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About Author: Carly Young

Posts by Carly Young

2

Chilling Out: Preserving a Rhino Legacy

The Frozen Zoo® at the San Diego Zoo Institute for Conservation Research is a unique resource playing a crucial role in current conservation efforts and will be an indispensable tool for the future.

Samples of Anglifu’s tissues and sperm were added to the Frozen Zoo at the San Diego Zoo Institute for Conservation Research, which holds specimens from more than 1,000 species.

 

It was a Sunday morning when I got the call that Angalifu, our male northern white rhino, had passed away. I asked over and over, “Are you sure it was a northern white rhino?” thinking to myself there are only six left in the world and maybe even five if what the pathologist was telling me was true. After she confirmed it indeed was Angalifu, our 44-year-old northern white rhino, I began to panic. My boss and co-worker are the only other two people who know how to freeze rhino sperm and they both were out of town!

Collecting and freezing sperm from this extremely rare animal was now all up to me. This would probably be one of the most important days of my career—no pressure! Knowing I couldn’t possibly do everything on my own, I contacted the rest of the Reproductive Physiology staff, and Kaitlin Croyle and Chelsea Mannie graciously gave up their Sunday to help me.

A complete post-mortem exam is conducted on all animals that die at the Zoo and the Safari Park to document the cause of death and to preserve tissues for histological examination and research. I picked up Angalifu’s gonadal tissue from the Zoo, then drove to the Institute for Conservation Research at the Safari Park to begin recovering the sperm and processing the testicular tissue.

Staining samples of Anglifu's sperm allowed the author to check its integrity before freezing.

Staining samples of Anglifu’s sperm allowed the author to check its integrity before freezing.

The first thing I did was check to see if his sperm was still motile, and luckily it was. I made a few different stains to check the viability, acrosome integrity, morphology and plasma membrane integrity. All of this told me the quality of the sperm before I cryopreserved it. Time was of the essence, so while I was processing the sperm, Kaitlin and Chelsea were labeling 275 small cryovials in which we would freeze the sperm and tissue; 200 for the sperm and 75 for the testicular tissue. Labeling vials is a long, tedious process. Without their help I would have been there longer and the sperm quality would have been compromised.

Our team had prepared a plan of action in advance of Angalifu’s passing, hoping we would not have to use it anytime soon. We decided to freeze his sperm using two different methods, so that if one technique did not result in good viability and motility after thaw, perhaps the other method would have fared better. After all the vials were labeled, I placed the sperm—diluted with a protective buffer—into the vials and placed them in a 39°F (4°C) cold room to cool for two and a half hours.

While the sperm was cooling, we began to mince the testicular tissue and distribute it among vials with a buffer and a cryoprotectant, which would protect the cells from the damage of ice crystal formation during the freezing procedure. The cryopreserved tissue can be used to isolate spermatogonial stem cells for future assisted reproductive technology. The tissue samples were slowly frozen in a controlled-rate programmable freezer. When it was finished, I submerged the vials in liquid nitrogen and placed them in a large liquid nitrogen storage tank.

A total of 275 vials of material were expertly processed and preserved.

A total of 275 vials of material were expertly processed and preserved.

By the time the Angalifu’s testicular tissue was safely stored in the Frozen Zoo®, the sperm had nearly finished cooling. It was then time to add the cryoprotectant. The sperm was now at 39°F (4°C), and we didn’t want it to experience a rise in temperature due to warm cryoprotectant or by doing the addition at room temperature. So, we bundled up in jackets, removed 200 vial caps, and pipeted the cold cryoprotectant to each vial in the cold. It may not sound that hard, but when you must do so standing inside a huge refrigerator, it’s pretty difficult. We took turns exiting the cold room for a few seconds at a time to we warm our hands. Once the cryoprotectant was added, we froze the sperm in liquid nitrogen vapor. The first step is to place the vials on a Styrofoam block floating on the surface of a container of liquid nitrogen. After 15 minutes, we submerged the vials completely into the liquid nitrogen and stored them in the large tank.

It was a very long day working non-stop to help preserve this critically important sperm, but in the end it felt very rewarding. It is great to know that we did all we could to conserve the northern white rhino species. I am happy to say that we have 200 vials of sperm and 75 vials of testicular tissue from Angalifu stored in the Frozen Zoo.

Angalifu’s sperm, along with previously collected semen, will be utilized in the future to fertilize eggs through artificial insemination, in vitro fertilization, and/or intracytoplasmic sperm injection. Stem cells will be isolated from his testicular tissue and cultured to stimulate the manufacture of sperm. Although Angalifu did not reproduce during his lifetime, there is hope that he will make a future genetic contribution to the preservation of his species through artificial reproduction.

Carly Young is a senior research technician for the San Diego Zoo Institute for Conservation Research. Read her previous blog, The Python Challenge.

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The Python Challenge

Burmese pythons are an invasive species in Florida's Everglades.

Burmese pythons are an invasive species in Florida’s Everglades.

