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endangered birds

4

When an Egg Won’t Hatch

Three San Clemente loggerhead shrike eggs are ready to be tested.

Three San Clemente loggerhead shrike eggs are ready to be tested.

When an egg doesn’t hatch, what went wrong? Captive breeding of critically endangered birds, where every egg counts, can be tricky. Here in the Reproductive Physiology Division of the San Diego Zoo Institute for Conservation Research, we are working hard to assist avian conservation programs by developing new fertility testing methods. One new method, which I am helping to develop, is in ovo sperm detection.

Barren eggs are not uncommon, and every year we receive hundreds of potentially infertile eggs from the
San Diego Zoo, San Diego Zoo Safari Park, and offsite breeding facilities. Numerous undeveloped eggs from a given breeding pair may indicate the pair’s inability to produce offspring. This can occur for a variety of reasons, including sterility, genetic incompatibility, or the absence of mating. Although discovering the absence of sperm within an egg does not provide all the answers, it is a key piece of the puzzle that can help narrow down the potential cause of a reproductive problem.

When eggs arrive in our lab, the yolk membranes are removed and stained for sperm. There is nothing more exciting than locating a sperm under the microscope! Not only does this indicate that the male is producing viable motile sperm, but their presence provides hope that offspring from that pair may be possible in the future. Most of the eggs received by our lab are partially incubated, and we know that sperm slowly degrade during the incubation process. The determination of a sperm-degradation timeline is especially helpful for kiwi conservation. The kiwi has an unusually long incubation period of almost three months, so their eggs spend a significant amount of time incubating before they are declared potentially infertile and sent our way for sperm detection. Although one of the female kiwis at the Zoo is producing eggs, none of them have hatched. But because we have been able to identify sperm in a few of her eggs, now we know that she is actively breeding!

Last year, I also analyzed eggs from the San Clemente loggerhead shrike, a subspecies for which we have been managing a breeding facility funded by the U.S. Navy since the early 1990s. One of the captive pairs had laid a clutch and then abandoned it. The male was one of the program’s oldest birds, and Susan Farabaugh, Ph.D., the conservation program manager, suspected that he might be ready for retirement. She brought several fresh eggs to the lab from the fruitless shrike pair, as well as a presumed fertile egg from a prolific pair that had accidentally broken during incubation. Upon inspection, I was unable to locate a single sperm on the membranes from the unsuccessful pair, while the cracked egg contained numerous glowing sperm. This shed light on the root of the older male’s reproductive problem, and Susan immediately re-paired the female with a younger male. Her decision paid off and resulted in three additional chicks in 2012!

While sperm detection is not the only method for assessing breeding potential, it is an important management approach for our avian conservation programs. This technique is yet another tool to more confidently assess breeding pairs, allowing us to hatch the greatest number of chicks before the end of the breeding season. For birds on the verge of extinction, every chick brings the species one step closer to sustainability.

Kaitlin Croyle is an research assistant with the San Diego Zoo Institute for Conservation Research.

3

Working in the Dark Sheds Light on Bird Conservation

Mandi performs the delicate job of injecting an embryo.

The first time I sanded a hole in a chicken egg, peeled back the shell, and looked at the embryo, I was amazed. I could watch the blood being pumped in and out of the heart, and once I looked through the dissecting microscope, I was even able to see the individual red blood cells flowing through the vessels. Now, having worked in the Reproductive Physiology Division for a year opening numerous eggshells for injections, I still take a moment to look in wonder at this spectacle.

I work as a research associate studying avian reproduction. We are currently working on a model system to aid in preserving endangered birds by transferring gonadal stem cells (self-renewing cells that are located in the gonads and produce sperm or eggs) into chicken embryos. The cells that I inject, or transfer, are adult gonadal stem cells collected from male quails. I inject quail cells to develop the method, because it is easier to get quail testes than testes from exotic birds.

