Evaluating the utility of toenail samples in elephant endocrinology

This semester I worked on a project to evaluate the utility of toenails in elephant endocrinology. Toenails are keratin tissues which are non-traditional samples in elephant endocrinology. Traditionally, samples like blood serum, urine, and feces have been used to monitor hormones in elephants. Traditional samples however are limited in the period of hormone content they reflect and can be difficult to collect and use. Tail hairs and toenails on the other hand could contain a history book of hormone variation from throughout the time they grew and can be collected and stored easily. To evaluate their utility, we looked at both the growth rate of the toenails to correlate samples to a period in time as well as their functionality in standard hormone assays. For growth rate we had keepers make a harmless groove in a toenail and track how far the groove moved every two weeks. We found variation in growth rate that could impact the accuracy of correlating samples to a time period. For testing the samples in hormone assays we converted our samples to a powder and utilized a methanol extraction to create hormone extracts. These hormone extracts were then tested in progesterone and cortisol assays. We found that elephant toenails worked in both progesterone and cortisol assays and were able to show variation in concentrations. Variation in progesterone concentrations have so far aligned with the reproductive statuses of their corresponding elephants. More work will be completed in the future to check the accuracy of hormone variation.

Hi, I’m Garrett Rich a senior Biology student here at George Mason University. Over the course of the Fall semester, I worked with Dr. Freeman, Dr. Hunt, along with another undergrad to look at the utility of keratin tissues in elephant endocrinology. But let’s define… what that even means. Keratin tissues are just tissues made of the protein keratin that extend outward from your skin. You would know them best as hairs and nails. Endocrinology on the other hand is just the study of hormones and their corresponding ties to physiology. So, this semester we looked at elephant toenails and hairs to see if there is any utility in being able to monitor hormones in elephants.

Endocrinology can be a really valuable tool when it comes to monitoring the wellbeing of endangered and at-risk species. Endocrinology allows conservationists to keep an eye on key hormones and what changes in those hormones mean for the organism. Keratin sampling is a bit newer, especially in elephant endocrinology. And we are looking at it to remedy some of the issues associated with traditional samples. Blood plasma, urine and feces are all traditionally used for elephant endocrinology but are limited in the hormone period they reflect. This means that some of the key variations in hormone concentration may not always be detectable. Take for example the graph on the right from Ganswindt, et al (2005) where they monitored fecal hormones in male African elephants. It required dozens of samples over the course of months to get variations in hormone concentrations. Whereas a tail hair, a keratin tissue, that grew alongside those variations could also contain those variations and only require the collection of one sample.

This semester our project was divided into two parts. Thanks to the hard-working keepers of the Smithsonian National Zoo and the keepers at the Maryland Zoo in Baltimore we got access to both tail hair and toenail samples from their elephant populations, as well as got to investigate the growth rate of those tissues. We got samples from the Smithsonian National Zoo’s Asian elephants and the Maryland Zoo in Baltimore’s African Savanah elephants. I mainly focused on toenails this semester so that’s what I’ll be mostly talking about.

Let’s talk about the growth rate first. We looked at growth rate because we wanted to correlate our samples and their hormones to the period in time in which they grew. This is really important for toenails. Toenails are regularly trimmed as a part of normal care. And we use these trimmings as our hormone samples. The problem is trimmings can only be collected from the bottom of the nail. Whereas the nail grows all the way at the top of the nail in the nail bed. So, you need the growth rate to determine how long it took a sample to leave the nail bed and get into a position where it can be trimmed. So how do you measure this growth rate? We had keepers make a harmless groove in the surface of the nail and then track how far that groove moved every two weeks. You can then take the differences in those measurements and extrapolate a growth rate.

Once you have the growth rate, you can begin to work backwards. You can take when a sample was collected, how big the toenail was at collection, and then use the growth rate to try and determine when that toenail grew. But what we noticed in the data was that the growth rate was not constant. It actually varied over time. This is important to account for as the longer it takes a sample to come available, say from a longer toenail, the more variations can lead to inaccuracy. This semester we looked at temperature, as elephants are not allowed to go outside if temperatures are low enough. To generalize for the Mid-Atlantic, we divided the year in half with October-March being the colder part of the year and April-September being a warm part of the year. This gave us this graph. So, while we can’t say anything definitive about trends with growth rate over the year, we can definitively say that there are variations. These values may look indistinguishable. They are differences in the fractions of a millimeter. But when these growth rates are extrapolated over large periods of time it can result in differences of weeks to months for determining when a sample grew.

Growth rate is only half of what you need to track hormone variations. We also needed to have hormone concentrations to track. While keratin tissues have been used in endocrinology before, they have never been tested in elephant toenails specifically. So, the first thing we had to do was make sure they would work at all. To do this we would take an elephant toenail like the one on the right and convert it into a powder and then use that powder in a methanol extraction. A methanol extraction is just a process of using methanol to remove hormone from the sample. And then evaporating off that methanol to get us a hormone extract.

A lot of the work this semester was dedicated to us working with our toenail samples to determine a procedure that we not only liked but also produced samples that worked correctly in our hormone assays. Thankfully though, the samples worked quite well and we were able to get ideal assay plates that look like this.

This semester we were only able to test samples from Anna and Felix. Anna and Felix are female African elephants at the Maryland Zoo. Their samples were tested in progesterone and cortisol assays. We found that their toenail samples worked surprisingly well in our assays. And produced decent amounts of hormone concentration as well as variation that may align with physiological status. Female elephants enter what’s called an estrous period when they ovulate which is marked by the production of progesterone and progesterone like compounds. Some older female elephants will stop going into estrous and their corresponding progesterone concentrations will stay roughly around a baseline. When we tested our Anna samples, we found her samples were low in progesterone content. While Felix also had low samples, Felix samples would also occasionally spike up into higher progesterone concentrations. This is a great sign for us as Anna is considered to be post-reproductive and no longer entering estrous. While Felix is still believed to be entering estrus. So, while it isn’t definitive yet, these variations in concentrations are a good sign for us.

The goal of our research was to test this unutilized sample type and provide a base of information for other elephant endocrinologists and animal endocrinologists. In the spring we are looking to continue our work on this project by testing more samples, looking at different hormones, and continuing to try and match hormone variations to known physiological statuses. We hope that our research will be able to provide elephant endocrinologists and other conservationists with a tool to help them save species.

I first would like to the thank the Smithsonian National Zoo and the Maryland Zoo in Baltimore for giving us access to the growth rate and these samples. I would especially like to thank the keepers for all their hard work collecting samples and gathering growth rate measurements for us. Without them this project could not have happened. I would also like to thank Dr. Freeman, Dr. Hunt along with Trent Grasso for all their help this semester both in and out of the lab. And finally, I would also like to thank Dr. Lee, Oscar along with the Faculty Research Development Award for supporting both me and this project. And here are my sources.