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About Author: Dianne Van Dien

Posts by Dianne Van Dien

9

‘Alala: We’re Getting Closer

By
Dianne Van Dien
– May 12, 2010Posted in: Animal Stories, Animals and Plants, Birds, Conservation at the Zoo, Hawaiian Birds

'Alala Kinohi

“I know, I know, I know, I know –oh-oh–oh,” says the crow through the door.

“What do you know?” I ask.

“Why-oh-why-wah-hah-hah” is the reply.

Obviously, this is not ordinary crow speech, but this is no ordinary crow. This is Kinohi, an ‘alala (Hawaiian crow) hatched in captivity 20 years ago. Growing up, he lacked other crows to socialize with, and so he developed an unusual vocabulary. But while we may find his human-like babble amusing, there is nothing funny about the fact that he will not breed.

With only 70 known ‘alala left on the planet (see post ‘Alala Season: Encouraging Start), Kinohi’s genes are extremely important. Last spring, it was decided that intervention was necessary. Kinohi was shipped from the Zoo’s breeding facility in Hawaii to the Wild Animal Park, and for nearly an entire year now, Dr. Barbara Durrant and I have been visiting him almost every day to train him for semen collection (see post Training an ‘Alala for an Important Job).

We like to think Kinohi looks forward to our visits. When we enter the anteroom to his new indoor-outdoor enclosure, he calls out, letting us know he knows we are there. As we cut up his mouse into a cup (the pieces are his reward for cooperating with us), Kinohi waits at the door, peering at our feet through the small space at the bottom. To position his eye low enough to see us, he hangs his head upside down, the top of his head resting on the floor.

Spring marks the beginning of the breeding season, and while the ‘alala in Hawaii have been building nests, Kinohi also has been responding to the lengthening daylight. A few weeks ago, he began saving part of his food reward in his beak. It is now his routine to pick up the pieces of mouse one by one, but after swallowing a few, he holds the rest in the back of his mouth. Then, when he has emptied the cup, he takes the morsels to the box that serves as his nest. We think he is trying to bring food to an imaginary mate. He mumbles in a whiny tone, moving the food pieces up and down over his tongue, giving his voice a gurgling quality. Eventually he leaves the nest box, lines up the pieces of mouse on a perch, and eats them one by one as though savoring each bite.

Bird sperm is quite different from the typical sperm of mammals; instead of a round head, the head of bird sperm is long and oval, and in some birds it has a corkscrew shape.

But something more exciting is now underway. Kinohi has begun producing sperm again. Although we have not been able to collect a sample directly from his cloaca, we did find sperm another way. This may sound strange, but after each training session, we wait for him to poop. Because feces, urine, and reproductive fluids all pass through the cloacal opening, it is possible to find sperm in a bird’s droppings. As soon as Kinohi defecates, we use a pipet to suck up the clear portion and take it back to the lab. In the past week, we have finally started to see sperm under the microscope.

The first day there were only a few of these cells, but a few days later, we found dozens in just a tiny drop. Only one of these sperm was motile, however; the rest were not even twitching, but that is not surprising or discouraging, because the sperm were in the inhospitable environment of feces and urine. Over time, the number of sperm should keep increasing. With a little more persistence, perhaps Kinohi will give us a pure sample that we can send to Hawaii, and perhaps he will sire offspring at last (via artificial insemination).

Dianne Van Dien is a research technician for the San Diego Zoo’s Institute for Conservation Research.

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10

Training an Alala for an Important Job

By
Dianne Van Dien
– August 6, 2009Posted in: Animals and Plants, Birds, Conservation at the Zoo, Hawaiian Birds
'Alala Kinohi

Alala Kinohi

Kinohi, a male ‘alala (Hawaiian crow), has finally found a place to call home-away-from-home here at the Wild Animal Park. After flying in from Hawaii in May (see previous post, ‘Alala Takes Extraordinary Flight), he spent his first 30 days in quarantine at the San Diego Zoo’s Harter Veterinary Medical Center (HVMC). He was then moved to the off-exhibit Bird Breeding Center at the Wild Animal Park, where we thought he would stay.

