But the road from watching a hyena poop to having usable data is a long one. This spring, before heading out to the Mara for my two-year field season, fellow grad student Sarah Jones and I packed up about 550 hyena fecal samples and headed to the University of Nebraska to see what our hyenas’ various bowel movements over the past six years or so could tell us. Sarah was analyzing samples for androgen levels (a class of hormones including testosterone). I analyzed samples for corticosterone – a main mammalian stress hormone.
After our precious cargo made it safely to Omaha (driving around with a back seat full of coolers of valuable poop is a bit unnerving), we learned from endocrine god Dr. Jeff French how to transform our stinky poop samples into PhD gold. Dr. French normally spends his time exploring the connections between hormones and behavior in marmoset monkeys and humans but takes a break every few years to help us process these more exotic poops transported all the way from the wilds of Africa.
Before even doing hormone assays to determine the concentrations of hormones in our samples, we first had to extract the hormone from the feces. This proved the most labor-intensive part of the whole process and amounted to about five days of thawing poop, weighing poop, heating poop, drying poop, grinding poop with a mortar and pestle, picking hairs out of poop, and…weighing it again. The goal is to get a pure sample of dried feces that, when combined in a known mass with a known volume of liquid, will give us a homogenous solution of hormones. Here are some of the steps we went through to turn hyena diarrhea into some pretty science-y looking tubes of extracted hormone…
Weighing out a precise quantity of feces
Drying out a hundred-or-so fecal samples overnight in an incubator
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DO NOT drop this tray of incubated samples! Aren’t they colorful!? Dr. French and I think they look like spices set out in a Middle Eastern market.
In the beginning, this process was super cool to me. Five years after some hyena ate a zebra, here I was sitting in Nebraska seeing all those tiny black and white hairs in its poop and picking them out with the attention of a surgeon. But after doing this for more or less 12 hours a day, Sarah and I were starting to think our morning coffee grounds looked like poop (and wow, so perfectly mortared-and-pesteled!) and had the urge to weigh out a perfect .2 grams of that pepper before adding it our pasta. We were ready to reach the end of the extracting phase….
Voila! We do some chemistry and centrifuging magic with those dried samples and here’s that solution of hormones on the left. The rest of the stuff in our sample – all the stuff we don’t want – remains in the tube on the right.
After using some more fancy science tools that us field biologists don’t usually lay our hands on, like this multi-channel pipetter on the left, we are left with our final extracted product on the right. I swear, these little holders filled with just the precise quantity of yellowish sample, perfectly in solution, looked more beautiful than liquid gold. Now, on to the exciting part…finding out the concentration of corticosterone in each of those little tubes. This will help us determine how a hyena’s stress level is shaped by ecological, social, and physiological factors, as well as conservation management decisions.
And for this, us field folk get to feel even more like real, honest-to-god scientists by utilizing the properties of radioactive substances. We add an antibody to our extracted hormone as well as corticosterone tagged with a radioactive Iodine tracer. What this amounts to in the lab is adding various brightly-colored radioactive liquids to our precious tubes of extracted hormone in specific timed succession, giving us pretty test-tube racks like this…
Radioactive poop!
Then, the hormone in our sample and this radioactive antigen compete for binding sights on the antibody. This is one of the highlights of the process because as the magic of “competitive binding” occurs within each of these tubes, you get to take a 2-hr break from pipetting and go grab some lunch. When you return, chemistry magic has happened… and I have never been this excited about basic chemistry principles in my life. We can then add another brightly colored liquid to our tubes to separate the radioactive-labeled hormone that has bound to the antibody from the hormone that has not. After adding this precipitant and centrifuging our tubes, we’ve successfully completed this separation.
