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

What has cheap genome sequencing done for dog science lately?

So you have the full sequence of a couple few dog genomes, and a wolf genome to boot. (Yes, these days sequencing is cheap enough that genomics researchers can do this. There are more errors in these less expensive, “shallowly sequenced” genomes than the one that we use for the standard canine reference, but even with errors, you can still get the whole genome to play with.) So you have these genomes. And you are curious about domestication. What makes a dog different from a wolf? These genomes each are made up of millions of nucelotides, so when you do a straightforward comparison between dog and wolf, you get hundreds of thousands of differences in nucleotides, ranging from single nucleotides that are different to long stretches where chunks of the genome are repeated in one species but not the other. And what to make of the differences between pairs of dogs — are those important too? It can seem an overwhelming problem.

Luckily, in addition to having fast and cheap access to full genome sequences, we also have powerful computers for analyzing these sequences (and one of my favorite parts of my PhD program is that I get to use my programming skills in addition to my biomedical skills). What people do is think of patterns that suggest that certain areas are the interesting ones, and tell computers to look for those areas. It turns out that if you have a couple of genomes of animals of the same species, you can compare them to find regions where there is very little variation between animals. This suggests that this area is important — everyone has to have exactly the same sequence here, because anyone who has any differences is less fit and less likely to survive to pass on their genes. This is called a selective sweep, because at some point in the past, this change swept through the genome and everyone eventually got the identical copy of this region.

For an added bonus, if you have the ancestral species — in this case I am obviously talking about wolves, which are ancestral to dogs — you can compare this region in that species. If you find that this region is the same in all the dogs but different in the wolves, you have an area which is highly suspicious for being involved in domestication. So you can ask a computer to go find some of these low variation regions for you,

There are a lot of statistical tests that you can do to convince yourself that this area has sufficiently low variation to be interesting, but that area doesn’t, and it has been my pleasure this week to be reading about those in great detail. (Being a grad student rocks, but then sometimes there is statistics.) But the most recent papers I have been reading have pretty much done away with statistical tests to convince themselves that certain areas are involved in domestication. What they have done is to use stats to find areas that are just potentially interesting, and then they actually go look at the areas and see what they see. What known genes are in that area? Anything that could have to do with domestication? Yes? So let’s see how that gene differs in a whole bunch of dogs and wolves. The same in all the dogs, and different from that in all the wolves? Awesome. So what does this gene actually do? Can we understand how the genomic change between dogs and wolves — the mutation — changed the protein? Did it change the protein’s function? Or maybe dogs make more, or less, of that protein. Labs have just been selecting specific genes from these areas and investigating them intensely and seeing what they find out.

The best example of this approach (and the one I find the most interesting, because it was done in dogs, not pigs or chickens like the other papers I have been reading) was published early this year. You have probably heard it if you are interested in dog domestication, because it made a big stir by declaring that dogs had evolved to be better at digesting starch than wolves.

Axelsson E., Ratnakumar A., Arendt M.L., Maqbool K., Webster M.T., Perloski M., Liberg O., Arnemo J.M., Hedhammar Å. & Lindblad-Toh K. (2013). The genomic signature of dog domestication reveals adaptation to a starch-rich diet, Nature, 495 (7441) 360-364. DOI: 10.1038/nature11837

But when you read about this paper, did you know how they figured out that dogs are better at digesting starch? They did one of these low-variation genomic scans. They found some interesting regions. They looked at what genes were in these regions. They found a lot of genes involved in digestion, so they decided to chase that first. (They also found some interesting genes that work in the brain, and hopefully we will see a followup paper on that soon.) They focused on a few genes and tried to figure out what they did and how they had changed between dog and wolf. In at least one case they found that dogs just expressed a lot more of a particular protein than wolves do, and that protein is involved in digesting starch.

There are a lot more regions to look at in dogs, and there are some interesting things to hunt down in tame foxes, too, of course. We are in a fascinating time for genomics. The technology is becoming so inexpensive that we can actually look at the code of genomes belonging to individual animals more more readily than we could just a few years ago, and this is a game-changer. There should be many more discoveries to come soon about the mechanics of canid domestication!

Looking at dog brains

Today I was privileged to visit Dr. Greg Berns' laboratory to see awake dogs in an fMRI. In vet school, of course I saw dogs getting MRIs of their brains as part of medical diagnostics, in hunts for cancer, stroke, inflammation, etc. But because an MRI requires that the subject hold perfectly still for several minutes at a time, these dogs were under general anesthesia, which is both expensive for the owner and physically difficult on the dog.

In humans, we can use the related technology, functional MRI (fMRI), to see changes in brain activity in response to different stimuli, such as music, smells, or looking at pictures. This is a useful tool in research, for example as we try to figure out which brain areas perform which tasks. In dogs, we haven't been able to do such studies, because the only way to keep dogs still enough for an fMRI has been to anesthetize them, and obviously a sleeping dog isn't going to have a meaningful reaction to external stimuli.

At Dr. Berns' lab, they have trained dogs to hold still in an fMRI machine while resting their chins on a chin rest. Can your dog hold its head perfectly still for minutes at a time? What about in a strange room, with loud machine noises all around, with ear muffs on to protect their hearing? It's an impressive feat, and done using entirely positive methods. (The training protocol was developed by Mark Spivak of Comprehensive Pet Therapy, Inc.)

I was most impressed by the dogs' relaxed body language. They entered the machine willingly, when their owners asked them to. They lay down with their chins on the rest and waited. As I watched from behind, I could see that many of the dogs were lying on one hip or even frog-legged, in very relaxed postures, suggesting that they were comfortable being in the machine. (Have you ever had an MRI? It is a claustrophobic experience. Humans getting MRIs would benefit from the extensive conditioning preparation that these dogs had, as well as having a loved one present to feed them treats periodically!) Some dogs would balk at some points and exit the machine, at which point their handler would ask them to return and they would. Dogs always had the opportunity to leave. At the end of the test, they came out happy and wriggly.

Highlights of the day for me:

• The Boston terrier who hurled himself into the fMRI at full speed and then became rock-still for as long as his owner asked him to. That dog was committed to his fMRI experience! (Who would expect the Boston to be the calmest dog in the magnet?)
• The dogs with their ear protectors wrapped onto their heads with an elastic material normally used to attach catheters and the like. They looked hilarious.
• The treats fed to dogs on the end of long sticks so that they're easier to deliver inside the magnet. Ingenious.
• Personally getting to participate in experiments by giving hand signals to dogs who were in the magnet, watching me intently as they waited for their treats.

The joke around the lab is that these tests will tell us why our dogs really love us: are we best friends or just food dispensers? It is a joke because of course fMRI is not a test for love; science has some trouble testing for squishy concepts like that. But fMRI does give us a new  tool for guessing at what goes on in doggy heads, in addition to having to muck around with hormones like cortisol (as I have done) or strange little cognition tests like separation experiments or pointing experiments, as others have done. We have never been able to use this tool on awake animals before, so this is a huge step forward.