As crows are highly susceptible to West Nile virus, his enclosure had to be lined with fine-mesh mosquito netting. Unfortunately, the netting not only kept out the mosquitoes but also reduced airflow through the enclosure, and when summer turned up the heat, not even the water spraying down from the misters could keep his area cool. So, Kinohi was moved back to the HVMC. He now has a large room with a big skylight, two cardboard “nest” boxes, many perches, a bath, several toys, and, of course, air conditioning. His room has a camera, and the keepers can monitor his activity throughout the day. From what they can see, Kinohi likes this quiet and cool room; he appears calm and quite content.

And calm is much better for those of us working with him. Kinohi was sent here so that Dr. Barbara Durrant, director of Reproductive Physiology at the San Diego Zoo’s Institute for Conservation Research, and her staff could train him for semen collection. As one of only 60 ‘alala left in the world, Kinohi is very important to that population; unfortunately, he is behaviorally compromised and will not breed. His genes will be lost unless we intervene. Our goal is to collect semen to artificially inseminate a compatible female. We also hope to store some of the ‘alala’ semen in the Frozen Zoo. (See previous post, Freezing and Thawing: Not so Easy)

Working with Kinohi certainly is a challenge. Crows are very smart, and if we are not careful, he’ll end up training us instead of the other way around! Luckily, we have had the help of the keepers at both the HVMC and the Bird Breeding Center. They have shared their insights with us as well as making sure all of Kinohi’s needs are met, from food and water to appropriate perches and toys. Park animal trainer Kim Caldwell also gave us advice and taught us the proper way to reward Kinohi during training.

We’ve had to spend time getting to know Kinohi’s personality and gaining his trust. In the beginning, he would never sit still for long, and we felt successful if we had just one solid minute to pet him and left without any new bruises from his beak pounding on our ankles. But little by little we have seen Kinohi transform from an anxious bird, constantly hopping from perch to ground, to one who sits contentedly to get his head scratched. He now perches with his feathers so fluffed that he almost looks like a youngster, and he will even close his eyes while I rub his head feathers and Barbara pets his back and tail, abdomen and cloaca. (The cloaca is the opening through which birds secrete sperm as well as feces and urine.) Eventually he sidles away, hops over to his cardboard box, and fusses with the grasses inside, mumbling to himself. Then we try to lure him back. When he cooperates, he gets one of his favorite treats: a piece of mouse or a waxworm. When he refuses to come back, the session is over.

We feel positive about the progress we’ve made, but our time is running out. Last week, Kinohi started molting. When birds start expending energy to replace their feathers, it is a good indication that the breeding season is over and sperm production is ending. Thus, in our case, Kinohi’s molt tells us that we are not going to collect any sperm this year. Kinohi, however, will be spending the entire year here at the Wild Animal Park, and Barbara and I will be visiting him every day so that next year, when breeding season rolls around again, he should be ready to donate sperm samples to help save his species.

Dianne Van Dien is a research technician for the San Diego Zoo’s Institute for Conservation Research. Read Dianne’s previous post, Frozen Zoo: One Step Forward.

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0

Frozen Zoo: One Small Step Forward

By
Dianne Van Dien
– July 10, 2009Posted in: Conservation at the Zoo

Okapi

Okapi

A few weeks ago a female okapi died after complications giving birth. The death of such a beautiful animal is always a heartbreaking event, but it is also a time when we in the San Diego Zoo’s Reproductive Physiology Lab can do our part to preserve the gametes for possible re-introduction into a population at a later date. And so, as soon as they could, the pathology technicians performing the necropsy gave us the okapi’s ovaries.

