Wildlife Conservation: Breeding Eastern Tiger Salamanders

Amphibian breeding season is here! That means it’s time to start helping amphibians get in the mood for love and romance.

The Institution of Museum and Library Sciences (IMLS) recently awarded a $500,000 National Leadership Grant for the purpose of improving reproduction within captive assurance colonies of imperiled salamanders. The Detroit Zoological Society is one of the primary partners on this grant. My doctoral research focused on salamanders, their reproductive physiology and techniques to help them breed, and that very research was the basis for the recent IMLS grant proposal.

Other partners on the grant include Mississippi State University and Omaha’s Henry Doorly Zoo and Aquarium. Now that the grant was awarded, techniques must be taught to the other partners so we can all work together in salamander conservation efforts. I recently visited Mississippi State University in order to train the other principal investigators and the graduate students involved on this salamander grant in principles of natural salamander reproduction and performing assisted reproduction techniques with salamanders.

The training session involved eastern tiger salamanders (Ambystoma tigrinum), which are regionally threatened and endangered in various areas of North America. Like many species of salamanders, the tiger salamander is very difficult to breed in human care. Similar to other species in the “mole salamander” family, tiger salamanders respond to changes in air pressure and temperature when seasonal rain storms occur. These storms are what cue breeding behavior and are very difficult to replicate in human care. Without the ability to provide the appropriate “mood”, tiger salamanders in human care will not often feel romantically inclined. Natural breeding is always the first goal when breeding animals for conservation, but sometimes this is extremely challenging. In these cases, we use alternative techniques while we perfect replicating the natural breeding environment.

In vitro fertilization is a technique used to assist salamanders and other amphibians in breeding. Most salamander species undergo internal fertilization, in which the female picks up a capsule of male sperm, called a spermatophore, which the male has deposited into the environment. The female holds the sperm in an internal pouch that she later empties over her eggs as the eggs are laid. In in vitro fertilization of salamanders, sperm is collected from males by giving them a massage. Eggs are then collected from the female into a small dish where sperm is placed on top of the eggs. Just add water, and presto, you have salamander babies. Of course, it is never just that easy, but the concept is straightforward.

I trained the other primary investigators and students in other techniques as well, including cryopreservation – or freezing and long-term storage – of salamander sperm and sperm quality assessment. The training was very successful, with nearly 100 tiger salamander babies produced. Now the trainees can go on to teach more amphibian conservationists, and we can save more species by assisting with breeding!

– Dr. Ruth Marcec is the director of the National Amphibian Conservation Center at the Detroit Zoo.

Wildlife Conservation: Lending Amphibian “Sexpertise” to Other Accredited Zoos

I recently participated in a partnership to save an incredible species of amphibian: the blue spotted salamander (Ambystoma laterale). This animal is regionally endangered in parts of the Midwest and about 10 years ago, the Iowa Department of Natural Resources started a partnership with Omaha’s Henry Doorly Zoo. They wanted to breed these salamanders for release at locations where they once thrived but have now disappeared. Having had little success in doing so thus far, the organizations reached out and requested my amphibian “sexpertise”. I travelled to Omaha to deploy some of my special reproductive techniques with the blue spotted salamander assurance colony living there.

It comes as no surprise that these salamanders proved very difficult to breed. Blue spotted salamanders need an extraordinary amount of “salamander romance” in order to breed naturally. These animals undergo winter brumation (a hibernation-like state). In the spring, they will emerge from brumation when it begins to rain and when the temperature rises in order to migrate to breeding ponds. Their migrations can be over several miles, and most salamanders return to the same pond every breeding season. In captive breeding situations, we attempt to recreate the cues that the salamanders take from the wild in order to get them “in the mood” for breeding. We cool them down and offer them the opportunity to undergo brumation. We place them in rain chambers so they can experience an “indoor storm”. We also attempt to create a naturalistic pond for them to breed in with proper vegetation and leaf litter.