It was a fascinating day. I am deeply happy to see non-invasive research going on which takes the welfare of its canine participants into account, and waiting with bated breath to find out the results of the experiments I saw.


Further reading
 

• Greg Berns' home page at Emory

• Functional fMRI in awake unrestrained dogs (open access)
•  

Would you cut off your dog’s leg?

Yesterday we had a lecture on osteosarcoma, a cancer of the bone. Osteosarcoma is not a good cancer, guys. It is liable to occur in younger dogs, it is extremely painful, and no matter what you do, it is almost certainly going to come back.

But there are things you can do to reduce or remove your dog’s pain, and to get more time with him (as much as a few years, sometimes). Because this tumor is so painful and aggressive, you really want to cut it out. But that is awfully hard to do as it usually appears in the long bones of the leg. So the surgical answer is almost always amputation of the limb. Alternatively, you could just do a course of chemotherapy to knock it back for a while, and deal with the pain using analgesics.

Who would want to cut off their dog’s leg? Chemo + painkillers is the obvious answer, right? And yet it is not. Chemo is much less effective than surgery, so you will have less time with your dog if you choose this option. And oral painkillers just don’t seem to help very much with this tumor, so your dog’s quality of life is likely to be pretty poor during that remaining time.

Amputation is actually a pretty good option. It just removes all the pain. And dogs do great with three legs. Dr. Glace said, with his typical deadpan delivery, “Some people say it’s like they don’t know they have lost a leg. That’s stupid. Dogs aren’t that dumb. They know they’ve lost a leg.” But they don’t care about it the way we do. They relearn to walk and then they do fine. Three legs is still one more than most of you have. I have seen three-legged dogs (“tripods”) in a flat-out run. No problems.

Dr. Glace says he won’t amputate a leg from one of the truly giant-size breeds (Great Pyrenees, Saint Bernard, Newfoundland), but noted that he amputated a leg from a mastiff (those are very big dogs!) a few months ago and the dog did extremely well. To test if the dog would manage successfully on only three legs, he employed the high tech test of picking up one leg and making the dog walk around the room on the remaining ones. Success.

The biggest problem, Dr. Glace says, is that owners really don’t want to amputate their dog’s leg. There is something viscerally upsetting about it. It’s one of those situations where your instincts might lead you wrong, leaving your dog with less time to live and more pain. Personally, I can report that I saw a tripod running an agility course, and she did just fine, even over the jumps. I direct you to this blog post about Serena, an agility tripod. Go tripods!

Living with a shy dog

I adopted a shy dog two days ago. You can see how tense she is in my house in the first picture. I’m including a second picture to prove that she isn’t like that all the time! (Also: doesn’t she look just like a domesticated fox that was dipped in yellow paint?)

 

This is Jenny. Jenny spent her first ten months on the same property on which she was born. She got to live with other dogs and knows a lot about how to interact with them. However, she doesn’t know a whole lot about interacting with humans, and we are pretty scary to her. She also doesn’t have much experience with the world in general. She’s been with me for about two days now and is just getting to the point where she’s willing to eat while I am in the room.

When Jenny is really alarmed by something I do, she pees. This is known as submissive urination; she’s sending a social cue to say “I’m harmless; please don’t eat me!” I can mostly avoid doing things to her that are this scary, but sometimes I do have to put a leash on her to get her outside, and then she is liable to pee. I’m taking various management measures to preserve my furniture, but this afternoon Jenny started being interested in eating treats that I tossed her, so I saw the opportunity to engage in some counter-conditioning with her.

The problem

When I approach Jenny purposefully and pull out a leash, she is scared, and pees.

Conditioning a new emotional response

The goal is for Jenny to see the cue (my purposeful approach, leash in hand) and feel good about it instead of scared. The solution is to break the scary cue down into cues that are smaller and less scary, and help her work through each of those with the help of something positive (treats).

It’s not just one thing that tells Jenny that I am about to grope around for the clip on her harness and attach a leash. It is my approach; the way I look straight at her; the purposeful way I walk towards her; the display of the leash in my hand. Each of these things is really a separate cue, and each should be worked on individually.

Working with Jenny

Jenny was on the couch downstairs. I wanted to be able to walk down the stairs and approach her with the leash. First, I tried it without the leash. I walked down the stairs more slowly than usual, stopped farther from her than usual, and avoided eye contact. I tossed her a treat. She thought about it, then ate it.

I repeated exactly the same sequence of events. This time, she ate the treat  promptly, suggesting that she was comfortable with the sequence.

I tried it again, and this time walked a little bit closer to her. That was okay. I tried again, making eye contact and walking faster. This scared her; she wriggled away from me on the couch. I stopped and backed up, looked away, threw a treat. She waited for me to go upstairs before she ate it. I tried again, this time backing up to something that she had previously accepted — stopping a ways from the couch and not making eye contact. This was still successful (she ate the treat without appearing alarmed). Phew. I started progressing again, but more slowly.

Counter-conditioning is extremely simple, but it can be really hard to implement properly in practice. We tend to get impatient. Why do we have to take such small steps? Can’t we go faster? Unfortunately, if the protocol you’re trying isn’t working, the answer is almost always to break the sequence you’re conditioning into smaller events and add new challenges more slowly (or maybe give better rewards; I could explore different types of treats to see if there is something more exciting for Jenny). But that is really hard for most humans. That’s the challenge of counter-conditioning and why it is often best to do it with the help of an experienced trainer until you get the hang of it.

Hopefully I will be able to teach Jenny over the next few days that the leash isn’t scary. For tonight, I stopped while I was ahead and didn’t push things too far.

What could poison ivy possibly have to do with the stress response?

Hyperactive immune system + big back yard + hard-to-find poison ivy + dog who likes to roll in plants in back yard + dog zombie who likes to let her dog sleep on her bed... It’s all bad, people. I’ve thought of various ways I could turn my bad fortune into blog material, but I don’t have the energy due to side effects from various medications (prednisone can upset your stomach; it can also make it hard to sleep, and overdosing on sleeping pills will apparently also upset your stomach), so I’m just going to distill out some facts, inspired by the joyful weekend I’ve had.