Let me give you a little background in the development of birds as it relates to our project. Their gonadal stem cells circulate throughout the bloodstream before they migrate to the gonad, where they mature and produce either sperm or eggs. Because we inject the adult male quail gonadal stem cells into early stage chicken embryos, they will also migrate to the gonadal ridge. When this rooster reaches maturity, he will produce both chicken sperm and quail sperm derived from the injected cells. The goal is to be able to use this technique to collect and inject gonadal stem cells from valuable exotic birds that have died of natural causes, so that even after death the sperm of that valuable bird can still be used for artificial insemination.

Let me take you through a typical day of embryo injections. The first thing that needs to be looked after are the needles. As you can imagine, the blood vessels of a chicken embryo three days after it is laid are very tiny. Our needles are smaller than a strand of hair at the sharpened tip. It takes a lot of time and patience to learn how to grind these glass capillary tubes (small, hollow glass tubes) into a fine, sharp point. The needles are mounted onto a micromanipulator apparatus that holds the needle steady during the injections. I load the needle with quail gonadal stem cells that are stained a fluorescent red so that we know which cells we injected and which are the chicken embryo’s own gonadal stem cells.

I sand a hole about the size of a quarter into the shell of a chicken embryo using your everyday belt sander. Gently, I peel off the shell to expose the embryo. Next, I very carefully inject the cells into one of the blood vessels while looking through the microscope. I seal the egg up using melted parafilm (a kind of scientist’s cling wrap) and a hot glue gun (minus the hot glue) and put the embryo back into the warm incubator.

Did I mention that with the exception of needle sharpening, all of this is done in the dark? The fluorescent-stained cells will lose their color if exposed to long periods of light. To keep this from happening, we use only one light directed away from where we are injecting, and the injected eggs are kept in a darkened incubator. At the end of our experiments, we identify the injected cells within the gonads of the chicken embryo by looking for red fluorescent cells using a microscope or a laser cell counter. This tells us whether the quail gonadal stem cells migrated to the gonads as predicted.

Our next step will be to use gonadal stem cells from exotic birds to assess whether they will migrate in the same way as the quail gonadal stem cells.

Mandi Roe is a research associate for the San Diego Zoo Institute for Conservation Research.

2

Puaiohi: Released and Breeding

A released puaiohi

Winter in Hawaii is usually thought of as a “snowbirds” paradise; people flock to the islands to get away from the nasty storms associated with mainland winters. This year, here on the islands, we got to experience our own sort of winter storm. Throughout the months of February and March we were pelted with winds and rain, leaving everyone quite soggy and begging for sun. With some of the rainiest months in recent history came some new inhabitants for the island of Kauai. The crews at the Keauhou and Maui Bird Conservation Centers transferred 22 puaiohi Myadestes palmeri to the Alaka’i Wilderness Preserve for release, with special thanks to Hawaiian Airlines for giving the endangered birds their VIP seats on the plane.

This year marked the 14th release of puaiohi to the Alaka’i Wilderness Preserve. On February 13, the first 12 birds made their journey from the Keauhou Bird Conservation Center on the Big Island to Kauai and were released one week later. We apply a “soft release” principle, where the birds are housed in a hack tower for one week to get accustomed to their surroundings before having to fend for themselves; after release, we offer them supplemental food near the release site. Before these birds are released, we fit them with a small radio transmitter, attached by means of a backpack. Using radio telemetry, we can track the individuals and find out how they are doing. Our partner, the Kauai Forest Bird Recovery Project, does most of these tracking efforts, helping us evaluate the movements and survival of the release birds.

This wild bird is paired with one of the HEBCP's released birds, making their nest at an artificial nest site, evidence of successful conservation measures in action. Photo credit: Mitch Walters

The first group was lucky to have nice weather for the first few days after release, enabling the birds to explore their new home in suitable weather. This being said, quite a few dispersed farther than anticipated. The second group of 10 birds traveled from the Maui Bird Conservation Center (MBCC) on February 28, delayed by a day due to storms. From then on, this group struggled just to stay dry. The puaiohi were released on March 8, again a few days late, but seemed to do quite well, considering the unusual weather. Several of them were spotted feeding at the supplemental feeding stations, which was a relief to the crews.

