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. [caption id="attachment_34530" align="aligncenter" width="1000"] Staining samples of Anglifu's sperm allowed the author to check its integrity before freezing.[/caption] 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. [caption id="attachment_34532" align="aligncenter" width="1000"] A total of 275 vials of material were expertly processed and preserved.[/caption] 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.