• It isn’t the urushiol oil itself on the poison ivy which makes you itch; it is your immune system’s extreme response to it. This may seem like splitting hairs, but it is an important distinction when the rash continues to spread for days (in my case, more than a week). Is the problem that you are being continually exposed? (Dog + yard — this was possible in my case.) Or is the problem that your immune system has become so overstimulated that it is simply continuing to make rashes here and there, whether or not there is any good reason to?
• If the rash just continues to spread, it is worth trying to convince a doctor to give you prednisone. Prednisone is an artificial imitation of our very favorite hormone, cortisol. Because chronic stress suppresses the immune system, when we need to suppress the immune system we can do so by telling the body that it is under extreme amounts of stress. The doses of prednisone that are given in this case are really large compared to the amount of cortisol you might normally expect to see circulating around your blood system. This is why it is important to taper off your dose of prednisone. Your body notices that it is pumped brim-full of glucocorticoid substitute, and stops making cortisol itself. So if you stop taking prednisone suddenly, you could suffer from the effects of a sudden deficiency of glucocorticoids in your system. They are stress hormones, but we have a little bit of stress every day, and so they are actually vital for proper bodily functioning.
• What are the side effects of so much stress hormone in your body likely to be? A lot of the background reading for my thesis involved the effects of endogenous (natural) glucocorticoids on health outcomes, and I did some side reading about the effects of artificial glucocorticoids. So, as you can imagine, I asked my doctor what side effects I might see. He allowed as how my immune system would be suppressed (that was the whole point, after all), and so if I had a fever or some such, I should let him know. For the next few days, I was hyperactive, almost manic, and had extreme difficulty sleeping. I wondered if this was just psychosomatic, due to my knowledge that I was full of stress hormones, and my personal obsession with them. I finally did some research online and discovered that no, these were side effects common to this medication, about which my doctor had failed to warn me. When you are very stressed, you need lots of energy (to run away from the predator which your body assumes is pursuing you). Therefore, your body elevates your blood sugar, mobilizing storage reserves if necessary. This may account for my jumpiness.
• A side note about poison ivy and life with dogs... I know how reactive I am to the stuff, and I am very careful not to touch it. If I had seen any of it in my yard, I would have noticed it. When you live with dogs, it’s important to remember that they may get it on their fur. They may not show signs themselves, but will carry it into the house and give it to you as a present. I don’t actually know that this is what happened, but I suspect. Today I made a hand-made haz-mat suit and toured my yard, killing anything that had three leaflets, then washed everything I could think of, including my dog. Apparently Palmolive is a great way to cut urushiol oil. I also use Tecnu, which is marketed for the purpose, though I hate the smell of it. I was tempted to put this task off until my boyfriend was in town, as he is less reactive to poison ivy, but decided it was best to deal with it while I was still on high doses of prednisone, in case of a reaction.

If this post sounded slightly manic, thank the pred. I have about another week to taper down.

Links post

• Are clicks better markers than words? (The Other End of the Leash): Patricia McConnell gets nerdy about the mechanics of operant conditioning (in this case, animal training).
• Massachusetts Animal Coalition’s Pit Bull Spay/Neuter Task Force: “The task force recognizes that Pit Bulls are the most common breed surrendered to adoption centers and found as strays and that spaying and neutering that is affordable is key to reducing the number of homeless pit bulls in our state.” A constructive approach to the pit bull problem.
•  “Animal connection” helps separate humans from other species (Nobel Intent): Nice post reviewing a journal article about our domestication of various species, with a particular emphasis on how we domesticated dogs. If you want to read more about how humans and dogs developed together, and what the process of canid domestication might have looked like, I recommend reading Dogs: a new understanding of canine origin, behavior, and evolution, by Coppinger and Coppinger.

Why do other measurements of stress suck worse than cortisol?

After an overwhelming number of requests (2) for a sequel to my post Why cortisol sucks as a measurement of stress, I am obliging. The fact that I am in the middle of writing this particular section of my thesis and need some high-level perspective on it might also have something to do with it. So: why do other measurements of stress suck worse than cortisol?

When I left you, you were trying to design a study of stress in hospitalized dogs using cortisol as your marker of psychological distress. You were confounded by the fact that cortisol measures both psychological and physiological distress, and that it varies a lot between individuals. I haven’t been around to keep an eye on you lately, so you have started investigating other approaches to measuring stress other than cortisol.

Cortisol is a messenger used by the HPA (hypothalamic-pituitary-adrenal) axis, for the brain to send a message about stress levels out to the body, for the body to pass that message along to the organs that need to change their operations as a result, and for the body to then report back to the brain that the message has been received, so the brain can stop yelling about it. There are multiple levels in this axis; cortisol comes from the bottom-most level, the adrenals. Why not go up one level, to the pituitary? It is actually in the brain, so it is closer to the source of the message and might be less distorted by the game of telephone.

The hormone that the pituitary gland releases as part of the HPA axis is ACTH (adrenocorticotropic hormone, or “the hormone that makes the adrenal cortex change”). ACTH causes cortisol release. Why don’t you measure ACTH release directly? Unfortunately, ACTH can only be measured in the blood; it doesn’t get into the saliva. (Or urine, hair, or feces, three other places you can go to get an estimate of cortisol levels.) The owners of your hospitalized dogs aren’t going to be happy if you tell them you need to draw blood from their dogs for your study. And remember, you’d have to draw the blood pretty quickly in order to get it before the brain mounted a stress response as a result of having a needle stuck into the body. Cortisol levels change in under three minutes. I don’t actually know how long it takes ACTH levels to change, but I will hazard a guess that since they are farther up the telephone chain, they change faster.

What about farther down the chain? CBG (corticosteroid binding globulin, a.k.a. transcortin) is a protein that carries cortisol around in the blood. The body uses CBG as a way of regulating the stress response. When there is less CBG, cortisol is more able to jump inside cells and do its work. OK, no one actually uses CBG to measure stress levels, because we have no real idea how it works. But it is a very cool system that I’m really curious about. And stress researchers would do well to remember that it is there. If the dogs you are studying are very sick, they might not be able to make as much CBG as a healthy dog would, and that would affect their cortisol levels.

That pretty much exhausts using the HPA. Luckily there is an entire second axis for you to mine: the SAM (sympatho-adrenomedullary) axis. This is the series of chemicals that regulate the well-known “fight or flight” response. This particular game of telephone includes adrenaline (epinephrine), the effects of which which many people enjoy abusing when they go on roller coasters. This axis works much more quickly than the HPA. If you hear a sudden loud noise, you will get an adrenaline rush within a second. So you can try to measure adrenaline levels in the blood, but there is just no way you will be able to get the blood out fast enough to not have the stress of the needle (damn needle) affecting them. If you had a very controlled population of animals, with catheters already placed that they were used to, so that you could draw out blood without stressing them, that might work, assuming you could catch the animals without stress. (Catch a mouse without stressing it: difficult. Catch a dog without stressing it: actually, when I went into the runs with the hospitalized dogs I was studying, they definitely experienced eustress, or happy stress.)

You can also measure adrenaline levels in pee! This turns out not to be useful, though. Adrenaline levels go up and down, as we’ve said, very quickly, in response to individual stressors. Pee collects all those changes and averages them out over however many hours (say six). So this approach is definitely not good for measuring responses to specific stressors, like a sudden loud noise. It might be better at measuring something longer term (hey, like the response to being in a hospital!) but initial studies haven’t shown it to work very well at that, either. Adrenaline is just the most interesting when you can map it as it goes up and down, not when you have to look at an average and guess about what was smoothed out.