Tubes of radioactive poop about to be centrifuged
And finally comes the most nerve-wracking step of the entire procedure…decanting. Imagine, after spending days extracting the hormones, and then many hours carefully pipetting small amounts of somewhat dangerous substances in and out of small containers, you manage to get each of these precious tubes safely in and out of the massive centrifuge. You’ve also managed to not spill anything and are trying not to think about just how much each of these little tubes actually costs if you were to sum up the price of all the different liquids you’ve added. Now comes the time when you put these tubes in a special holder, turn them upside down, and literally pour all the fancy chemicals you’ve added down the drain. For me, this was the most stressful step in the process. I always had a mental flash of all those valuable tubes crashing into the sink and having to start the whole thing over again!
Pouring it all down the drain...
...but not everything goes down the drain. What we are left with is a small pellet in each tube that can be read in a Gamma Counter to measure the amount of radioactivity. The higher the radioactivity, the more the radioactive hormone won out in our competitive binding battle and the less corticosterone there actually is in our fecal sample. By comparing our samples with various solutions of known standard concentration, we can determine precisely the amount of stress hormone in each of our samples.
In the coming months, we will be using these new data to address several questions in addition to the hypothesis that spotted hyenas can serve as an "indicator species" for the Mara ecosystem. For example, I will be asking whether hyenas living in areas within the Mara with different management strategies show different stress profiles. I will also be looking at the behavioral, physiological, and demographic consequences of maternal stress for mother-offspring relations and cub development. As these questions form the heart of my PhD research, you'll hear more about them in future blog posts...
My first foray into endocrine lab work would not have been possible without the help of the French Lab. Thanks to Dr. French for opening his doors to smelly hyena poop samples and all his guidance. Also, thanks to undergrad Benjamin Hochfelder in the French lab. Can you believe Ben came in on a weekend to help us grind and weigh poop!? Finally, Sarah and I want to give a huge thank you to Drew Bernie. Drew’s title is “lab tech” but his endless help and trouble-shooting abilities during our stay in Omaha quickly earned him the nickname “Superman.” Drew turned me from a pipette-naïve hyena watcher into an assay-machine and seemed to be able to put out any fire that Sarah and I started. Thanks, Drew!
Left: undergrad-extraordinaire Ben Hochfelder
Right: Drew didn’t want a commemorative photograph taken, but this picture pretty much sums him up.
Nice explanation! I have a great memory of spotting a high-ranking male produce a tiny sample for us right in the middle of the road. We pulled up next to it and politely waited for the comby behind us to pass. They then pulled up really, really close. We were worried about them running over the poo, so we kept staring down at it. Then all of the tourists in the comby stared down at it, and the driver said, "Is THAT what you're looking at?". That driver didn't follow our vehicle around after that.
ReplyDeleteReally enjoyed this post AND learned from it. Nice job! After watching Dr. Holekamp go through "the fires of hell" to keep the samples safe and then get them from the Mara to the lab, it's great to know that they are producing what you guys need.
ReplyDeleteHw's Target II doing?
I'm very surprised that at the most crucial step—pouring off the fluids to get at the now radioactive pellet in the tube—there isn't a sieve or other barrier you use to prevent the processed pellet from falling into the drain. I would think you don't want to contaminate one sample with another, so you can't use the same sieve again and again, but what about a paper coffee filter? or even just some tissue? Use once and dispose. And then it'd only be to catch the pellet if it falls out; If your hand is steady enough to keep the pellet in the tube, the process ends up exactly as before. The barrier is there just in case the pellet falls.
ReplyDeleteIf coffee filters or tissues contain substances that you don't want on the pellets, are there lab-grade filtration papers you can use? Or are the costs for such items prohibitive (i.e., the intended market is pharmaceutical or chemical research, studies with money to burn, as opposed to zoologists who must watch every cent spent on supplies)?
It's amazing not only that there's a link between old-fashioned field observation of animals and ultra-modern, highest of high technology biochemistry but that the link is something as humble as animal droppings! Thanks for the fascinating post, and good luck in your research. I'll be interested to know your results on stress levels among different populations, and—if there are differing stress levels by population—your hypotheses or findings on why this might be so.