Sometimes, despite our best efforts, the natural cues we provide artificially are not enough to trigger breeding. Many amphibians are having difficulty breeding in the wild, due to changes in the climate and their habitat. Given that the natural environment is not currently providing ideal cues for breeding, it is especially difficult to perfectly recreate environmental breeding cues for animals in human care. When captive animals do not breed in response to the cues we provide, we often use hormone treatments to give them a little boost. These hormone protocols are meant to trigger breeding behaviors as well as the release of sperm and eggs. In the best-case scenario, the application of hormones will result in the animals breeding on their own. In the worst-case scenario, the animals will not breed on their own after the application of hormones and an in vitro fertilization (an artificial fertilization of the eggs with the sperm in a petri dish) will take place.

We used a combination of natural cues and hormones to stimulate breeding in the blue spotted salamanders. The amount of hormones I gave to the female blue spotted salamanders was determined by how developed their eggs looked on ultrasound. While giving the animals hormones resulted in many interesting behaviors, the salamanders did not breed on their own. After giving them time to breed on their own without success, I attempted in vitro fertilization. In order to perform this technique, I needed to collect eggs from the females and sperm from the males. There was one big problem with attempting this technique – the male blue spotted salamanders were not producing sperm. Fortunately, I had a back-up plan!

The blue spotted salamander is no ordinary salamander. In the wild, many female blue spotted salamanders breed through a special adaptation called kleptogenesis, meaning that they steal sperm. You read that correctly: sperm thieves. Most salamanders undergo internal fertilization. This occurs when the male deposits the sperm in a ball – called a spermatophore – into the environment. Through a courtship dance, the spermatophore is collected by the female. These special female blue spotted salamanders will find and collect spermatophores deposited by males from other, closely related, species (including Jefferson’s salamanders, tiger salamanders, and small-mouth salamanders). This is the part where it becomes complicated. Rather than using the sperm to fertilize their eggs and have hybrid offspring, an enzyme in the sperm activates the egg which begins to grow into an embryo. The rest of the sperm is discarded, and the offspring which develop are essentially clones of the mother. Knowing that blue spotted salamander females are often sperm thieves, I had the Omaha Zoo’s resident male tiger salamanders (Ambystoma tigrinum) on standby. I was able to collect sperm from the tiger salamanders in order to use in the in vitro fertilization of the blue spotted salamander eggs.

Unfortunately, the eggs did not fertilize with the tiger salamander sperm. While we did not achieve offspring, we learned what we need to do differently next time around. We certainly will not give up on these amazing animals. We will try to breed the blue spotted salamanders again later this year, using slightly different techniques.

Thank you to our partners at Omaha’s Henry Doorly Zoo and the Iowa DNR.

– Dr. Ruth Marcec is the director of the National Amphibian Conservation Center at the Detroit Zoo.

Veterinary Care: Ultrasound Exams for Frogs

Spring is in the air, and as I hike and bike the trails in southeast Michigan I find myself excited to be immersed in the lovely chorus of spring peepers and tree frogs as they prepare for this year’s breeding season. This also means it won’t be long until it is amphibian breeding season at the Detroit Zoo! Each year, the veterinary team works closely with our amphibian staff to provide support for our amphibian breeding programs. Our curators and keepers set up special breeding areas and adjust the temperature, light cycles, humidity and water access to simulate conditions in the wild and encourage natural breeding. They even set up simulated rainfall and play tape-recorded sounds of breeding calls collected from the wild.

Even with all of this, some of our endangered amphibians need a little more help to breed successfully.   At the Detroit Zoo, we have over a decade of experience administering special hormones to Wyoming toads and Puerto Rican crested toads to help with breeding. We have been very successful with these programs; two years ago the amphibian staff sent 3,914 Wyoming toad tadpoles to Wyoming and last year they sent 22,571 Puerto Rican crested toad tadpoles to Puerto Rico for release into their native habitat. This is important work and we are proud to be able to help.