What about the other end of the SAM? When you get an adrenaline rush, you have some physical changes. Among many other things, your heart rate gets faster. Can you measure that? Well, again, good luck measuring that in a dog without having the excitement of interacting with a human confound your measurement! And heart rate is very sensitive to physical changes; you might be measuring whether the dog is standing up versus lying down, rather than its level of distress.

It turns out that what is a better way to measure physiologic changes from SAM activation is heart rate variability. Your heart rate normally speeds up a little when you breathe in, and slows down a little when you breathe out. (I actually did notice this in a dog once, in a lab where I was supposed to be learning how to find abnormal heart rhythms, and I had to call a vet over to ask if it was actually normal, because it sounded so weird once I noticed it.) When you are stressed (physically or psychologically), this variability goes away. This is not a bad way to measure stress, but you can’t measure it with a stethoscope; you have to hook up equipment to the dog in the form of a little vest with a monitor attached. This is expensive (too expensive for you to use, because your project is on a shoestring budget!). You would also have to get the dog used to the vest, so that you were sure you wouldn’t be measuring stress from having clothing on when the dog is used to being naked. It is therefore not a good measurement for hospitalized dogs on their first day in the hospital, but it is a good measurement for some studies. It’s best when used in conjunction with cortisol, so that the two measurements can catch each other’s mistakes.

That uses up the SAM, but there is a system that is the opposite of the SAM. When your body is not in “fight or flight” mode, it is in “rest and digest” mode. This mode is regulated by the parasympathetic branch of the ANS (autonomic nervous system). (The SAM is the sympathetic branch of the ANS.) Can you measure parasympathetic activity? It should increase when stress decreases, and vice versa. It turns out that when your body is thinking it’s time to rest and digest, it releases a digestive protein into your saliva, known as α-amylase. This protein is useful for pre-digesting carbohydrates. More α-amylase suggests less stress. And it’s even in the saliva, so it can be measured non-invasively! You are very excited until you find a paper from the 1950s (I am not kidding) which is the last time anyone bothered to look for α-amylase in dog saliva. Dogs don’t make it. Because they are not meant to eat lots of carbs? Oh wait, this isn’t a post about nutrition.

(For those of you who say “OK, but what about measuring stress via α-amylase in humans?” — I didn’t delve any deeper into this one after I learned it wasn’t useful in dogs. My guess is that it suffers from similar problems to measuring cortisol: it measures more than just [lack of] distress. It also has been less widely used than cortisol, so we understand its pitfalls less. This would be another good measurement to use as a complement to measuring cortisol. If you want to use it in humans, read lots studies that have used it before you commit.)

So much for the ANS. But you know that increases in stress cause decreases in parts of the immune system. In fact, that’s partly why we care about stress in hospitalized dogs — stressed dogs may not heal as quickly or as well. Can we measure the immune system?

We can. Your saliva normally contains a kind of antibody called IgA. This presumably provides a first line of defense against the bugs on your food. When you are stressed, you make less of it. (At a guess, this is because when you’re running from a lion, you’re not likely to be eating. You’re more likely to be getting bitten, so your immune system needs to focus on defenses against open wounds instead of microbes in food.) Salivary IgA is known as “sIgA.” Can you measure that in dogs? You can, and it is being fairly widely used in humans, in fact. Only some initial work has been done on it in dogs, though. It seems to be prey to some of the same issues cortisol is — varying regularly throughout the day, varying irregularly between individuals — so it’s not yet clear if it’s really a better option. It might be a good way to go for a long term project. For something short, though, it might be better to stick with what is well-understood.

Are there any other ways to measure immune system function as it relates to stress? As I said, your immune system reorients when it thinks you’re running from a lion, to protect against open wounds. It does this in part by packing the blood full of a kind of white blood cell called a neutrophil. Neuts are the first line of defense against microbes coming in through open wounds. You can measure their ratio to another kind of white blood cell, a lymphocyte, to measure stress. A greater N : L (neutrophil : lymphocyte) ratio implies greater stress levels. In some ways, this is a really great measure of stress, because it takes a little while — an hour or so — for the N : L ratio to change after a stressor. So when dogs first come in to the hospital, if you can get blood right away, you could actually measure their unstressed baseline. A later blood sample could provide a comparison. Then you could ignore all that annoying individual variability, because you would be measuring the difference pre- and post-stressor in the same individual. I would have loved to have use this measurement.

But, as always, good luck getting an owner to consent to not one but two unnecessary blood draws. I am not sure I would have felt good about adding that much stress to an already stressed dog’s hospital visit, either. For a different kind of study, this might be a really good option, though as always, it measures the effects of multiple systems, so there is going to be some extra variability to account for.

And that is why, though cortisol is a really appalling way to try to measure stress (looking at my salivary cortisol data right now, I keep saying “why does anyone use this hormone?!”), it is still the most widely used approach. As we learn more about how all these systems interact, it is possible that some day we will develop a method of taking multiple kinds of measurements and basically triangulating distress. Or maybe we’ll develop hand-held fMRI scanners and be able to directly measure activation of specific parts of the brain. For now, we are stuck with spit.

Responsibility for dogs who bite

My school has a large, fenced field. Traditionally, people have been free to let their dogs run off leash in this field. I’ve taken advantage of this resource myself. However, my school is considering the possibility of closing the field, because some dogs have menaced or attacked people while in the field. My school is fearful of liability — if a dog severely bites someone while loose in the field, the school could be sued. In this economy, simply being sued is something for a cash-strapped school to be very cautious of, even if they are found not to be liable in the end.

In my opinion, the responsibility for a dog which might bite falls squarely on the shoulders of that dog’s owner. And there are currently some potential consequences. The owner of a dog which bites may be sued; the dog may be destroyed if it is judged to be a public menace. I imagine that these consquences do deter some people from letting their unsafe dogs run free in public, but apparently not enough people.

In a perfect world, owners of reliable, safe dogs would have full access to resources such as large fields, and would not lose access to these resources because of the behaviors of owners who do not manage their dogs responsibly. I don’t know the specifics about what happened in this particular field, but it is my strong suspicion that the owners of the dogs in question suspected that the dogs were not completely reliable, and took them to the field anyway, hoping for the best.

This is a bigger issue than just setting a policy for my school’s field. I believe that keeping large spaces open to the public is important. It’s good for humans to have green spaces to walk in. It’s good for dogs to have large spaces to run in. It’s good for humans and their dogs to spend quality time together, for walks to not be a chore. Neither we nor they get enough exercise as it is. It’s also good public relations for institutions which own these spaces to let the public use them.

How can we make the owners of unsafe dogs take responsibility for their pets? It’s a problem society is really struggling with right now. Some people feel that the right answer is to ban particular breeds. I don’t believe breed specific legislation is effective, because I believe it’s not the breed that’s the problem, it’s the owner. How do you ban irresponsibility? How do the owners of a privately owned space control who uses the space, short of disallowing all access?

