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Frozen Zoo

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Northern White Rhinos in Peril

Nola is one of two northern white rhinos living at the Safari Park and one of just six in the world.

Nola is one of two northern white rhinos living at the Safari Park and one of just six in the world.

Rhino-lovers worldwide suffered a tragic loss last week. It is with a heavy heart that I report the passing of Suni, a male northern white rhino living at the Ol Pejeta Conservancy in Kenya. Suni died a natural death at age 34 on October 18, 2014, leaving only six northern white rhinos in the world. This subspecies is critically endangered and is extinct in the wild: three remain at the conservancy in Kenya, a zoo in the Czech Republic houses one, and two live at the San Diego Zoo Safari Park.

Northern white rhinos are in peril because of poaching. Some cultures believe that rhino horn is medicine, which drives the price per ounce higher than that of gold. Rhino horn is actually made of keratin, which is the same substance that your nails and hair are made of. In addition, there are sustainable, FDA-approved medicinal alternatives to rhino horn, such as aspirin and Viagra. But that has not stopped the terrorist groups and organized crime syndicates who use poaching as a means to fund their illicit activities.

Northern white rhinos have had an exceptionally troublesome history. Their cousins, the southern white rhinos, are also highly poached for their horns. However, in 1929, the South African government interceded on behalf of these rhinos and hired the poachers as game wardens to protect the rhinos. The poachers at the time were impoverished farmers, so offering them an alternative source of income meant that they no longer needed to poach to supplement their livelihoods. This strategy worked: 40 years later, the number of rhinos in South Africa increased tenfold. North Africa was unable to employ a similar strategy to help the northern white rhinos because North African countries at the time were fraught with civil war, poverty, and disease. Governments were so worried about keeping their citizens alive that they had little time or money to spare for the rhinos. And, until recently, scientists thought northern and southern white rhinos were the same species, so this lack of funds did not seem important.

Our Frozen Zoo houses the genetic material of 12 northern white rhinos.

Our Frozen Zoo houses the genetic material of 12 northern white rhinos.

Dr. Oliver Ryder at the San Diego Zoo Institute for Conservation Research discovered that northern white rhinos are a separate subspecies by examining mitochondrial DNA. Even though this subspecies will go extinct in our lifetime, the Institute for Conservation Research has created a ray of hope for the future in its Frozen Zoo®.

The Frozen Zoo contains viable cell cultures from many different species that have been cryogenically frozen in liquid nitrogen (think Han Solo in Star Wars). The Frozen Zoo houses the genetic material of 12 northern white rhinos; from these samples, scientists like Dr. Ryder can generate pluripotent stem cells. These cells can be triggered to create any tissue in the body. Such technical advances make southern white rhino surrogacy and cloning possibilities for the future of northern white rhinos.

In the meantime, guests can visit two of the world’s remaining six northern white rhinos at the Safari Park. Nola, a female born in 1974, lives in the South Africa field exhibit; Angalifu, a male born in 1972, lives in the Central Africa field exhibit. Both of them are past breeding age, so they are living quiet lives of retirement with the other wildlife in their field habitats. Guests can see these two unique rhinos by taking the Africa Tram tour, a Cart Safari, or a Caravan Safari.

Elise Newman is a Caravan Safari guide. Read her previous post, Who Likes Rain: Giraffes, Rhinos, or Elephants?

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Fascinating Fibroblasts: New Uses for Frozen Zoo Cells

We use fibroblasts to examine how environmental chemicals interfere with reproduction in endangered species.

We use fibroblasts to examine how environmental chemicals interfere with reproduction in endangered species.