As zoo husbandry staff and amphibian reproductive experts have gained expertise, it has become clear that not all amphibians respond the same way to changes in their environment and established hormone protocols. Two years ago, we purchased a high frequency ultrasound probe, and we have found that we can monitor the appearance of the ovaries and follicles as they develop and mature within female frogs and toads. This provides a very powerful tool for understanding the impact of the husbandry and hormone treatments that we apply, and will allow us to establish assisted reproduction methods for other endangered species.

This season we have some exciting things planned, and have started the early work to check the females we are hoping to breed. Last week, we performed ultrasound exams on our three female giant waxy tree frogs, and were able to see that they are all developing large, follicle-filled ovaries. Based on the appearance of the follicles, we think that they will be ready to breed in the next few weeks to a month.

This week, we will be conducting ultrasound exams on our endangered Mississippi gopher frog females. Once we see their degree of follicle development, we’ll be able to plan a hormone and husbandry strategy for this year’s breeding season. Hopefully we will have exciting news to share in the coming months!

– Dr. Ann Duncan is the Director of Animal Health for the Detroit Zoological Society and oversees the Ruth Roby Glancy Animal Health Complex.

Amphibian Conservation: Breeding Puerto Rican Crested Toads

April is an important time for the Detroit Zoological Society’s amphibian conservation programs. Three of the four animals we work with at the National Amphibian Conservation Center (NACC) that are a part of Species Survival Plans (SSP) are preparing to take part in precisely planned and scheduled breeding events. The outcome is the release of captive-born offspring into the wild to aid in the increase of their populations.

First up for the season is the Puerto Rican crested toad, Peltophryne lemur. The PRC toad, as we like to call them, has been part of a well-managed SSP since 1984. Habitat loss and competition from the invasive cane or marine toad (Rhinella marina) are believed to have been primary causes for the toad’s decline. Listed as critically endangered by the International Union of the Conservation of Nature (IUCN), collaborative efforts by the Associations of Zoos and Aquariums, U.S. Fish and Wildlife Service and the Puerto Rico Department of Natural and Environmental Resources have aided the recovery of this toad in the wild. More than 300,000 captive-bred tadpoles produced by 20 zoos have been released to the wild since 1982. Since the year 2000, the Detroit Zoological Society has produced more than 45,000 tadpoles for this program. In 2015, three clutches of eggs laid produced 22,571 tadpoles for release – the largest amount the Detroit Zoo had ever sent. Ongoing research and the creation and protection of pond habitats have also assisted in the recovery of this toad in its natural habitat.

You may be wondering what it takes to produce thousands of tadpoles from toads half the size of the palm of your hand. The breeding season starts with the assignment of four breeding pairs from the SSP. Each toad in captivity is identified and tracked using a studbook. Specialized software chooses pairs of toads that will produce the most genetically diverse offspring. By mid-March, we know who these eight toads are and we can begin preparing them for breeding.

Each toad must be easily identifiable – if you think all toads look alike you’ll be surprised to know that wart patterns and throat markings are very unique, although reading glasses are sometimes needed to make the proper ID. Toads are conditioned by slightly cooling and drying their environments out for a month-long period. A thermostat-controlled refrigeration unit keeps the toads cooled precisely at 66 degrees Fahrenheit. While the toads are “sleeping”, their primary zookeeper works diligently on preparing the breeding tanks where the pairs will breed and the eggs will develop. At this same time, veterinarians prepare a schedule of specialized breeding hormones that will further assist in ensuring that eggs are laid and fertilization happens at the right moment.

Upon their exit from cooling, they return to normal husbandry to warm up and eat for a few days. Breeding calls of male toads are played to encourage breeding behaviors. Males are first to go into the breeding tanks, followed hours later by the females. If all the preparation works, pairs of toads will be in amplexus – which happens when a male is positioned on top of a female and he squeezes her to encourage egg-laying – before we leave for the evening, followed by a morning of tanks full of eggs.

