepack on top of the vinyl lunch bag. 

  

SECTION: Shipping
16. Close the lid to the styrofoam container. Enclose all paperwork to the top, including the Biopsy Form. Close the shipping box and place the prepaid shipping label to the top. 

  



SECTION: Preparation
1. Pre-chill icepacks in the freezer the day before planning to collect and ship samples (testicular tissues only).
SECTION: Shipping
15. 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: Shipping
17. Contact the receiving facility when you have shipped your sample and provide the tracking number so that personnel know when to expect your shipment.