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About Author: Kaitlin Croyle

Posts by Kaitlin Croyle

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One Day in Suzhou for the Yangtze Giant Softshell Turtle

With great skill, the author carefully opens a turtle egg to remove a sample for examination under the microscope.

To uncover clues to the mystery of the lack of Rafetus hatchings, the author removes a sample from an egg to view under a microscope. Photo: Gerald Kuchling

With the last remaining breeding pair in captivity, the Yangtze giant softshell turtle Rafetus swinhoei is one of the world’s most critically endangered species. Housed together at China’s Suzhou Zoo, the pair’s breeding success is crucial to the survival of their species.

Although the female has laid hundreds of eggs since 2008, none of her eggs have hatched and the lack of Rafetus hatchlings over the last several years has raised more questions than answers. My involvement in the Rafetus breeding program at the Suzhou Zoo began this past summer with the trip of a lifetime to help save the Yangtze giant softshell turtle from extinction.

As part of my Master’s degree, I am working in the Reproductive Physiology Division at the San Diego Zoo Institute for Conservation Research, developing a technique for oocyte membrane-bound sperm detection (OMSD) for use in the management of endangered turtles and tortoises. OMSD tests for the presence of sperm in eggs that fail to develop an embryo. This information, in combination with behavioral observations, reproductive history, and veterinary examination, can be used to make educated decisions about breeding pairs to increase the chance for future offspring. For example, an egg containing sperm indicates that the male is producing functional sperm capable of progressing up the female’s oviduct to the oocyte. Sperm absence, on the other hand, can point toward a reproductive problem.

Although the female giant softshell turtle has laid numerous eggs, non have been fertile.

Although the female giant softshell turtle has laid numerous eggs, none have been fertile.

With the help of the Turtle Conservancy’s Behler Chelonian Center, we tested OMSD on a variety of turtle and tortoise eggs. This exciting innovation led to a whirlwind trip, organized by the Turtle Survival Alliance and the Wildlife Conservation Society, to fly me to the Suzhou Zoo so I could examine eggs of the critically endangered Rafetus. Within days of the female Rafetus nesting, I was on my way to Shanghai. After almost 36 hours of planes, trains, and automobiles, I was greeted in Suzhou by turtle conservationist and my personal hero, Dr. Gerald Kuchling. We went straight to work, as I only had one full day in China before I needed to take a flight back to San Diego. We picked up the eggs at the Suzhou Zoo, where I got a quick glimpse of the female Rafetus, then drove straight to Suzhou University and I spent the afternoon examining as many Rafetus eggs as possible. After several hours hunched over a microscope, I made the disheartening announcement that I was unable to confirm the presence of sperm.

So what is happening between the pair of Yangtze giant softshell turtles? My results indicate that the male Rafetus is either not capable of successful copulation and insemination of the female, not producing sperm, or that he is making low quality sperm that cannot fertilize the female’s oocytes. Although discouraging, this new piece of information provides valuable insight into the potential cause of past reproductive failure. What’s next for the Rafetus pair in Suzhou? First and foremost, a comprehensive reproductive exam is needed to evaluate the fertility status of the male Rafetus. If the male is producing viable sperm, but is not capable of breeding with the female, collection of his sperm followed by artificial insemination could be used to circumvent the problem. In the event that the male Rafetus is deemed infertile, the female will require a new breeding partner.

At this point, it appears that the greatest chance of bringing another male Rafetus to the Suzhou Zoo lies in the hands of Dr. Kuchling, who has been working with the Kunming Institute of Zoology to locate other Yangtze giant softshell turtles in the wild. In the meantime, we continue to hold our breath as we wait for the results of the male’s reproductive exam. Although the odds seem stacked against the Rafetus, only time will tell whether this species can be saved.

Kaitlin Croyle is a research assistant with the San Diego Zoo Institute for Conservation Research. Read her previous blog, When an Egg Won’t Hatch.

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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.