It takes two to three days for eggs to hatch and another couple of days for tadpoles to begin swimming around and actively eating. Tadpoles can be some of the hungriest creatures you will ever encounter. Keepers spend the next 10 days keeping them fully fed by offering them algae pellets, powdered diets and romaine lettuce sometimes three times a day. At the same time, all those foraging tadpoles create a lot of waste, so keepers spend the rest of their time keeping their water clean with frequent water changes.

During all of this, we keep a very important date in the backs of our minds – the last big event in our PRC toad breeding season. Any facility that breeds the PRC toad needs to ship them to Puerto Rico for release on the same date. The release of the tadpoles is timed with the season in which the tads would grow and develop the best in the wild. Tadpoles also need to be shipped at a certain age, before they get too big and begin to develop appendages.

Amphibian staff can spend well over 24 hours collectively counting and packing the tadpoles into Styrofoam-protected shipping boxes. Heavy-duty fish shipping bags are used, doubled up and filled with oxygen to keep tadpoles healthy and safe on their trip to Puerto Rico. Approximately 24 hours later, they will reach their new home in a pond located in a well-protected forest in Puerto Rico. As they develop and grow, they will add to the wild population and one day, hopefully, participate in creating many more thousands of tadpoles!

Rebecca Johnson is the associate curator of amphibians for the Detroit Zoological Society.

Education: What is a Species Survival Plan?

Red pandas Ta-Shi and Shifu have produced several adorable cubs at the Detroit Zoo, most recently little Tofu. North American river otters Whisker and Lucius have sired a couple pups and reticulated giraffes Kivuli and Jabari are well known for their now 13-foot-tall calf Mpenzi. Pairings like these and the offspring that follows are not by chance; each is carefully planned out and managed through what is known as a Species Survival Plan (SSP).

The 230 accredited zoological institutions that comprise the Association of Zoos & Aquariums (AZA) work together through these cooperative management programs to ensure genetically healthy, diverse and self-sustaining populations of threatened and endangered species. More than 450 species are apart of an SSP throughout zoological institutions in North America, overseen through a comprehensive population management system, which includes a Studbook and a Breeding and Transfer Plan. Each of these identifies population management goals and makes recommendations to ensure the sustainability of a healthy, genetically diverse and demographically varied animal population.

The Detroit Zoo has individuals from 98 of these species under its care, including 38 birds, 30 mammals, 24 reptiles, four amphibians, one fish and one invertebrate. Many of these species are animals that require immediate attention to save the remaining wild populations. Our cooperative breeding efforts have proven extremely successful – for example, the Detroit Zoo has been credited with restoring the population of a Tahitian land snail called partula nodosa, once extinct in the wild. Additionally, in May of last year, 22,571 Puerto Rican crested toad tadpoles bred at the Detroit Zoo were released into the wild of Cabo Rojo, Puerto Rico. In 2014, a record 3,945 Wyoming toad tadpoles bred at the Detroit Zoo were released into the wild. This long-running effort was previously recognized as No. 1 on the AZA’s list of the Top 10 wildlife conservation success stories.

AZA institutions and partners work together to carefully monitor SSP species both in the wild and in zoo populations. Organizations will often move SSP animals to other zoos and aquariums so they can mate with individuals to ensure a long-term healthy future for the species. Breeding recommendations are made with consideration given to each animal’s social and biological needs as well as transfer feasibility.

Be sure to look for the SSP logo on animal signage as you explore the Detroit Zoo on your next visit. Each time you see the logo, you’ll know that there are countless individuals working at zoos, aquariums and in the field around the world to do everything we can to save and rebuild the remaining populations of these species.