As a graduate student, I first learned the story of Henrietta Lacks from a professor of mine. She is a woman that forever changed biological research without being a scientist, physician, or high-level researcher. As told in The Immortal Life of Henrietta Lacks, she was a patient suffering from an aggressive form of cervical cancer. While receiving medical treatment, her tumor was biopsied and researchers tried growing her cells in a petri dish. They discovered that her cells possessed the unique ability to grow almost as aggressively outside of her body as they did inside. As Henrietta’s cells continued to grow in the lab, they were named using the first two letters of her first and last name. And with that the world’s first immortal cell line, would be called HeLa. Today, HeLa cells are still growing in research labs around the world, nearly 60 years after Henrietta’s passing.

HeLa cells became workhorses for biomedical advancement by being used to determine the amount of DNA in human cells, discover new effective cancer treatments, and develop the polio vaccine. Each of these research breakthroughs was possible because of the extraordinary ability of HeLa cells to be kept alive outside of the body. Thanks to a greater understanding of proper cell culture conditions, there are now thousands of cell lines in existence from all kinds of different species.

An unparalleled collection of animal cell lines lives within the Frozen Zoo® as part of the San Diego Zoo Institute for Conservation Research. Currently there are cells, called fibroblasts, from approximately 900 species represented by over 9,000 individuals. It is the largest “zoo” of its kind, harboring samples from some of the world’s most endangered animals. There are even fibroblasts in there from extinct species.

The ability to work with cells growing in culture opens the door to many research possibilities. For example, our Reproductive Physiology lab is interested in testing the effects of environmental chemicals on endangered species reproduction. Let’s say you wanted to conduct this work in mice. All you would need to do is simply get a colony of mice, expose them to whichever chemicals you were interested in testing, and measure how reproduction was affected. But we don’t work with mice. We work with animals like rhinos. We will never have, nor would we ever want, an experimental rhino colony. However, we do take advantage of cell lines to determine how environmental chemicals might affect rhino reproduction.

To do this we take copies of rhino DNA and stick them into primate cells in petri dishes so that they make the rhino proteins that regulate reproduction. Then we expose those rhino proteins to different chemicals and measure how the two interact by measuring the production of a “reporter,” which (are you ready for this?) happens to be an insect enzyme. It sounds absolutely bizarre, but believe it or not, this is a well-accepted, common approach. The problem is, we can’t help but also ask, “How well does what is happening in this petri dish reflect what happens in a 5,000-pound rhino?” An answer to that question may lie in the Frozen Zoo.

In collaboration with the Institute’s Genetics Division, we are exploring new ways to use Frozen Zoo fibroblast lines to get a more accurate idea of how environmental chemicals may affect endangered species reproduction. This idea all started with our interesting discovery that many fibroblasts in the Frozen Zoo make proteins that regulate reproduction, even though they are usually taken from nonreproductive tissues. We have also found that, like HeLa cells, individual fibroblast lines possess their own unique qualities, and we can take advantage of this to address specific research questions. So instead of the science fiction-sounding rhino/primate/insect system, we can treat rhino cells directly with chemicals to which rhinos may be exposed. Everything about those cells, from the membrane to the DNA to protein, is made in the same way it would be in a rhino. In other words, we think what happens in those rhino cells paints a much more accurate picture of what happens in a whole animal. Sure, they are still cells growing in a dish, but cells alone can be extremely powerful tools. Just look at what Henrietta and her cells have accomplished.

Christopher Tubbs is a scientist in the Reproductive Physiology Division of the San Diego Zoo Institute for Conservation Research.

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Ear Notches: Trash or Treasure?

Our newest reindeer calf’s DNA will soon be part of our Frozen Zoo.

We have over 8,500 individual animal cell lines represented in our Frozen Zoo®, a cell line collection started in 1975 by San Diego Zoo Institute for Conservation Research founder Kurt Benirschke and his laboratory technician, Arlene Kumamoto. They started establishing cell lines on what was an easy source of material at that time: ear notches. The San Diego Zoo Safari Park had just opened a few years earlier and was home to many hoofed animals (deer, gazelles, antelope, oryx). All of these animals were ear notched as a way to identify individuals out in the field (see post Picking One from the Herd). These small ear-notch pieces were put into vials with a transport media, instead of being disposed of, and taken back to our Genetics Laboratory.