One solution that comes to mind is that owners of private spaces (or towns with public spaces) require some proof that a dog is reliable before it is allowed off leash in the space in question. For example, a dog might have to pass the Canine Good Citizen test, administered by the American Kennel Club, to prove that it has basic obedience skills. The CGC isn’t an off-leash test, but it’s a start, and more appropriate tests could easily be designed. However, obviously the overhead of such a system would be prohibitive. The owner of the space would have to maintain some sort of registration system, perhaps even give out tags with proof that access is allowed. They would also have to police the space to make sure unregistered dogs weren’t being allowed into it.

The other extreme is to push for punishment, after the fact, of owners whose dogs dangerously misbehave. The space owner could sue such owners themselves. (Is there any precedent for this, I wonder? What grounds would they have?) They could declare that the owners of dogs which menace or bite while in the space will be fined. (How would collection of such fines be enforced?) Perhaps simply posting that the owners of the space are not responsible for any altercations, and then hoping not to get sued if something happens, is the only practical course of action besides closing the space.

I like the idea of having consequences for irresponsible owners. Hopefully such consequences would encourage owners to think before they act, so that no one else gets hurt. Public spaces where dogs are let off leash might be very good places to post advertisements for off leash training classes! But I just can’t figure out the mechanism for what these consequences would be, or how they would be applied. I’m very sad to see open spaces gradually closing, as people are unable to behave responsibly in them.

What about you, Blogosphere? Any ideas?

Why cortisol sucks as a measurement of stress

Standing in the ward of a veterinary hospital, you see a dog jumping up and down in his run, barking. Is he distressed at being here? Or is he just barking to get attention? Obviously, you decide to perform a research study on dogs in the hospital, to measure their stress levels. How are you going to go about getting some sort of numeric measurement of stress, so that you can perform statistics on your data and publish it in a journal?

People have approached similar problems in a lot of ways. One of the most common answers is to measure the dog’s cortisol levels. (Or corticosterone, if it’s actually a rat, mouse, rabbit, or bird.) This is the approach I’m using; cortisol is in saliva, which is why I spent so much time over the last year trying to get dogs to drool more.

What is cortisol, actually? There was an excellent post on mindhacks.com recently about cortisol and how science journalists sometimes misrepresent it. I’m going to use dogs as my examples, but what I have to say is just as relevant to studies on humans, if that is your cup of tea. It might help you to understand some of the news stories floating around about various things which “raise cortisol levels.”

Cortisol is a hormone made by your adrenal glands. Your adrenals sit next to your kidneys, but they produce cortisol in response to hormones released from your brain in stressful situations. So we like to measure cortisol levels because they tend to increase when the brain is sending out “I’m stressed” messages.

OK, but what is stress? What I care about, and what many people who measure cortisol care about, is psychological distress — being yelled at, being scared you’re going to be eaten by a predator, being left in a loud veterinary hospital with no familiar faces around you. Stress is a lot of other things as well, however. It is hunger, illness, feeling too cold, having exercised recently. In fact, cortisol has a normal rise and fall over the course of the day to help your body know that it is time to be awake or to go to sleep. Your adrenals also produce it to help you deal with anything which requires some extra energy. You may need that extra energy for a good reason, such as competing in an athletic event. “Good” stressors like that are known as eustressors. So if you’re going to use cortisol to measure stress, you are going to be measuring both eustress and normal daily stress like hunger, in addition to whatever source of distress you may be interested in.

Knowing this, you’ll try to design your experiment to work around the problem. You’re interested in whether dogs find their time in a veterinary hospital to be distressing. So you will try to remove eustressors from the equation — you will make sure that none of our dogs have exciting things like getting fed or taken for walks happen while we’re studying them. You will also make sure that all of your study dogs are healthy, since illness can raise cortisol levels. And you will measure the dogs’ cortisol levels at exactly the same time of day, because of cortisol’s diurnal cycle. (There is some very interesting debate about whether dogs, unlike all other mammals which have been studied, actually don’t have a daily cycle of cortisol. One theory is that they don’t because they sleep most of the day.) Now you believe you're just measuring distress.

Cortisol is still an awfully bad way to measure distress! Males and females react to stressors in different ways. (This has mainly been reported in humans, but it’s been said that studies of stress in rats which are limited to males miss an important segment of the population.) Age has something to do with cortisol levels as well, though mainly just in the very young and very old. In dogs, it is an open question of whether breed matters, but I’m guessing it does, since personality affects cortisol responses to stress in humans.

So you control for that, too. You get a bunch of dogs of the exact same age, gender, and breed. They are all laboratory animals, so you can be reasonably sure their histories are the same, and you aren’t going to find out at the end that half of them have spent more time in a veterinary hospital than others. (This wasn’t the direction I chose, but some studies do give it a go, using laboratory beagles of similar ages and only one gender. There are obvious ethical implications here, but that’s a post for another time.) You put these dogs in a veterinary hospital and measure their cortisol levels. Now are you measuring their reaction to the hospital setting?

Maybe. The next problem is that all these animals have their baseline “unstressed” cortisol level set at a different point. We don't understand all the genetics having to do with how this system works, but we are learning. We do know that a cortisol level that indicates stress (good or bad) in one individual might indicate total relaxation in another. Many studies deal with this problem by looking only at changes in cortisol levels. They measure cortisol before and after the stressor, and look at the difference, rather than at absolute levels. So let’s assume you can do this in your hospitalized dogs. You keep them in one environment for a few weeks or months, until they have time to settle in and relax, and you keep track of their average cortisol levels there. Then you put them in the hospital and look for a difference. Now are you measuring their reaction to the hospital?

You probably are, but what exactly are they reacting to? Something which is a stressor for one individual isn’t necessarily a big deal for another. For example, the Trier Social Stress Test (TSST) is a test specifically designed to raise cortisol levels in humans. It’s used to study things like how gender affects responses to stress. You stress the person out by making them do some public speaking and public arithmetic. But only 70% of people who take the TSST actually have increased cortisol levels compared to just before they took the test. Doing arithmetic in front of a hostile audience just isn't alarming for some people.

In the case of your hospitalized dogs, some don’t like the noise, and some don’t like being in a cage, and some don’t like having other dogs around, and some don't like all of the above. But some think it’s awesome to be in such an exciting environment with so much going on. The hospital isn’t just one big stressor, it is a lot of different little ones.

If cortisol is such a bad way to measure distress, why do we use it? Unfortunately, it is still the best understood method we have. There are lots of other methods, but they all have their own problems. It’s a good idea to use at least two methods together, actually.

So what do you do? Give up? My approach has been to cross my fingers (maybe close my eyes) and just proceed. I think a lot of research involves just circling around a problem, picking away at it until it starts to give in. Studies of stress may not be able to give precise answers to questions about stressors. But if enough of them are done, our picture of how the stress response works will continue to get clearer and clearer. It’s really hard to know what is going on in the mind of a member of a different species; it can even be hard to know what’s going on inside the mind of a member of your own species. We just have to keep trying.

