Hellbender Conservation Program at Nashville Zoo

Nashville Zoo is pleased to announce the first captive breeding of Eastern hellbenders, and the first controlled breeding of any hellbenders using biotechnology. Hellbenders and their close cousins the Japanese and Chinese giant salamanders are the only living members of an evolutionary and morphologically distinct family of salamanders that have remained unchanged since 60 million years ago during the age of the dinosaurs. All three species are now in decline and may be threatened with extinction unless conservation programs are developed. The St. Louis Zoo, a leader in hellbender conservation, reproduced Ozark hellbenders in an artificial stream system for the first time in 2011.

The baby Eastern hellbenders born at Nashville Zoo were produced using assisted reproductive technologies supported by research with a small group of 3 males and 1 female maintained at the zoo, in excellent health, for over 5 years in a laboratory setting. This conservation milestone is a result of a long term collaborative research project with a team of International researchers. Nashville Zoo Herpetology and Veterinary Departments have worked closely with Dr. Robert Brown, an Australian cryobiologist, to develop cryopreservation techniques and to develop a gene bank for the species. Dr. Vance Trudeau, a Canadian endocrinologist, helped develop techniques to stimulate reproduction of captive hellbenders through temperature cycling and hormonal induction in a laboratory setting. Bill Reeves, with the Tennessee Wildlife Resources Agency (TWRA) have also collaborated on and helped fund this project.

The adult hellbenders at Nashville Zoo have been maintained individually in 100 gallon aquariums with temperature and day length adjusted seasonally to simulate the hellbender’s natural environment. These hellbenders have been monitored monthly over the last 4 years with portable ultrasound equipment to determine the status and development of their reproductive organs throughout each season. While the female produced ovarian follicles and the males developed enlarged testes during the breeding season for the first two years in captivity, no sperm or eggs were produced. In 2009, hormonal induction was attempted with hCG (human Chorionic Gonadotropin). This hormone treatment stimulated the males to produce viable milt, but did not stimulate the female to produce eggs. At this time, Zoo staff worked with Dr. Robert Brown and developed cryopreservation techniques for hellbenders using captive animals.

Viability of the thawed sperm was assessed at Michigan State University with electron microscopy and staining techniques, both of which indicated that the thawed motile sperm was viable. In 2011, Zoo staff collaborated with Dr. Vance Trudeau at the University of Ottawa to utilize a new hormone protocol that he had developed for breeding frogs called Amphiplex. With the timing of the Amphiplex injection based on ultrasound determination of gonadal development, the Zoo staff was able to collect milt and eggs from our captive hellbenders for the first time at the Zoo in 2011. The milt was stripped from the male hellbenders and then placed on the eggs, but none of the eggs developed. In 2012, the Amphiplex injections again successfully stimulated the hellbenders to produce milt and eggs. We altered our fertilization and egg incubation techniques and have been able to successfully hatch our first baby hellbenders from eggs produced and artificially fertilized from our long-term captive animals. Only two offspring resulted from this breeding. This success is an important first step, and is line with Nashville Zoo’s commitment to conservation and propagation of endangered species.

It has taken five years to develop the techniques to reliably collect milt and eggs from captive hellbenders in a laboratory setting. Further refinement of incubation and fertilization techniques over the next few years will enable hellbenders to be reliably reproduced in captivity, and genetically fit offspring produced to suit various conservation needs, including eventual reintroduction into the wild.

For as yet unknown reasons, hellbenders have gone through rapid declines across most of their range over the last 30 years. The problem may be related to recruitment of young animals into the adult population. There may be a problem with larval survival in streams and rivers and only a few older animals from declining populations are found each year. Larvae are more sensitive to some diseases, stream siltation and pollutants. Another threat is dams that have resulted in isolated populations in feeder streams and rivers creating inbreeding problems through a lack of gene flow between populations.

Once assisted reproduction technologies are fully developed, they can be utilized to increase the genetic diversity in small isolated populations and repopulate extinct populations with genetically fit stock. Nashville Zoo staff have already cryopreserved milt from several watersheds and created the first practical gene bank for any amphibian species. Cryopreserved sperm in milt can be used to fertilize eggs anytime in the foreseeable future as it can remain viable for thousands of years.

Dale McGinnity, the Curator of Ectotherms, and project leader stated that this early success is the result of a broad international collaboration. Our Zoo’s Amphibian Specialist, Sherri Reinsch, and our Veterinary staff made the project possible. In addition, valuable collaborators include Dr. Robert Brown, Dr. Vance Trudeau, Dr. Heather Robertson, Joe Greathouse, Dr. Michael Freak, Dr. Brian Miller, Dr. Dalen Agnew, Dr. Carla Carelton, and Dr. Sally Nofs. Finally, a special thanks to Bill Reeves and TWRA for their support and for funding a collaborative SWG grant that helped fund this work along with statewide surveys, disease testing, and genetic work on Tennessee Hellbenders.