A vial containing an ear notch sample in transport media.

We still receive many ear-notch samples each year on hoofed animals new to our collection. Once we have the samples at the laboratory, the ear notch is processed and set up. We log in all of the information on the animal—species, common name, identification number, and birth date—into the Frozen Zoo database. Then the sample is taken into our tissue culture laboratory and, while working in a biological safety cabinet, or “hood,” the sample is set up. To do this, we first clean up the ear notch by removing any hair or debris. Then we cut the sample into very small pieces, and the pieces are covered with an enzyme and put into an incubator for around four hours to digest.

An ear-notch sample is processed in our Genetics Lab.

Next, when the sample looks digested, it is put into a flask (a sterile tissue culture vessel), and a special media is added and then incubated. In the next 72 hours, we hope to see cells attached to the bottom of the flask. These cells will then start to divide and grow for about three to four weeks until we have enough cells to freeze and make this animal part of the Frozen Zoo’s cell line collection. At this point, we have this animal’s DNA saved as a living cell line. At any time we can thaw a vial of cells and put them back into the incubator, and they will start to grow and divide again.

Fibroblast cells from a Siberian reindeer.

Recently, we received an ear notch from our Siberian reindeer calf. We have his cells growing, and soon his DNA will be part of the Frozen Zoo. Even though I have seen the start of many cell lines, it still seems like magic when you see those first cells start to grow and divide. What species would you choose to add to the Frozen Zoo?

Suellen Charter is a research coordinator for the San Diego Zoo Institute for Conservation Research. Read her previous post, Counting Chromosomes.

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Counting Chromosomes

Lowland gorilla karyotype

On any given day, I never know what species I will be working with in the Genetics Laboratory of the San Diego Zoo Institute for Conservation Research. Today it was a western lowland gorilla (48 chromosomes), an eastern red kangaroo (20 chromosomes), and a hooded pitta (too many micro-chromosomes to count, but we can confirm the species and gender).

Fibroblast cells

In 2011, we added 23 new species to our Frozen Zoo® fibroblast cell line collection: 13 birds, 4 reptiles, 5 mammals, and 1 amphibian. This is amazing, as we already have about 900 species/subspecies represented in the Frozen Zoo, so therefore it is difficult to get samples from species we do not already have represented. We also looked at the chromosomes and did karyotype case studies on 270 different individual animals this past year.

We start with a small skin biopsy (about the size of a pea), and from that we are usually able to establish a cell line. These cells are then frozen in liquid nitrogen (-321 degrees Fahrenheit or -196 degrees Celsius) and placed into boxes and racks that go into the freezers that are the Frozen Zoo. Every cell line in the Frozen Zoo is then checked for quality control by thawing one vial of cells. We see how well the cells recover from freezing and also harvest the cells in metaphase and look at the chromosomes.

Hooded pitta metaphase spread

Karyotyping is the best part of my day. It’s like doing a jigsaw or crossword puzzle: you never know how it’s going to work out or what your picture will look like, but when you finish the puzzle, it is really great! Chromosomes are lined up by shape and size, and then their karyotype, or genetic map, is compared to published karyotypes or ones that we have done previously on the same species. We can see any chromosomal abnormalities, and we can confirm the gender and species of the animal.

By the way, the hooded pitta was a female, and the eastern red kangaroo had an inversion in pair two.

Suellen Charter is a research coordinator for the San Diego Zoo Institute for Conservation Research.

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Freezing Cells: Out-of-the-Box Ideas

Here’s an example of Bornean bearded pig sperm, stained blue for ease of viewing.

On a Monday afternoon last month, my coworker Carly walked into the lab and said to me, “Acacia Animal Health Center called, and they just neutered an adult male pot-bellied pig. Do we want it?” My answer was, “Yes! I’ll pick the sperm sample up in the morning.” At this point you may be checking your web address to make sure you are still on the San Diego Zoo’s website. I assure you that you are!

