Designing stress studies, part 3: how do you get the pee?

Having discussed how to choose what substance to test for cortisol (blood, saliva, urine, feces, hair), and how to get the blood or saliva, I now move on to how to collect the —

Urine

I don’t have any personal experience with collecting urine for stress studies. How hard can it be, though, right? I certainly was sent to collect urine from patients fairly frequently as a vet student, and have fond memories of chasing male dogs around a yard with a cup while they would spray just two or three drops at a time. The best vet clinics have long-handled soup ladles which you can use to collect the pee. I have certainly never used my own soup ladle to collect pee from my own dogs to take in for analysis when they were doing poorly.

One of my professors this past semester analyzed estrogen in baboon urine. Apparently one waits on the ground while the baboon is in a tree and watches. Eventually the baboon pees out of the tree. It falls on the ground and voila. Confused, I asked, “But doesn’t it soak into the ground? How do you collect it?” She explained that usually it fell onto a leaf and you could use a syringe to get it from there. I thought to myself: your world is not my world.

Getting pee from cats is a whole separate story. You provide them with a litter box with nonabsorbable pellets, and collect the pee from that. It sounds simple in practice, but in my experience many cats will refuse to pee on a non-absorbable surface.

Of course, if all else fails, you can extract urine directly from the bladder of a dog or cat using a needle. This procedure, called a cystocentesis, obviously requires trained personnel, who may not be available to all studies.

No post on pee would be complete without input from the queen of pee, Julie Hecht. When asked, Julie had quite a bit of advice about urine collection in dogs. She pointed out that when the study in question is being performed using laboratory animals rather than pets, you can teach the dogs to pee on command. This is super convenient, but you’re less likely to have that option with pet dogs. She listed some pitfalls that she found with colleting pee from pet dogs:

• Timing! If you need to collect pee before and after the particular event that you’re studying, it is problematic if the animal doesn’t feel the need to go at the right time.
• If you are out walking with the owner and the dog, try not to act weird. Dogs notice when you act weird. Then they don’t feel like peeing. So make casual conversation, even though all you are thinking about is collecting that lovely, lovely pee.
• Wind sucks. Wear plastic gloves.
• If a dog has a lot of fur, finding the urine stream can be hard. She says succinctly: “That stinks.”

So that is the lowdown on pee collection, and the conclusion of my series on designing stress studies!

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.

Blood

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

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.

Sorbettes

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.

References

[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: 10.1016/j.jveb.2009.09.027

Designing stress studies, part 1: what do you sample?

Apparently I am an expert in designing stress studies in dogs using cortisol, because I have published one paper about it. Here are some of the words of wisdom I have to share from my extensive experience. You may also be interested in my previous post from several years ago, Why cortisol sucks as a measurement of stress. As I have so many words of wisdom to share, I am going to start with a post just on what you should sample in order to get some cortisol levels. (I intend more posts to follow. But you know how these things go.)

You can measure cortisol in blood, saliva, urine, feces, or hair. We consider the blood (plasma) measurement to be the gold standard: when the adrenals release cortisol, they release it into the blood. This is the hardest to get (you have to stick a needle into the dog) and the fastest to change. Blood cortisol starts increasing only 3 minutes after the onset of a stressor. Practically, this means that since sticking a needle into a dog is likely to stress the dog, you have to complete the blood draw (probably including catching and restraining the dog, unless it is a very mellow dog) in under 3 minutes! [1] This can be possible to do with some dogs and impossible with others. Either way, it requires someone who is very competent at blood draws.

After cortisol is released into the blood, it diffuses into the saliva. This process takes about a minute, so you should collect the saliva less than 4 minutes after you stress the dog by restraining it. [1] If the dog really doesn’t mind the restraint, you can take longer, but I found that sticking things in a dog’s mouth to collect saliva tended to get them excited. In a hospital, just walking into the dog’s run got most dogs excited!

Blood and saliva are the best ways to measure the immediate response to a stressor: take a baseline measurement (in under 3-4 minutes), stress the dog, wait some period of time, then take the post-stress measurement (in under 3-4 minutes, in order to be sure you’re measuring the correct stressor). Taking a single measurement of blood or saliva is not going to tell you as much: there is no known baseline of cortisol for any species, including dogs. It varies too much hour to hour, not to mention that some individuals just start at a different level when they are unstressed. [2]

So take one sample before the stressor starts. After the stressor starts, how long do you wait to sample again? Definitely the same amount of time for each dog. Studies have mapped the time course of cortisol’s rise and fall after a stressor: it seems to go up for an hour or so and then come back down [3]. This is almost certainly dependent on the stressor, of course. My personal rule of thumb is that 20 minutes is a good amount of time to wait to make sure that the cortisol levels have come up enough to be a good reflection of the dog’s reaction to the stressor you’re measuring. (So, just to be super clear: the 3-4 minute rule is just about the beginning of the rise in cortisol levels. The rise will continue for a while.)