When a male reptile in the San Diego Zoo collection passes away, it is my job to freeze his sperm. Unfortunately, there has been so little research done on freezing reptile sperm that there are no guidelines in the scientific literature. So, we have to develop the protocols for ourselves, which requires a great deal of research and a lot of sperm samples. This scenario plays out all too often in the Reproductive Physiology Lab of the San Diego Zoo Institute for Conservation Research. We always need more samples! How could we know how to freeze golden lancehead sperm without any practice on this or any related snake species?

Our lab group has struggled with this problem for years and has come up with some creative solutions to the sperm shortage problem. My colleagues Nicole Ravida, Dr. Barbara Durrant, and I began scouring the Internet to find a way to collect large numbers of reptile sperm samples in a short period of time to use as models for endangered reptile species. That’s when we learned about the Python Challenge in the Everglades.

Carly and Barbara got an early start in the Everglades.

Carly and Barbara got an early start in the Everglades.

The Florida Fish and Wildlife Conservation Commission (FWC) launched the Python Challenge to raise awareness about Burmese pythons and how this invasive species is a threat to the Everglades ecosystem and its native wildlife. The Burmese python is one of the deadliest and most competitive predators in South Florida. With no known natural predators, population estimates for the python range from the thousands to hundreds of thousands. A severe decline in a variety of mammal populations in the Everglades over the last eight years coincides with the proliferation of the invasive Burmese python. Necropsies on the captured snakes reveal what pythons are eating, their reproductive status, and location data from the hunters that will help scientists figure out where the snakes are living—valuable data for researchers working to stop their spread.

The Python Challenge was a month-long harvest open to anyone, and we knew this would be our opportunity to collect many snake sperm samples. We immediately contacted the Invasive Species Program staff at the University of Florida, one of the Python Challenge partners, and the project all started to fall into place. Barbara and I arrived in Florida and immediately collected all the supplies and equipment we had sent ahead to Zoo Miami and then purchased thick sheets of dry ice. Lining a large Styrofoam box with the cold sheets, we fabricated a minus 112-degrees-Fahrenheit (-80 degrees Celsius) freezer for one of our freezing protocols. With everything we would need piled into our rental car, we finally made it to the hotel room and organized our temporary lab.

The Python Challenge check-in station was simple but effective.

The Python Challenge check-in station was simple but effective.

The next day we drove to the Python Challenge check station, which consisted of a pickup truck and a tent. Hunters came to the check station to have their snake(s) measured and documented by the Invasive Species Program staff. Prizes were awarded to the hunter who harvested the longest snake and the one who brought in the most snakes. We anxiously waited with the people from the University of Florida for a male snake to be brought in. Unfortunately, the first snake to arrive had been frozen the previous day. We needed fresh, cooled samples, not frozen, so we continued to wait for another snake, which came in a few hours later. We dissected out the vas deferens, where the sperm is stored, on the back of a pickup truck as the sun set over the Everglades. We immediately put the tissue in saline in a cooler and raced back to the hotel to process the sample. But it was a bust—no motile sperm. We just had to hope for better luck the next day.

The vas defrens were taken back to the makeshift lab in the hotel room for processing.

The vas defrens were taken back to the makeshift lab in the hotel room for processing.

The next morning we got a call from our colleagues at the University of Florida saying that they had two live snakes. This was fantastic news, because we would be able to obtain fresh sperm samples. During the snakes’ necropsies, we collected the vas deferens and drove an hour back to our hotel room to process the samples. Fortunately, both males had motile sperm. More sperm, in fact, than we had ever seen and certainly more than we could ship back to San Diego. After several hours of freezing the sperm in our homemade dry-ice box or in liquid nitrogen vapor, we received a call that another male snake was available. We drove back to the Check Station, arriving after dark. We removed the vas deferens in the back of the truck using my phone as our light source. We made it back to the hotel room for another five hours of processing and freezing, falling into bed at 1a.m. It was a very long day but a successful one, with sperm from three snakes safely stored in our shippers.

Our luck continued the next day, with an interesting twist. This time the live snakes had been brought to another checkpoint, and we would need to transport them to the University of Florida lab. It was a bit surreal to be driving down the highway with three large pythons in snake bags in the trunk. We wondered if we had violated the rental agreement when we promised not to carry pets in the car. It was worth the risk; snakes and humans arrived safely at the university, and we froze three more sperm samples back in our hotel room lab.

Overall, it was a successful trip to the Python Challenge in the Everglades. We froze 130 vials of sperm, shipping them back to San Diego. Then began the long process of thawing and evaluating each sample, comparing three different freezing protocols to determine which one resulted in the best post-thaw viability. We have analyzed the data, and we have an early winner among the protocols we tested. However, we will need to repeat the experiment with improved protocols to maximize sperm motility and membrane integrity, both of which are essential for potential fertility.

Although we will never use the sperm of this invasive species for artificial insemination (we certainly don’t want more Burmese pythons in the United States!), we have taken a big step forward in the development of sperm-freezing methods for its endangered relatives such as the Indian python and the Cropan’s boa.

Carly Young is a senior research technician for the San Diego Zoo Institute for Conservation Research.