[ETA: See the follow-up post, “Why do other measurements of stress suck worse than cortisol?”]

Greeting dogs

After reading my assertion that children should learn the appropriate way to approach a strange dog, Nathan commented: It seems strange to put the responsibility there. Why is it OK for us to have creatures around that are dangerous to children who have not memorized this bit of trivia?

It is an excellent question, which I’d like to break down into several questions.

What is the purpose of learning how to approach a strange dog?

It could certainly be beneficial to the human to learn how to approach a strange dog. However, I originally posted a link to the lovely How Not to Greet a Dog comic with the benefit of the dog in mind. I’d like to see more people be aware that their approach to a strange dog can be stressful to the dog. Luckily, most of the time, the dog’s stress at an impolite approach doesn’t have consequences for the human (no one gets bitten or even growled at). But it does have consequences for the dog, who might feel some measure of alarm.

A few days ago, a child visited my dog in my back yard. She put her arms around him and hugged him while making high pitched noises. Jack looked away from her in alarm and licked his nose three times in rapid succession to signal his discomfort. Because he did not growl at her, however, I’m guessing that she assumed he enjoyed the interaction. Doesn’t everyone like to be hugged?

I think Jack felt somewhat as you might if a stranger bodyslammed you on the street. (Remember, canines don’t hug the way we do. Patricia McConnell has made the excellent point that if a dog is pressing its stomach against another dog, it is probably mounting the other dog in a dominance display.) The girl had a good time, but I’m guessing that was mostly because she was assuming Jack was enjoying the hug. Hopefully she’d actually prefer for the dog to have a good time, too.

Whose responsibility is it to prevent dog bites?

OK, but some dogs do bite, and Nathan’s question remains: whose responsibility is it to make sure that that doesn’t happen? The child’s?

In my opinion, it is absolutely not the child’s responsibility to ensure their safety around a strange dog. It is the responsibility of the owner of the dog. If the dog cannot be trusted to put up with a hug, or a high pitched squeal, or a hand waved in its face, then the dog should not be around the child.

But this is the real world. Sometimes children approach dogs when the owner is looking the other way. Sometimes the owner is irresponsible and is letting an untrustworthy dog be around a child inadvisedly. And sometimes the owner thinks the dog is trustworthy, for very good reasons, but the dog is having an extremely stressful day and the child’s behavior is the last straw. You never know. I would say that Jack is “extremely unlikely” to bite, because I have never seen him growl at a child, or try to escape from the vicinity of a child, or even show stress behaviors around a child (until the child does something over the top like hug him — but as soon as she stopped, he relaxed again). But you never know. Any dog can bite. If I were a parent, I’d want my child to have some tools for dealing with a potentially dangerous situation, just in case.

Why should I learn how to politely approach a dog?

I think it would be a good thing for people to learn how to interact with dogs — to learn what is polite in the dog world. We have to learn what is polite in interacting with each other. (Don’t run up to total strangers on the street and hug them, even if they are extremely attractive!) Dogs have to learn what is polite in interacting with us. (Don’t leap up on humans and lick them in the face! ...Some of them don’t learn this, and most of us would agree that this is a real failing on the part of their owners.) So why shouldn’t we also learn how to greet a dog? It seems only fair.

If you don’t like dogs, and don’t intend to be greeting any, then it’s less worth it for you to learn this skill. However, in this case, learning a little canine body language can be helpful for keeping them away from you, too. I once saw a man in a park who encouraged every dog he met to jump on him by crouching down in front of them and raising his hands in front of his body. He meant to prevent them from jumping on him. He should have turned his shoulder to them and avoided eye contact.

A lot of people who don’t own dogs do like them and like to be able to greet strange ones on the street. A lot of young people are in this category, from what I can tell from my experiences walking dogs. Some dogs are social butterflies and don’t much care how you greet them (my old roommate’s dog Casey definitely falls into this category), but some are very sensitive. I think it makes sense for people to learn the polite way to greet a dog — both for the dog’s comfort and, very occasionally, for the human’s safety.

What can I do?

• If you’re the parent of a school-age child, support your child’s school in providing education about how to interact with animals. It’s useful.


• Of course, read that comic if you haven’t yet. It’s cute.


• Patricia McConnell’s book The Other End of the Leash is a great resource if you’re curious about learning more about how to see the world from the dog’s perspective.


• If you don’t want to read a book about this but have some specific questions about how to interact with dogs that you want answered, where can you go? It occurs to me that our society doesn’t really have a great solution to this sort of problem. Maybe the blogosphere can step up. I’d be happy to answer any questions people have — Translator for Dogs would be a fun job for me to have some day. Hopefully dog trainers who maintain blogs (and there are plenty of them out there) would enjoy answering specific questions, too.

The assessment of emotional expression in dogs

“The assessment of emotional expression in dogs using a Free Choice Profiling methodology” (Walker et. al., Animal Welfare).

Do different people tend to have overlapping or at least complementary ways of describing dog behaviors? And if they do, can a computer put together a behavior scale out of those descriptions, even without any understanding of what’s actually being described? Put a different way: Can we describe a group of observations of dog behavior using a fancy statistical technique which is hard for Dog Zombies to understand? Our intrepid investigators put a bunch of college women together with some dog videos and applied a lot of statistical processing to find out.

The mammals

The people: eighteen undergraduate women with varying levels of familiarity with dogs, but all currently studying animal behavior. (At a guess, they were all students in a class taught by one of the investigators.)

The dogs: ten Beagles trained as customs dogs.

The setup: The 18 women sit in a movie theater. They watch video clips of the beagles. They write down words that come to mind as useful in describing the dogs.

At the end of this first session, they hand in their terms. They then are sat down again (it doesn’t say if they’re all together in the movie theater this time, but I imagine them at individual desks for some reason) with the list of terms that they had generated. Each person gets their own list; there’s no collation yet at this point. They watch some of the beagle videos again. (I sympathize. I have watched a lot of Dog TV lately.) They rate each dog against each term using a visual analog scale. That’s a line, from zero to lots, and you put a mark on the line to rate the dog.

So if I were to have the word “energetic,” and be asked to rate my dog Jack at this moment, I’d draw a line and put a mark on it to rate how far along the “energetic” scale he is. Jack is currently fully lateral on the floor and twitching, so I’d put the mark on the far left of the scale. If you then asked me to rate him for “cute,” I’d put the mark on the far right. For “red,” I’d put the mark somewhere in the middle, based on my personal assessment of how red he is on the scale of blond to brick. (Jack’s sort of a strawberry blond, so the marker would be a little left of center.) So these are very individual sorts of assessments.

