Part of the project: The Pygmy Hippo

Pygmy Hippopotamus Labwork in Florida at SEZARC

Back in April of 2014, I travelled to 12 zoos around the USA to meet their pygmy hippos and initiate sample collection for a year-long hormone study using dung samples. The last year flew by, and April 2015 came a lot faster than expected! Now it was finally time to gather all my samples and start conducting analysis and determine some exciting new information about stress and reproduction in pygmy hippos.

I left the University of Western Australia in Perth and flew to Jacksonville, Florida, at the end of April and started working at the lab the first week in May. The lab that is hosting me for the next five months is called the South-East Zoo Alliance for Reproduction and Conservation (SEZARC), and is located at White Oak Conservation Center in far north-east corner of Florida. They also have a satellite lab facility at the University of North Florida (UNF) in Jacksonville, about 45 minutes south of White Oak. I split my time between both facilities, and at UNF I am lucky enough to have five dedicated undergraduate student interns who are assisting with my project.

It is time to find the data hidden inside those samples !

After an initial week of training in the lab, I headed out to the wilds of Florida to collect my samples from Zoo Miami, Tampa’s Lowry Park Zoo, Rum Creek Ranch, and Safari Wildlife/Giraffe Ranch. The samples coming from the various zoos outside of Florida would be shipped overnight to the lab via Fed Ex to ensure the dung would remain frozen in transit. Meanwhile, I loaded up my car with six giant coolers and hundreds of ice packs and hit the road, driving over 1500 miles in less than a week.

At the Lowry Park Zoo I was able to meet Zuri, a young female pygmy hippo born in October 2014. Her mother, Zsa Zsa, is one of the animals in my study. Rum Creek Ranch had seven animals in my study, so after loading up all my samples at their facility the shock absorbers on the vehicle were having to work extra hard. I found myself wondering what I would say if I were stopped by the police for some minor traffic infringement and they noticed the multitude of giant coolers. Imagine the police officer’s surprise upon opening these coolers and being greeted by thousands of smelly samples of frozen hippoo when they were probably expecting some kind of illicit contraband!

Fitri & Zuri at Lowry Park Zoo

Zuri & Zsa Zsa at Lowry Park Zoo

Fitri & Zuri at Lowry Park Zoo

Zsa Zsa & Zuri at Lowry Park Zoo

Summer in Florida brings with it plenty of heat, humidity and insectivorous life forms, but it also brings massive afternoon thunderstorms. Sometimes while I was driving it was raining so hard I could barely see the road. Lightning and thunder crashing all around, gale force winds and pouring rain, quite an adventure! However, an hour later it generally blows over and it’s more humid than ever, but it has cooled off a bit and steam rises from the road as the water evaporates off the hot tar surface.

the poo need to be smashed

The poo samples need to be smashed and thoroughly mixed

After returning to Jacksonville I hit the ground running with my dung samples and have been spending 12 to 14 hours per day in the lab, six or seven days per week, ever since. The process of determining hormone data from a lump of frozen pygmy hippo dung is quite involved and encompasses a multitude of tedious steps. First, the frozen dung sample is smashed to smithereens with a hammer, breaking the poo into tiny pieces. Then, the sample is thoroughly mixed and 0.5000 grams of samples is measured out into a test tube using an analytical balance, the type used in chemistry laboratories to measure out incredible precise amounts of chemical substances.

the analytical balance

The analytical balance

Next, the 0.5 grams of poo is mixed with water and ethanol and is the shaken vigorously in a specialized machine that shakes the crap out the samples! But seriously, it mixes the alcohol, water and poo and ‘extracts’ the hormones from the poo into the liquid. Then the mixture is centrifuged, the liquid is removed and diluted in assay buffer. Whew! I’m tired already. Now repeat that process approximately 3,500 times – that’s how many samples I have, about 125 samples per animal from 28 pygmy hippos.

DSC_0398

Mixing 3500 tubes is quite time consuming !

Then the liquid is diluted to an appropriate concentration, and each sample is loaded in duplicate into wells (like tiny itsy bitsy cups) on an assay plate that has 96 wells total and can hold up to 30 samples at a time. Loading all the wells on a plate with all the various reagents and standards and controls and samples takes about 2 hours from start to finish. Then the plate incubates for 2 hours to allow all the components to ‘come to equilibrium,’ next it is rinsed, and finally a substrate is added to each well that causes a color change reaction.

wells ready for the final analysis

Assay plate with 96 wells during the final analysis

Now the exciting part happens. Each sample will have a different amount of hormone in it, and the amount of hormone affects the degree of color change in the well. Lots of hormone = light blue color. Very little hormone = dark blue color. And millions of variations of shades of blue in between. These myriad of color variations are then read by a specialized machine that can detect microscopic differences in optical density (color) at a certain wavelength of light. The machine then spits out your data, telling you the concentration of color (i.e. hormone) in each well.

Finally, you take all your data from all your samples from each hippo, and you can look at hormone trends over time. You can follow estrus cycles and diagnose pregnancy by measuring estrogen and progesterone metabolites; you can look at physiological reactions to potentially stressful situations by measuring cortisol metabolites; and you can look at reproductive activity in males by measuring testosterone metabolites.

