Monday, January 6, 2014

Designing stress studies, part 2: how do you get your sample?

I recently posted about how to choose what bodily substance to use to test for cortisol in a stress study: blood, saliva, urine, feces, or hair. Once you have your substance of choice, though, you have to actually extract it from the dog. This can present more or fewer challenges, you know, depending.


When people first started measuring cortisol, they used blood to do it. Blood is where cortisol shows up first. All the other substances that we measure cortisol in have had their cortisol levels compared to blood cortisol levels, to make sure that they correlate strongly. Researchers had to do studies to prove that these other substances worked for this measurement, which cost a lot of effort and money. They did this because blood is pretty hard to get hold of, in most cases. Sticking a needle in a dog will usually stress it out, and it's hard to get the blood extracted before the stress of the restraint starts changing the blood cortisol levels.

But even aside from that, sometimes a blood draw is simply out of the question. For my Master’s work, I had to cold-call hospital clients and convince them to let me enroll their dog (already in the hospital for some procedure or other, in other words, already having a bad day) in my study. If I had told them that the dog would need a blood draw too, I guarantee that most of them would have said no.

In a comment on the previous post in this series, Tegan pointed out that animals can be trained to submit calmly to blood draws. For some studies, this approach would be invaluable. For my study, again, it wouldn’t have worked. Training an animal to accept a needle is an arduous process, and I had access to those dogs once, on one night. For most shelter dog studies, this would also be an impossible hurdle. But it’s a pretty cool thing to do, if you can do it.

I wish I had a video of another approach to stress-free blood draws. I have seen other vets slide a needle into the lateral saphenous vein, the vein that bulges out of the side of a dog’s hind leg just above the hock. If the dog is distracted (say by someone feeding it), a competent venipuncturist can get it done using this vein with little to no stress. I have seen this technique used in shelter dogs who would not allow restraint for a more traditional draw. But it takes a dog with short, smooth fur and a particularly lovely bulgey vein. It does not work in little dogs. And it definitely requires a competent person to do the draw. After a few years of practice in blood draws, I was just getting to the point during my internship where I could do this one. There can’t be too much poking around to find the vein, or the game is up.

(I did find a video of a technician drawing from the lateral saphenous of a dog who is lying on his side, with an assistant holding off. This is the same vein as the one I am talking about, but in the procedure I’ve seen, the dog can be standing and you actually don’t need someone else to hold off the vein. You come at the vein from above, not below, in a standing dog. Just in case any of you blood-drawers out there want to try this yourself.)

Since blood was such a pain to get, people started trying other substances, figuring anything had to be easier than a blood draw.


Saliva is now used much more often than blood in human cortisol studies. You hand a person a cup and they drool into it. No needles, no added stress. Dogs are not so easy. You can’t ask a dog to drool into a cup; you have to get the drool out yourself.

For my study, I used Sorbettes, also known as eye sponges. The instructions say to put one Sorbette into the dog’s mouth for 30-60 seconds, and voila, it has enough saliva on it for an assay. You then put the Sorbette into a tube and spin the tube in a centrifuge to get the saliva out. You only need 25µg, which is hardly anything! What could go wrong.


First of all, when you are analyzing the saliva later on, you use 25µg per well in the plate of saliva samples, and you get one cortisol value per well. But it turns out that the assay is fairly imprecise, and gets it wrong a decent percent of the time, sometimes close to 10% of the time. So it makes sense to use two wells per sample (now we are at 50 µg per dog). This way, if you get two very different answers for your two wells, you know that the assay went wrong and not to use one of the samples. Wait, which sample is good and which sample is bad? To avoid that problem, just use three wells per sample (now 75µg per dog). Then you can throw out the bad one and keep the two good ones. I had to do this maybe 4-5 times total out of my 90-odd samples. Every time, I was really glad that I had three wells. With two wells I would have had to discard that sample (and that dog) from the study. With one well I would have included bad data in my results.

So 75µg is still not all that much saliva, but it turns out that it is enough to be pretty difficult to get, especially from dogs who are stressed out in a hospital. I used three Sorbettes and rolled them around in the dogs’ mouths for up to four minutes, at which point I had to stop in case the stress of restraint was affecting the cortisol levels. Even then, I had a lot of dry sponges. It was incredibly disheartening. In the end, we saved most of my samples by a) diluting them and changing our calculations, and b) showing the dogs cans of cat food to make them salivate.

I am currently engaged in an email exchange with other researchers who are having similar problems, particularly in small breed dogs and puppies. These days, the new tech to use to get saliva out of dogs is a small rope which the dog can chew on. I like that better than the little sponge-on-a-stick, which dogs could possibly break off and swallow (I had one come perilously close to doing just that). But even so, the problem of getting enough spit remains.

Could you give the dogs food? There is a study suggesting that cheese will not interfere with the cortisol assay, and would be safe to give. [1] It makes me nervous, though.

Could you condition the dogs to salivate when you present the little rope? This is currently under discussion, but some of us are concerned that messing around with the dog’s experience of sampling would invalidate the sample. It’s worth a small study to test it out, though, for sure. I hope someone does it.

By the way: I heard a story, which may be apocryphal, but I will repeat it anyways (and maybe someone out there can corroborate): supposedly a rhino salivary cortisol study used the procedure of collecting saliva with a very long-handled spoon. If true, it is awesome.

To come: urine, feces, and hair, oh my.


[1] Ligout S., Wright H., van Driel K., Gladwell F., Mills D.S. & Cooper J.J. (2010). Reliability of salivary cortisol measures in dogs in training context, Journal of Veterinary Behavior: Clinical Applications and Research, 5 (1) 49. DOI:

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