The computers

The investigators then took all this data and turned it into numbers by measuring the distance of the marks in terms of millimeters. And they handed it to a computer, which performed Generalized Procrustes Analysis (GPA) on it. I love that this technique involves the name “Procrustes.”

So, this is where I sort of want a companion blogger who is an expert in statistics to take over for a few paragraphs. I am not an expert in statistics by any means, but I will give explaining what happens next a shot. Please take it all with a grain of salt; I may be completely inaccurate.

Basically, I imagine the computer spreading all these scores out and seeing which ones match. I think the idea is, if one dog gets a 3-4 mm score by lots of observers, then the computer guesses that those people are all measuring the same thing with those particular terms. The computer checks to see if other dogs also rank similarly with those terms. So if one dog scores 1-2 mm on Term 2 from Observer 5, and Term 3 from Observer 7, then Observer 5’s Term 2 might be describing the same thing as Observer 7’s Term 3. It would then be worth checking a second dog. Is its score on Observer 5’s Term 2 (say 4.4 mm) similar to its score on Term 3 from Observer 7 (say 4.6 mm)? If so, and if other dogs are also similar, those two terms might be describing the same thing (“nervous” vs “shy,” for example).

In addition to seeing which terms might be measuring the same thing, the computer is also trying to figure out which terms are related to each other in other ways. If this dog scores high on Term 1 from Observer 13, does he also score low on Term 3 from Observer 13? Maybe those terms are opposites. (“Outgoing” vs “shy,” for example.) If this dog scores very high on Term 2 from Observer 10, does he also score mid-range to high on Term 7 from Observer 12? Maybe those terms have some sort of relationship, but aren’t exactly the same thing (“outgoing” vs “friendly,” for example).

From all this, the computer comes up with “dimensions.” Although the eighteen women had just ranked each dog in terms of “how much of this term does it have?”, the dimensions are paired, so that a group of positive terms are at one end, and a group of negative (opposite) terms are at the other. In this case, they got three dimensions:

• playful/happy/confident versus nervous/unsure/tense
• alert/inquisitive/investigative versus attention-seeking/quiet/unsure
• playful/nervous/boisterous versus calm/relaxed/confident

So each of the observers’ original terms were allocated to one (or more?) of these dimensions. “GPA thus transforms the 18 different dog-scoring configurations into one multidimensional consensus profile, entirely independently of any interpretation by the experimenter.” In other words, a computer has done all the assignments, and it does not understand what the terms mean. The assignments were done in the complete absence of semantics. There is a point in the process where a check for “satisfactory semantic convergence between observer word charts” is done, checking to see if the grouped terms are reasonable concepts to put together. It’s not clear if a computer or a human performs that check. (How would a computer do it? Using a digital thesaurus? I love the idea of a database with weights for how similar each English word is to every other English word.)

They assure us that the eighteen observers, when their terms are applied to this scale, score the dogs very similarly. I started to lose the thread of the statistics at this point, but they did helpfully provide an image of a bullseye. Thirteen of the observers were inside the bullseye (scored dogs similarly). Five were outside.

The meaning

So what have they actually done here? It looks like these observers tended to pick up on similar traits. So if you show a dog to a bunch of people, they will have similar ideas about it. They may use different words, but whether they say “nervous” or “shy,” they will have comparable amounts of that trait in mind. And that is really interesting.

Of course, I also want to say what this is not. These people may all agree about how shy a dog is, but that doesn’t mean that they are any good at telling if the dog actually is shy. The scale generated here, and the scores these observers made, has not been tested for its predictive power. I would love to see something like a test of the scale on dogs placed in new and strange surroundings. Can a dog’s score on this scale predict how it will respond to a friendly stranger, or how much it will explore versus hide in a strange room? I am not criticizing this technique; it doesn’t claim to answer that question. But I think it’s worth keeping in mind what its limits are.

It does claim to tell us whether different people have similar perceptions, and I would love to see it tested on people of different cultural backgrounds, especially people who speak different native languages. The terms that the observers chose didn’t have to be the same in order to be grouped together, but they seemed to all choose similar concepts. Would people who were the native speakers of a variety of languages have such a strong overlap of chosen concepts?

And, as the researchers point out, the observers were all women. Is it possible that women tend to have similar perceptions about dogs, versus the perceptions men tend to have? Do women assign different levels of importance to different behavioral traits, and are they therefore more likely to choose different traits as important enough to score?

Looking more closely at the dimensions that were constructed causes me to suspect that the dimensions are indeed pulling together different traits. For example, one dimension has “alert/inquisitive/investigative” vs “attention-seeking/quiet/unsure.” Outgoing dogs on one side, insecure dogs on the other — makes sense. But the insecure dogs are also “attention-seeking.” That makes sense logically, as insecure dogs may be more likely to seek reassurance from humans. However, it is a somewhat different trait than “quiet.” In fact, I can imagine that a quiet dog might tend to be less attention-seeking, by virtue of being, well, quiet. So it’s interesting that these traits were pulled together into one dimension, even when people who labeled dogs “quiet” probably didn’t necessarily think of those same dogs as “attention-seeking.” Different behaviors, but one interpretation that pulls them together.

On the other hand, you get the “playful/nervous/boisterous” dimension. That may well be different characterizations of the same trait — high energy. Some people think high energy is good (playfulness) and some think it’s less good (boisterousness, what we call “freshness” in this house). But it’s the same thing, whether it’s something you look for in a family pet or not. So it’s also interesting that these traits were pulled together.

I’m particularly impressed that the computer was able to correctly pull positive and negative ends of dimensions together. It was able to determine that “nervous” is the opposite of “confident,” even though no human ever explicitly told it that. Good job.

On the other hand, it did less well on other terms. Looking at the table which details which terms were pulled into which dimensions, I have to say: “aloof/disinterested” is on the same end of dimension 2 with “curious/explorative”? Really, computer?

And there is significant overlap between the dimensions. “Nervous” and “unsure” each show up in two different dimensions. Lots of other terms overlap, too. If there’s so much overlap, shouldn’t the dimensions be constructed differently, more cleanly, somehow?

The investigators suggest that the resulting scale does describe real behavior, because “the dogs are distributed reasonably evenly over the three dimensions, which suggests that these dimensions effectively characterise observed variances in behavioral expression.” I’m not sure about this argument. Wouldn’t you expect to see clumping of some behaviors? Some things that aren’t desirable for customs dogs, or are unusual for beagles, but show up anyways? Why should behaviors be naturally evenly distributed?

So what does it mean? Could it just mean that computers are able to find meaning in any data set? All that semantic overlap makes me feel “nervous” and “unsure.” What are they really describing?

The investigators address this (I think) in some beautiful but very dense prose: “descriptors are not meant to designate separate, sharply delineated, causal factors, but complementary, overlapping, mutually-enhancing aspects of the whole organism. Rather than be confused by the multitude of terms, the idea is to perceive the meaning expressed through them.” That’s actually really lovely, but I am not yet sure it really works.