Notes from the Field: Saving the Wyoming Toad

For the past few summers, I have traveled to Laramie, Wyoming for the Wyoming Toad Species Survival Plan (SSP) meeting, which is an effort by the Association of Zoos & Aquariums (AZA) to cooperatively manage species populations within accredited zoos and aquariums.

First, I fly into the Denver International Airport, which is followed by a 140-mile road trip to Laramie. The trip is full of spectacular views of mountains, canyons, vast plains, huge clouds and pronghorn antelopes. Sometimes I drive the distance alone but this year I made the trip with three colleagues from different zoos. Each of us is a representative for our respective institution, all sharing the same goal of saving one of North Americas most endangered amphibians, the Wyoming toad.

After a stop for groceries and gas, we arrived at our destination for the next five days – a cabin in this remote area of Wyoming. We’re joined there by most of the other members of the SSP – this year, 10 people stayed at the cabin. Most of us have known each other for some time, so sleeping together in a room full of triple-layered bunk beds seems like a week at camp with old pals. But it’s far more than summer camp – we take part in discussions and updates on a number of important topics such as husbandry, health, management, fieldwork and research on this toad.

The next morning, representatives from the U.S. Fish and Wildlife Service (USFWS) arrived along with the AZA SSP coordinator to begin the meetings. Besides the zoos and aquariums involved, there are two USFWS facilities in Wyoming that hold, breed and release the toads. Many important topics are discussed with the last being choosing captive breeding pairs for 2016.

The Detroit Zoological Society (DZS) breeds these critically endangered Wyoming toads at the National Amphibian Conservation Center (NACC). Since 2001, the DZS has released more than 6,500 Wyoming toads into the wild as tadpoles, toadlets and adults. In 2007, the Detroit Zoo earned the highest honor on the AZA’s list of the Top 10 wildlife conservation success stories for that year.

The population of toads is maintained in special bio-secure off-exhibit rooms of the NACC. We have enough room to breed four pairings each year, and any tadpoles produced by the pairings are shipped out to Wyoming for release into protected wetlands in efforts to create self-sustaining populations.

Like each of the organizations present, I brought detailed notes on every adult toad in our resident population so that the best pairs could be chosen. Captive Wyoming toads need to be of ideal weight and health to take part in a month-long hibernation, followed by a June breeding event. Through the AZA, there is an identified “studbook keeper” who matches toads pairs that are least related and most likely to produce offspring that will be the most genetically fit.

On the second day in Laramie, we received training for the USFWS field surveys. Field surveys are done three times during the warmer months of the year around Mortenson Lake. This lake is the last known area the Wyoming toad lived before it was removed from the wild to protect the species from extinction. It is also the site of past and present releases of captive-produced tadpoles and toadlets. Present-day releases are more protected and provide several topics for research. Members of the SSP have been assisting with the mid-summer field surveys for more than five years and the training is just the beginning of a two- to three-day process to see how many Wyoming toads can be found around the lake.

The third and fourth day of my stay is spent almost entirely out in the field surveying for toads around the lake. Teams of two or three people carry backpacks full of equipment for data collection. Each plot must be surveyed in a specific allotted amount of time and by walking in an S-shaped pattern. Toads can be found hiding under grass, sitting on hard-packed sandy areas or swimming near the shore of the lake. If a toad is located, the timer is stopped, and data collection starts. Toads are photographed for identification, weighed, swabbed for disease testing and, if large enough, “microchipped”. It is particularly exciting when an older toad or a toad with an existing microchip is found. This usually means the toad has survived one or more very cold winters and may potentially breed in the lake.

Weeks later, back at the Detroit Zoo, I receive the compiled results of the survey. This year, we located and collected data on 224 toads – 129 toadlets that were captive-born and released into the lake this spring; 29 toads that were captive-born, released last year and survived the winter; 33 adult females and 33 adult males. Until next July, go toads!

– Rebecca Johnson is the associate curator of amphibians for the Detroit Zoological Society.