DSC_0385

The samples are weighed and identified

Needless to say, all of these steps are quite time consuming, and so far after almost two months of labwork I have only finished hormone profiles for two animals! But from here forward things should be proceeding much more quickly as I have ironed out many of the details in the procedures, and I have become more adept at doing things efficiently. It was a steep learning curve at first!

My undergraduate interns and volunteers have been instrumental in making all of this possible because without them I would still be sitting and weighing out dung samples and would be nowhere near as far along in the process. They are all eager, young women scientists with future careers in veterinary medicine, zoo-keeping, pharmacy, and animal management. I would like to whole-heartedly thank Sarah Allred, Kim Daly-Crews, Saleha Khan, Kayla Weller, Paige Pickering, and Joanne Wozniak.

We’ve got those University of North Florida (Official Fan Page) students working hard in the SEZARC lab on campus! Here Kim and Sarah are working on pygmy hippo samples! Learn more about SEZARC at www.sezarc.org

post from SEZARC Facebook Page  16 june 2015

I would also like to once again express my sincere appreciation to the dedicated zookeepers who literally spent an entire year diligently collecting hippo dung samples twice weekly for this project. I certainly could not have done it without them, and they have done an excellent job of keeping everything organized and recording the behavioral data that helps explain the reason behind the different hormone trends.

Zuri & Zsa Zsa at Lowry Park Zoo

Zuri & Zsa Zsa at Lowry Park Zoo

Meanwhile, the world has welcomed at least one new pygmy hippo here in the USA. There was a little calf born at the Brownsville Gladys Porter Zoo, home to Clover and Juanito, early the morning of 24 June 2015. It has not yet had its first neonatal exam, so we don’t know yet if it’s male or female, but stay tuned for updated, or check the zoo’s Facebook page in the near future.

OK, back to the lab, my samples are calling!

more samples to analyse !

More samples to analyse !

After the labwork is complete (estimated late October), I will return to Perth and start my data analysis and thesis chapter writing process, with the aim to complete my PhD mid to late 2016. This portion of my PhD research has been financially supported by the American Association of Zoo Veterinarians Wild Animal Health Fund, the Center for Conservation of Tropical Ungulates, Omaha’s Henry Doorly Zoo, the University of Western Australia (UWA) Postgraduate Student’s Association, the UWA Convocation, and IBREAM.

Wild Animal Health Fund Logo

Extra pictures

the smashing station

The smashing station

sample tube preparation station

Sample tube preparation station

Latest project news

Background and highlights

Team members

monique

Monique Paris

IBREAM Research Director

Gabriella Flacke

Gabriella Flacke

IBREAM PhD Student, University of Western Australia

inzakone

Inza Kone

IBREAM Advisory Board member, Ivorian based project partner (CSRS)

markvanheukelum

Mark van Heukelum

Masters Student, Wageningen University

karimouatarra

Karim Ouatarra

Ivorian based project partner (CSRS)

Bogui Elie Bandama

Bogui Elie Bandama

Ivorian based PhD student, Université Félix Houphouët Boigny

henkeshuis

Henk Eshuis

Masters Student, Wageningen University

Captivity Studies European Collaborators
Professor Franz Schwarzenberger, Sue Walker, Aalborg Zoo (Denmark), Zoo Basel (Switzerland), Kerkrade Zoo (Netherlands), Overloon and Wissel (Zodiac Zoos), Burgers Zoo (Netherlands), Rotterdam Zoo (Netherlands), Berlin Zoo (Germany), Paris Zoo (France), Colchester Zoo (UK), Bristol Zoo (UK), Zoological Society of London (UK), Givskud Zoo (Denmark), Zurich Zoo (Switzerland), Edinburgh Zoo (UK), Chester Zoo (UK).

Captivity Studies USA Collaborators
Baton Rouge Zoo, Brookfield Zoo, Brownsville's Gladys Porter Zoo, Chicago Zoological Society, Chicago's Lincoln Park Zoo Giraffe Ranch/Safari Wilderness Ranch, Jackson Zoo, Louisville Zoo, Oklahoma City Zoo, Omaha's Henry Doorly Zoo, Rum Creek Ranch, Southeast Zoo Alliance for Reproduction and Conservation (SEZARC), Tampa's Lowry Park Zoo, Zoo Miami.

Captivity Studies USA Sponsors
American Association of Zoo Veterinarians (AAZV) Wild Animal Health Fund, Center for Conservation of Tropical Ungulates (CCTU), Omaha's Henry Doorly Zoo, University of Western Australia Postgraduate Student's Association, UWA Convocation.

Ivory Coast Project Work Study Sponsors:
Al Bustan Zoological Centre (UAE), Al Wabra Wildlife Preservation (QATAR), Auckland Zoo (New Zealand), Banham Zoo (UK), CB Films (UK), EI Medical Imaging (USA), Fundación Bioparc (Spain), Mammal Research Institute (South Africa), Mohamed bin Zayed Species Conservation Fund (UAE), Omaha's Henry Doorly Zoo (USA), Rum Creek Ranch, Parken Zoo (Sweden), Phoenix Zoo (USA), Prince Bernard Foundation (Netherlands), Seoul Zoo (South Korea), University of Western Australia, WWF Netherlands, The Royal Zoological Society of Scotland, Zoo Plock (Poland), Zoo New England, Toronto Zoo (Canada).