Riding On A Wave of Chickens

I've been riding on a high for the past couple of weeks. And I have to chalk it all up to the chickens.

I'm planning on conducting a research project next summer using tissues from laying hens to study ovarian cancer. No need to go into details here, but laying hens are considered a robust animal model for this disease. Considering that synapsids, from which mammals arose, and sauropsids, from which avians remain, split apart around 320 million years ago, that's pretty amazing.

Figure from University of California at Berkeley.

My chosen summer project has some unusual constraints. To cut right to the point, 18-month-old laying hens take, well, eighteen months to make. I could not wait until summer to begin "making" my tissues. I had access to appropriate laying hens last fall and I had to start collecting my data then, somehow finding time between all the studying I was doing.

First, I collected blood samples from the hens at monthly intervals.

In case you were wondering, there are three locations that can be used for venous blood collection from avians, just like in small mammals: external jugular vein, saphenous vein in the lower leg, and wing vein. I chose to collect from the wing vein. The skin in this location is paper-thin and transparent--you can see the veins clearly. The vessels tend to be rather delicate too and tear easily. When you pull back on the plunger of the syringe, even a tiny bit, you can see the veins collapse completely. It's a delicate balance to pull the plunger back between the heartbeats that fill the vein. We needed about 2 ml from each bird, which doesn't sound like a lot until you actually have to collect it. I invited other first-year vet students and vet tech friends to come with me each time. If you can successfully do a blood draw from the wing vein of a chicken, you can claim considerable bragging rights about your blood draw skills.

Second, I had to collect the tissues. The nutritional trial that included these birds was ending in December. Of course, it was during finals week that I had to euthanize and collect the tissues for my study: complete ovaries and oviducts and samples of liver. No reason for any of this to be convenient or simple!

The types of analyses that I want to perform on the tissues in the summer meant that I couldn't keep them in formalin for too long, certainly not until the summer. But the next step required some money to pay for specialized lab work. Sections of the tissues needed to be embedded in paraffin blocks. From these, we can cut thin slices that can be stained for various purposes such as microscopic or immunohistochemical analysis.

The college has an established program to provide funds to students for summer research projects that pays a small amount for supplies and a bit more for a stipend for the student. I of course submitted my application to this program but none of this money would be released before the summer. I had also applied for a scholarship from the American Association of Avian Pathologists but wasn't going to hear about that until early February. And Gita, my MS advisor, had encouraged me to prepare a proposal to the Agricultural Research Foundation of the university. She has received numerous grants from them in the past for her own work, including for the project that I did for my MS. The grants pay for student salaries and equipment and other supplies. We submitted the proposal back in December. It was a real crush for me to find time to write the draft but we made the deadline. I didn't expect to hear about that until late February. So there were plenty of potential funding sources up in the air but none of them were assured or even available to me so I could complete this critical step.

In desperation, I turned to another of my mentors, Jean. She allowed me to use her discretionary account, a sort of slush fund each faculty member can use for small expenses not directly related to specific grants, to cover the costs of having the tissues embedded in paraffin.

I went down to the Histology lab to get information. They gave me a tour, and showed me how to load tissues into the little cassettes that would be used to form then hold the paraffin blocks. I learned that if I trimmed the tissues and loaded the cassettes myself, I would save a lot of money.

So instead of spending more time with the cadaver dogs and cats on the Saturday before the gross anatomy midterm, I spent four hours in the gross anatomy lab trimming pieces of laying hen ovaries and oviducts. In our lab, we have special dissection tables with downdraft circulation systems. By using one of these tables, I didn't have to use a fume hood to do the work--formalin is nasty. It was delicate and slow work, made slower by my inexperience.

Here's what a loaded cassette looks like:

The two round bits are slices of oviduct near the ovary (upper left) and about 4 cm away from the ovary (upper right). The lower bit and the bits on the paper towel are pieces of the hen's ovary. Based on gross observation of tumors on her oviduct and abnormalities of the ovary, this hen had ovarian cancer. In fact, I think that eight of the eighteen hens had ovarian and/or oviductal cancer. That is always a risk with projects of this sort: will enough of the study animals have the disease of interest to make the results statistically significant? The minimum number of disease cases that you need out of your study population can of course be determined before you even begin the experiment, and I had already made those calculations. But I had no control over the outcome. So it was really exciting to see the tissue with my own eyes.

This is what the Histology lab emailed to me after I returned the cassettes to them:

Also, we would like to tell you how wonderful your samples were! The tissues were the appropriate size and thickness and easy to embed. Your cassettes were not too full or difficult to read and your sample numbers were simple. Many thanks.

Well, this was a confidence boost! Maybe it was beginner's luck. But I did receive detailed instructions that I saw no reason not to follow to the letter.

Whew. I was able to breathe a bit easier knowing that at least select bits of the tissues were now resting safely in paraffin. I was still stressed about funding though. The analyses I want to run on those tissues are pretty expensive--and that's just for the chemicals. I was wondering how I could pay for additional lab work too.

Well, it turns out there's good money in chickens, even when you use them to study human disease. 

Less than a week later, I received two emails one day apart. Gita and I got the ARF grant! We asked for, and were awarded, $12,500--with that level of funding, I will run out of avian-specific proteins to run on the tissues before I run out of money! And I got the AAAP scholarship, which I can use however I see fit.

Chickens. They got me into vet school. Here's hoping they get me a good job when I finish.