BD Accuri News

Preserving the Endangered Asian Elephant

Dennis Schmitt, DVM, PhD, Wendy Kiso, PhD, and Tracy Northcutt, MNAS (pictured left to right with elephants Kelly Ann, Rudy, Sundara, and Nichole), are researchers for the Ringling Bros. and Barnum & Bailey Center for Elephant Conservation in Polk City, FL. (Dr. Schmitt, who bred the first elephant from artificial insemination in 1999, won a 2011 Accuri Creativity Award for the project, “Using Flow Cytometry to Improve Assisted Reproductive Technologies in the Endangered Asian Elephant.”) They spoke to us about Asian elephant conservation, the challenges of freezing Asian elephant sperm, and their experience with the BD Accuri™ C6 flow cytometer.

Q: What is the ultimate goal of your research program?

A: The Asian elephant (Elephas maximus) is listed as an endangered species, with an estimated 30,000–50,000 individuals worldwide. Our goal is to enhance their breeding efforts by optimizing semen storage and sperm cryopreservation techniques with the simultaneous use of artificial insemination.

More importantly, the development of sperm cryopreservation technology will allow us to establish a genome resource bank (GRB) for Asian elephants and provide genetic insurance for this endangered species. Although Asian elephants in the wild are not poached for ivory like African elephants (only the males have tusks), the major threats are human encroachment and habitat destruction. Wild populations are becoming more fragmented, threatening their genetic diversity. The creation of a GRB will allow us to preserve the genetic diversity of the elephant population and ultimately prevent a genetic bottleneck.

Q: What is the role of captive elephants in the preservation of the wild population?

A: Captive animals are our ambassadors for scientific research. We learn as much as we can from them. Currently, the captive Asian elephant population of about 15,000 is not reproducing at a sufficient rate to maintain the population. Assisted reproductive technologies are essential in preventing its extinction.

Since captive males are dispersed throughout North America, and moving them is prohibitively expensive, semen cryopreservation has become a research priority to help propagate this endangered species. But as a species, unlike African elephants, their sperm is very sensitive to storage and freezing. So the ultimate goal of our research is to optimize Asian elephant semen freezing techniques by evaluating various compounds' ability to maintain acrosome integrity and sperm motility during cryopreservation. Although we plan to optimize the techniques with Asian elephants in the US, we hope to eventually utilize the same techniques in bull elephants in their native range countries.

Q: What's the role of flow cytometry?

A: Previous research utilized Spermac™ stains and light microscopes to determine acrosome status. With a flow cytometer, what once required hours can be accomplished in a few minutes with more objective and accurate results.

Q: Can you describe a typical study and the kinds of challenges you are encountering?

A: We are validating protocols for evaluation of fresh and frozen-thawed elephant sperm using SYBR® 14 and propidium iodide (PI) to assess membrane integrity, and FITC-conjugated Pisum sativum agglutinin (FITC/PSA) to evaluate acrosome status. We are currently focused on sperm membrane and acrosome integrity, but plan in the future to also investigate mitochondrial activity as related to motility.

Due to inconsistent sample quality and variable sperm concentration in elephant semen samples, fresh samples are more challenging to prepare for staining and analysis. We have also been working to resolve issues of debris, non-motile cells, and extremely high and low concentrations.

Q: Any results to report so far?

A: Recently we've had success loading cholesterol into the sperm membrane through the use of cholesterol-loaded cyclodextrins (CLCs). Cholesterol is a highly hydrophobic molecule and is not soluble when you just add it to the medium in which the sperm is swimming, so it will not be integrated into the sperm membrane by itself. Instead, we incorporate the cholesterol into a cyclodextrin structure, which encapsulates the cholesterol inside its hydrophobic core. This CLC molecule then acts as a vehicle that intercalates cholesterol into the sperm membrane, resulting in membrane stabilization during cooling and freezing. This has ultimately led to better sperm survival after freezing and thawing.

Q: Which features of the BD Accuri C6 are important in your research?

A: Immediate evaluation of the samples is crucial, to see how the sperm is doing when it's fresh. Unfortunately, we can't just ship the samples to our laboratory. Since the elephants cannot come to us, we have to go to the bulls. So we wanted a transportable unit like the BD Accuri C6, which we could transport to other bull-holding facilities as well as to range countries as we develop a sperm bank of frozen semen. This allows us to use the same system in wild populations, which keeps our methodology consistent.

In addition, by taking a machine with us, we can look at the sperm when it's immediately collected, after dilution and treatment, during cooling, before freezing, and after thawing. The BD Accuri C6 allows us to easily evaluate each step of the sperm cryopreservation process to see where sperm damage is occurring and optimize our techniques accordingly.

The BD Accuri C6 is easy to use and can be unpacked and set up very quickly. And our students speak highly of its user-friendly software.

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*Dr. Schmitt, who is chair of veterinary care and director of research and conservation for Ringling Bros., is also a professor of agriculture at Missouri State University. Dr. Kiso is the resident research scientist at the Center for Elephant Conservation. Ms. Northcutt is a junior research scientist and Dr. Schmitt’s laboratory manager at Missouri State University.