Once we had the ovary tissue in the lab, we sliced and then minced it to release any oocytes (eggs). Our goal was to extract the oocytes so we could mature them, fertilize them, and if all went well, cryopreserve the resulting embryos in our Frozen Zoo®. We found only three eggs that were big enough to develop in vitro with current technology. Because ovaries also contain small, undeveloped oocytes, we always cryopreserve the tissue so our frozen samples can be utilized when the appropriate technology is developed in the future.

Working with oocytes is rather complicated for us because oocytes extracted from tissue have not yet matured (i.e., their chromatin [DNA of the cell nucleus] is not yet at the right stage for fertilization). We must put them in culture to mature them, a process called in vitro maturation (IVM). The oocytes of different species require different culture conditions, which presents another challenge because we work with a wide assortment of species and for most no IVM protocol has been established. We had never had the opportunity to work with okapi eggs before, and so for these three eggs we used a model protocol that was developed for domestic cattle. Okapis, of course, are not in the bovid (cattle) family (they are most closely related to giraffes), but since there is no giraffe IVM protocol, we started with a protocol we know works for other hoofed mammals.

After 24 hours in culture, bovine eggs are ready to be fertilized, but would the okapi eggs be ready? If we had a large number of eggs, we could stain a few to see if the chromatin had matured, but with only three, staining wasn’t an option, so we decided to use the same timing required for cattle oocytes. There are two basic ways we can go about fertilizing an oocyte in vitro. If the sperm are relatively robust, fertilizing the eggs is quite easy: we just add a drop to the eggs in a Petri dish. Okapi sperm, however, are very sensitive to the freeze–thaw process, and no one has found an effective way to cryopreserve them. We knew if we thawed a vial, the sperm would be in poor condition and would not be able to break through the oocyte’s membrane. In these cases, we must fertilize by intracytoplasmic sperm injection (ICSI), in which sperm is actually injected into the egg.

In theory, ICSI is a simple procedure, but anyone who has ever attempted it can tell you that actually performing ICSI is no easy task. It involves tremendous hand–eye coordination and a certain amount of finesse. The people who perform ICSI at human fertility clinics practice daily for several months before they are allowed to do the procedure for a patient. Needless to say, with all the other projects we have going on here, we don’t get that much practice. But we do know people who are willing to volunteer their expertise in this area, and so we made some phone calls. None of the people who helped us in the past were available, but one of them asked a coworker and he was happy to help. And one full day after the eggs had been placed in culture, Bill from the San Diego Fertility Center came to our lab and injected the eggs with the thawed sperm. As we’d expected, the sperm was not very good, but no worse than the sperm Bill often sees at his job.

After the eggs were fertilized, we moved them into a medium that is good for embryo development and we waited. Would the eggs cleave? If they had matured properly and the sperm had successfully fertilized them, we would see some cell division after 36 hours. However, there was no cell division. Feeling rather disappointed, we took the eggs out of the culture and stained them to look for pronuclei (the DNA of the sperm or of the egg before the two have united). We did not find evidence that DNA from the sperm had decondensed after being injected into the eggs. We did, however, see the chromatin of the eggs, and we learned that we had fertilized the eggs a little too early. The eggs had been in the process of maturing but weren’t quite ready for the sperm.

From this we learned two important things: (1) okapi eggs take longer to mature in vitro than cow eggs and (2) the media in the bovine protocol were indeed appropriate choices for beginning work with okapi eggs. Now the next time we get ovaries from an okapi, we’ll be one step further in knowing what to do. I hope the rest of our okapis will stay healthy, and this event won’t repeat itself for a long, long time.

Dianne Van Dien is a research technician in the San Diego Zoo’s Reproductive Physiology Lab.

Read her previous post, Freezing and Thawing: Not So Easy

Read other posts about okapis: Exciting Times at the Okapi Barn! and How the Okapi Spends Its Day.

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Freezing and Thawing: Not so Easy

By
Dianne Van Dien
– June 16, 2009Posted in: Conservation at the Zoo

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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