The most common task my fellow technician Nicole Ravida and I perform is the cryopreservation of male gametes – that is, we freeze the sperm of endangered species. The majority is collected from animals after they have died at the Zoo or Wild Animal Park; sometimes even other zoos send us testes (or ovaries) from their animals so we can add those gametes to our bioresource bank, the Frozen Zoo®. To date, we have banked over 14,000 vials of sperm from more than 850 individuals representing 260 species. Because sperm from different species differ in how well they endure the freeze and thaw process, we constantly are tweaking our methods. We do these experiments with sperm from model animals (i.e., domestic or non-threatened relatives of the species we need to save), and the sperm from endangered animals stay in the Frozen Zoo until a circumstance arises when they are needed.
Although the factors for preparing sperm for cryopreservation are many and their interactions are complex, the actual freezing of the sperm is not high tech. Years before I began working here, technicians devised a freeze method utilizing a Styrofoam box (pictured below) of certain dimensions with a certain level of liquid nitrogen inside. All you have to do is tape the vials of semen on top of a Styrofoam platform and float it on the liquid nitrogen. For a fast freeze rate, use a thin platform, for a slower rate, a thick platform. Close the lid on the box to allow vapor to surround the vials. (Nitrogen is liquid at an icy -320 degrees Fahrenheit or -195 degrees Celsius, and its vapor is nearly as frigid.) After 15 minutes, the sperm are frozen solid and the vials are ready to be plunged into the nitrogen and stored in a tank until they are needed. This is the basic freeze method we use for almost all mammalian sperm, or, at least, it was…
When you use Styrofoam boxes year after year, eventually they begin to wear out, get beat up and dented, and maybe even a little warped. So a couple years ago we noted that ours (all two still in stock) were rather worn looking. It was time to replace them. A company here in Southern California had made them for us in the past, and so we thought getting new ones would be simple. The company, however, was no longer in business. We then assigned one of our summer students with the task of finding another company. She found a local one, gave them the required dimensions, and when the boxes were made (we had to buy 18 at a time), she brought them back to the lab.
All should have been good and well, but it wasn’t. The Styrofoam wasn’t dense enough, so the liquid nitrogen was able to escape through the sides of the boxes, evaporating too quickly for us to maintain a set level for sperm freezing. But no problem, the company had a denser type and they made us 18 more boxes with the densest stuff they had. And yet the nitrogen still found its way out through the sides. Our student then tried sealing the outside of the box with paint. It didn’t help. She covered one completely in duct tape (surely all-purpose duct tape could save the day) but even this did not work. After a few more futile attempts to seal the boxes, she gave up. Nicole and I resorted to searching the Internet and calling companies to find a maker of dense Styrofoam, but we found no one who could help us. We resigned ourselves to the fact that they just don’t make Styrofoam like they used to, and we had 36 boxes to prove it!
In the end, we had to find something else to hold the liquid nitrogen, and we decided this something had to be easy to replace and easily duplicated by other labs; something anyone could buy. The standard ice bucket, present in almost every lab and available from many scientific companies, was a good candidate (pictured at top). It holds nitrogen well. However, ice bucket dimensions are not the same as that of our Styrofoam box, so we weren’t done yet. We had to figure out how much nitrogen was needed for freezing different types of sperm. Another summer student began this task by using a temperature probe to find a few levels that produced freeze rates similar to that of the Styrofoam box.
The next step was to do some test freezes to see how sperm fared when frozen in the ice bucket compared to the Styrofoam box. To make sure results are consistent, we have to do many freeze-thaw experiments—the same thing over and over, and, yes, this takes a long time. So far we have found that freezing in the ice bucket is actually better for the sperm of deer and antelope species than freezing in the box. A fantastic find! Our work is not done, however, and we still are in the process of figuring out how to best freeze the sperm of other mammals in the ice bucket. And what happened to those 36 not-so-dense styrofoam boxes? Don’t worry, they didn’t end up in a landfill. They went to Hawaii. Maybe they can’t hold liquid nitrogen, but they can keep things cold, and now they are serving the Hawaii Endangered Bird Conservation Program!
Dianne Van Dien is a research technician for the San Diego Zoo’s Institute for Conservation Research.