If you are interested in how an animal is responding to a chronic stressor, like a few days or weeks in a shelter environment, you’ll be more interested in some measurement of cortisol which covers a longer time period than 20 minutes. Saliva and blood are awful for this kind of study, because their cortisol levels change so fast that you aren’t getting a good overall picture of daily cortisol level; you’re getting more of a snapshot. You could take hourly samples, but that would be difficult in terms of collection and expensive in terms of analysis.

For this kind of study, most people use urinary cortisol. Technically this is the cortisol to creatinine ratio: what is the ratio of cortisol to a standard urine molecule, creatinine? Measuring cortisol this way standardizes your measurement so that it isn’t affected by how dilute the urine is. Urinary cortisol levels will provide something like an average cortisol measurement over however long the dog has been filling up its bladder, probably about 4-6 hours. Urinary cortisol has  been used as a measurement for chronic stress in shelter dogs [4], where you are interested in average stress levels, not an immediate stress response. (For more on measuring stress in shelter dogs using cortisol, see the excellent recent review by Hennessy. [5])

One interesting study looked at elevations in urinary cortisol after dogs had had a trip to a veterinary clinic [6]. In this case, I worry that measuring a specific stressor that has a beginning and an end prior to urine collection is difficult with this method. When did the dogs start making that urine? Before they got stressed, while they were stressed, after they stopped being stressed? When you are comparing different dogs’ urinary cortisol, are you comparing the same thing?

I rarely see studies using fecal cortisol to assess stress in dogs, beyond the proof of concept study [2]; these studies are mostly done in wild animals, because poop is the only thing you can easily collect from them. I have always thought that fecal cortisol might actually be a really good approach to stress measurement in shelter dogs, though: easier to collect than urine, and measuring a longer period of time than urine (since dogs urinate more often than they defecate), so therefore presumably getting a better average. Today as I was looking on Mendeley for some references for this post, I encountered a new study using fecal cortisol to assess stress in cats. [7] Cool.

You can actually measure cortisol in hair as well! I have not seen this done in dogs. It would be a good measurement of even longer term stress levels, over months. One fascinating study measured cortisol levels in archaeological hair, to determine cortisol levels in prehistoric humans. [8]

So, in summary: saliva or blood are good samples to take for a response to an acute stressor, usually one you have control over. Take a sample before the stressor begins and then about 20 minutes after the stressor has begun. Be careful to take your samples very promptly to make sure you are not measuring the stress of the sampling. Urine and feces are better measurements for chronic stressors, and provide a several hour summary of what the cortisol has been doing in the dog’s blood. You can take just one sample of these to compare to your control group.

References

[1] Kobelt A.J., Hemsworth P.H., Barnett J.L. & Butler K.L. (2003). Sources of sampling variation in saliva cortisol in dogs, Research in Veterinary Science, 75 (2) 157-161. DOI: 10.1016/S0034-5288(03)00080-8

[2] Schatz S. & Palme R. Measurement of faecal cortisol metabolites in cats and dogs: a non-invasive method for evaluating adrenocortical function., Veterinary research communications, PMID: 11432429

[3] Vincent I.C. & Michell A.R. (1992). Comparison of cortisol concentrations in saliva and plasma of dogs, Research in Veterinary Science, 53 (3) 342-345. DOI: 10.1016/0034-5288(92)90137-Q

[4] Stephen J.M. & Ledger R.A. (2006). A longitudinal evaluation of urinary cortisol in kennelled dogs, Canis familiaris, Physiology & Behavior, 87 (5) 911-916. DOI: 10.1016/j.physbeh.2006.02.015

[5] Hennessy M.B. (2013). Using hypothalamic–pituitary–adrenal measures for assessing and reducing the stress of dogs in shelters: A review, Applied Animal Behaviour Science, 149 (1-4) 1-12. DOI: 10.1016/j.applanim.2013.09.004

[6] Vonderen I.K., Kooistra H.S. & Rijnberk A. (1998). Influence of Veterinary Care on the Urinary Corticoid: Creatinine Ratio in Dogs, Journal of Veterinary Internal Medicine, 12 (6) 431-435. DOI: 10.1111/j.1939-1676.1998.tb02146.x

[7] Gourkow N., LaVoy A., Dean G.A. & Phillips C.J.C. (2014). Associations of behaviour with secretory immunoglobulin A and cortisol in domestic cats during their first week in an animal shelter, Applied Animal Behaviour Science, 150 55-64. DOI: 10.1016/j.applanim.2013.11.006

[8] Webb E., Thomson S., Nelson A., White C., Koren G., Rieder M. & Van Uum S. (2010). Assessing individual systemic stress through cortisol analysis of archaeological hair, Journal of Archaeological Science, 37 (4) 807-812. DOI: 10.1016/j.jas.2009.11.010