Since this year spring’s release, one particular female has been of interest: puaiohi #345, a young bird who hatched at the MBCC in 2011. In the past month, this particular female has been observed paired with a wild male and, crucially, incubating eggs in a nest of her own; further proof that our released puaiohi adjust to life in the wild and are contributing to the survival of the wild population. We are hoping that this nest produces chicks and helps to ensure the population keeps growing.

Over the last 13 years, 222 puaiohi that were hatched and raised through the Hawaii Endangered Bird Conservation Program have been released back to the wild. Over the same time frame, the wild puaiohi population is estimated to have doubled to more than 500 birds; it is thought that numbers are currently remaining stable. While we love to report success stories such as this, our conservation partnership has decided to temporarily halt our puaiohi release effort. This partnership is now moving on to the next phase of species recovery, re-focusing efforts from captive propagation to protecting the species in its natural setting. This includes predator control, providing artificial nest boxes that are predator proof, and habitat restoration.

With efforts to protect the wild nests and habitat of puaiohi, as well as other critically endangered species on the island of Kauai, we hope to see many more nests in the future, just like that of #345’s.

Rachel Kingsley is a research associate at the San Diego Zoo Keauhou Bird Conservation Center in Hawaii.

1

Interns Help Endangered Birds

Ashley works with a Maui parrotbill.

We come from different backgrounds and all have different goals and aspirations. We take a three- to six-month break from our lives to travel to Maui and make the trek up the steep, windy road to the former minimum-security prison, now bird propagation facility for Hawaii’s endangered avifauna.

There is a lot to be gained as an intern at the San Diego Zoo Maui Bird Conservation Center. Our duties are essentially those of a staff member, and we get the opportunity to experience firsthand what it’s like to be an avian conservation biologist. With this role comes lots of responsibility and learning to ensure that these rare and exquisite birds get the best care possible. Our primary focus is avian husbandry and providing a stimulating environment to encourage reproductive success.

Each day begins at the early hour of 6:30, when we meet with the staff to discuss what’s going on with the birds and the facility. We then go our separate ways, based on our assignment for the day: `alala, nene, forest birds, or projects. Our first priority is to observe the birds’ behaviors and make sure they’re healthy. Then we clean their aviaries, feed out the diets that were prepared the day before, and possibly socialize breeding pairs, depending on the season and how the birds have been getting along. Each routine requires care and attention to detail.

While a typical day for interns involves a lot of cleaning, diet preparation, and detailed observation of the birds, there is still plenty of time for various projects and lots of scope for innovation. Many of the buildings here are old and in need of constant TLC. Therefore we get many opportunities to play mechanic/plumber/construction worker/landscaper. It’s pretty amazing what a bunch of bird nerds are able to accomplish! New ideas and projects are always welcome. The creation of new nest boxes by fellow intern Dustin Foote has proven to be an excellent enhancement for nene propagation, and the geese have happily begun laying eggs in these more private shelters.

Although only staff members are permitted to handle eggs and chicks, we take advantage of every opportunity to observe vital procedures such as egg candling, during which we can observe embryonic growth at each stage of development. It’s an exciting experience to watch an `alala egg’s development from the first day of candling to when it finally hatches

Interns play an important role in making the program the success that it is today. And while we contribute lots to the program, what we gain is every bit as important. We arrive as wide-eyed interns, eager to soak up every bit of the experience we can. We gain knowledge in captive bird care and artificial propagation, as well as an appreciation for Hawaii’s precious and endangered avifauna and the elaborate process to restore these birds’ populations in the wild.

No matter what our goals are in life—be it veterinary medicine, zoology, conservation, or even something entirely non-animal related—this internship provides us with a varied and enriching experience. Most importantly, when the birds bred by the program are thriving in the wild, contributing to the survival of their species, we will know that we helped make that happen…. And that’s pretty cool.

Ashley Higby is an intern at the San Diego Zoo’s Maui Bird Conservation Center.