Bornean bearded pig

I am the research coordinator for the Reproductive Physiology Division of the San Diego Zoo Institute for Conservation Research. One of my jobs is to find samples with which to try out new techniques for freezing reproductive cells. A large part of my job is to stock our Frozen Zoo® full of reproductive cells from the many exotic species we have in San Diego Zoo Global’s collection. We are fortunate to have many species of endangered pigs and hogs, including babirusa, Visayan warty pigs, and, my personal favorite, the red river hog. When one of these precious pigs or hogs passes away, we collect the sperm and freeze it for possible future use in artificial insemination or in-vitro fertilization.

Many advances in sperm freezing have been made over the years; however, what many people don’t realize is that one technique does not work for all sperm. So this is where we get to decide what new preparation techniques we are going to try to improve our sperm cell survivability. Since I was lucky enough to have sperm from a non-endangered species, the pot-bellied pig, I decided to go crazy and try three brand-new methods of freezing the cells while still freezing some of the cells using our old, reliable method for comparison.

I am always proud to recover reproductive cells from a very endangered species or genetically valuable animals, but there is just something very fun about working with the not-so-precious cells and having the opportunity to think outside of the box and try out new ideas. There is a real excitement in thinking that one of the new methods I use on the pot-bellied pig sperm might replace our old technique of freezing pig and hog sperm. It might even be the new best method or, even better, may one day revolutionize pig/hog sperm freezing and possibly make it to the cover of Animal Reproduction Science.

In the coming months I will be thawing out these vials of pot-bellied pig sperm to see if my out-of-the-box ideas will lead to recovering a sample of happily swimming sperm that are all racing to find the finish line, or if it is back to the box for me, with a list of new ideas to try.

Nicole Ravida is a research coordinator for the San Diego Zoo Institute for Conservation Research.

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We Can Learn from Nature

White rhinos are represented in our Frozen Zoo.

Here at the San Diego Zoo, we are uniquely situated to introduce the concept of biomimicry in an engaging, hands-on way using the thousands of species in our collection. Educational programs at the Zoo are teaching and inspiring students, and the expertise of our scientists, curators, horticulturists, veterinarians, and others is helping to guide entrepreneurs, investors, and companies to find sustainable solutions to design problems by looking to the animals and plants right here at the San Diego Zoo and San Diego Zoo Safari Park. With all the ways that the Zoo is using biomimicry to change the way that people approach challenges in business, we don’t often stop to think about how biomimicry has impacted our own efforts to conserve endangered species. As it turns out, we have learned a lot about how to conserve nature from nature!

Cryopreservation, the process where cells or whole tissues are preserved by cooling to sub-zero temperatures, is a technique that the San Diego Zoo Institute for Conservation Research utilizes daily. After all, we do house the world’s most diverse collection of frozen sperm, egg, and cell lines in a collection we call the Frozen Zoo®! This collection of cells represents more than 700 species of animals, from mammals like rhinos and tigers to reptiles and amphibians. Specimens in the Frozen Zoo can be stored indefinitely and used for procedures such as artificial insemination, in vitro fertilization, embryo transfer, and cloning.

This process of freezing cells to preserve them is pretty complicated! It requires a lot of work protecting the cells first, so that ice crystals won’t form and break open the cell membrane, and our scientists work hard to develop protocols for each animal’s cells. Many animals in nature have their own protocols for surviving at sub-zero temperatures. The wood frog Rana sylvantica is distributed throughout North America, including northeastern Canada and Alaska. As with other species of frogs that live in cold climates and hibernate close to the surface in soil or leaf litter, the wood frog can actually survive freezing! The frog starts storing urea and glucose in its tissues in preparation for winter, where these two compounds act as natural cryoprotectants that reduce ice crystals and osmotic shrinkage of cells.

The larvae of the Alaskan beetle Cucujus clavipes puniceus can survive up to -100 degrees Celsius (-148 degrees Fahrenheit) by using the freeze-avoiding compound glycerol. Our scientists have been able to learn from these “experts” in the field to perfect cryopreservation, thereby ensuring that we will have this indispensable resource for generations to come.

Robin Keith is a senior research coordinator at the San Diego Zoo Institute for Conservation Research. Read her previous post, Go Play Outside!

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Freezing and Thawing: Not so Easy

In reading over some of the blog posts here on the Zoo’s Web site, I could not help but notice that there are few, if any, about the laboratory work done at the San Diego Zoo’s Institute for Conservation Research. Lab work is a little tough to write about because there are no cute animals, no stories of climbing mountains or hiking through deserts. The work, however, is vital to our conservation efforts, and so I’ve decided to make an entry now and then to describe some of the things happening here in the Reproductive Physiology lab, including a new challenge we’re working on!

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.

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Preserving Hawaiian Bird Cell Lines

Palila cellsThere is another side to the Hawaii Endangered Bird Conservation Program that happens at the San Diego Zoo’s Beckman Center for Conservation Research. Our Genetics Division has preserved the genetic material of many Hawaiian birds in the Frozen Zoo®, a large collection of frozen samples, including thousands of cell lines. Most of the cell lines in the Frozen Zoo are grown using a piece of skin tissue from a small biopsy, which can be taken during an animal’s regular veterinary exam.

This is not as feasible for birds due to their fragile skin and small size, so our chance to obtain tissue from Hawaiian birds comes after they have died. Usually we receive an eyeball or a section of trachea. Then that piece of tissue is diced up into tiny pieces and put into an enzyme that digests the connective tissue, freeing up the individual cells.

Andrea places prepared flasks in an incubator.Once the diced tissue has been “digested” by the enzyme for a few hours, we put the remaining material in a tissue culture flask with the appropriate cell culture medium, which is a liquid containing the nutrients cells need to survive, and place them in a heated incubator. We will “feed” the cells every few days by emptying the flask and putting new medium in. If all goes as planned, the cells will attach to the bottom of the flask and will proceed to divide until they take up all the space. When there are enough cells, we will apply another enzyme, called trypsin, that breaks up the bonds holding the cells to the flask so they float freely, allowing us to move the cells into new, larger flasks for continued growth. Eventually the population of cells has doubled several times. It typically takes around a month for this to happen; tissue from younger animals tends to grow more rapidly than from aged animals.

Preserved cells in the Frozen ZooThen it’s time to put them in the Frozen Zoo. Ordinarily, cells die when frozen because the water they contain forms sharp ice crystals, tearing the fragile cell membranes apart. To inhibit crystal formation, we add a chemical called a cryoprotectant (in this case, dimethyl sulfoxide) to the cells. The cells are then placed into several tiny one-milliliter vials and put in a computerized cryogenic freezer that lowers the temperature at a carefully controlled rate. A little over an hour later the cells have reached 80 degrees below zero, and they’re ready to be put into boxes for storage in the Frozen Zoo, where liquid nitrogen keeps them frozen at an even colder temperature: 196 degrees below zero. At any time, they can be pulled out of the liquid nitrogen, thawed, and put back into cell culture medium and they will continue growing! Cells can be kept frozen for many years in this suspended but living state; nobody knows exactly how long because the technology has only been around for a few decades. The Frozen Zoo thus provides a self-renewing source of DNA for researchers studying the genetic makeup of particular species.

Currently there are cell lines from over 45 Hawaiian birds in the Frozen Zoo, including nene, ‘alala, Maui parrotbill, puaiohi, palila, and Hawaii creeper. Sadly, in 2005 the po’ouli became the first extinct species represented in the Frozen Zoo. It will doubtless not be the last, but thanks to all the people working hard on this project, there is reason to hope that no more Hawaiian birds will end up on that tragic list.

Andrea Johnson is a research technician for the San Diego Zoo.