The future directions

So what’s next? The investigators suggest using this FCP methodology in research into dog welfare. Sounds like a good idea to me. It’s notoriously hard to really tell if an animal’s welfare is good or not, so new tools might be helpful.

However, I note that the reason it’s hard to evaluate welfare is that our prejudices get in the way. We keep thinking what we would like in a particular situation, without having any way to understand what the animal would like. Good welfare science finds ways to ask the animal. FCP seems to me to be only about asking the human. But maybe it can be used to find new axes we hadn’t considered scoring dogs on, and then we can test what those axes correlate with. Good next steps would be comparing how a dog scores on this scale to physiologic parameters, like cortisol level, immune system function, sympathetic nervous system activation, and the like.

Also, as the researchers note, this method should be tested on a variety of breeds. Although they don’t specify this, I’d like to see the method tested on a group containing a variety of breeds. Just performing multiple tests on groups each containing a single breed doesn’t tell you if this method is any good at handling increased variability. Different breeds do have their different dialects; would that change this method’s effectiveness?

This article was a really fun read. Does it tell us more about dogs, or more about humans? I’m not yet sure how the approach will be used in dog welfare, but the article has some good ideas for things to try out. Good luck to them!

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J Walker, A Dale, N Waran, N Clarke, M Farnworth, & F Wemelsfelder (2010). The assessment of emotional expression in dogs using a Free Choice Profiling methodology
Animal Welfare, 19, 75-84

Links post

• How not to greet a dog. This poster should put posted everywhere. Children should have to learn it with their multiplication tables.
• Blogging in Academia: What Can It Do For You? (The Thoughtful Animal.)
• Whales, Dolphins, and Human Rights
• Guinea Pig domestication (The Thoughtful Animal). It makes me a little sad when the Thoughtful Animal has another excellent post about domestication, because I always intended to blog more about that. But I guess if I’m not going to get off my ass and do it, someone has to. Also, it’s possible his audience is slightly larger than mine.
  

Why won't they drool?

The saga of getting spit out of dogs continues. Why is it so hard? My roommate’s dog Stella will drool enormous strings of spit whilst contemplating our dinners, but I can’t get a sufficient sample out of her when I swab her mouth. I actually convinced someone who had written a peer-reviewed article about collecting saliva from dogs to have a phone conversation with me. She provided technique suggestions. These seemed to help quite a bit for a few weeks, but then I recently went through a run with about ten dry dogs in a row. Maybe it was all just chance?

My response to the run of dry dogs has been to frantically attempt to enroll more dogs. (The time when we need to be done collecting data is rapidly approaching.) Advisor feels that this is not the right approach, though, as she has to stay late after work to perform our stress-reduction intervention (or placebo) on these dogs, and she wishes not to stay late after work until the end of time. This is understandable.

On Advisor’s advice, I have constructed a mix of nasty-smelling cat food, placed in a tin with a cap that has airholes. I display this concoction to my subjects, who may or may not find it tasty-smelling. Some will become enthused and lick the tin. Others will turn their heads away. (I think this is stress and not lack of interest in nasty-smelling food per se. These are mostly labs we’re talking about, after all.)

This evening I tested the Concoction out on a few dogs in the hospital for whom I had permission to take saliva samples. Apparently it works on at least some dogs, because when I wandered back into A Ward a few minutes after testing the Concoction out on a Rottweiler, the techs said to me, “What did you just do to that dog? After you left she drooled a whole gob of spit on the floor!” Success? Or was that a reaction to being allowed to taste the Concoction as a reward?

It just floors me that after all this time and effort, I still seem to be doing only a little better than 50/50 on getting sufficient sample size from any given dog. Who knew dogs don’t like to drool?

Spring semester springs

This semester I have (almost) no classes. My one class is Journal Club, which mostly involves listening to other people present journal articles. One week in March I will have to present an article myself, which will involve a frenzied burst of activity, and then again a return to just research.

Just research! I am looking forward to it, although to be honest, last semester’s classes (biostatistics and bioethics) were surprisingly rewarding. The didactic part of this Master’s degree feels much more relevant and interesting to me than the majority of the didactic classes in the DVM coursework. That revelation is part of my increasing interest in pursuing a PhD in the future.

The distraction of classwork last semester caused a lot of dog videos to stack up, waiting to be analyzed. I was pulling in 2-3 videos of 20 minutes each per week; in theory, I would also analyze them every week, breaking each into 5 second bins in which I use a small web application which I wrote to note the dog’s location (front, middle, or back of the run), position (lying lateral, lying sternal, sitting, walking, etc), whether the dog is panting or not, whether the dog has moved at all in the last five seconds, etc. It is a somewhat tedious process, but on the up side, the good DVD player is in my bedroom, so I get to do the analysis in bed with my animals. I’m trying to analyze one or two videos a day for a while until the stack gets thinner. They take 1-2 hours to do, depending on how often the dog changes what it’s doing. The ideal video is of a dog lying still and not moving for 20 minutes. Two dogs have done that so far. I am fond of both of them.

Yesterday I scored two videos in the morning. The mid part of the day I spent reading about cortisol, for use in a summary paper about markers of stress. The summary papers provide useful background immediately, and should become part of my thesis paper this summer.

Then I went in to try to enroll dogs. I continue to work out the best timing strategy for this. There is a sweet spot somewhere around 5:30 pm. Too early, and I miss some dogs which haven’t yet filtered in unannounced. (It’s nice when dogs have appointments two days in advance, but sometimes people call to make surgery appointments and get scheduled for the next day, in which case the only way I have of discovering that dog is finding it in B Ward in the evening.) Too late, and the surgeons have all gone home, so I can’t find them to ask permission to use their patients. (I embarrassingly once said “awesome!” when told “Dr. Depardeau is still here even though it’s eight o’clock at night because his patient is bleeding out and he’s stuck in surgery.”)

Last night, I went in around 4 pm, and scouted. One dog looked like a great candidate. He was in the hospital for, I kid you not, a migrating foreign body in his foot. A tech and I discussed this as we stared at the anesthesia schedule for the next day: a migrating foreign body in his foot? His hospital record didn’t completely explain why they came up with this theory. I looked at the dog in B Ward, and indeed he had an open lesion on his foot, but he was mobile and alert and looked like a good candidiate. However, he had had imaging (radiographs and ultrasound) earlier in the afternoon, and had been sedated for that. It takes six hours for sedation to wear off to the point where I feel comfortable saying that it no longer affects their behavior, so I wouldn’t have been able to enroll him until 9 pm. That’s outside my window; enroll dogs at extremely different times of day and you risk seeing cortisol levels that differ because of cortisol’s diurnal cycle, not because of stress level differences. Oh, well.

I went to the gym for an hour, and came back around 5:30 in hopes that some new dog had shown up. No. I think the hospital is not quite back in full gear after the holidays, and also Mondays are typically slow. At this point I started looking more closely at the two smaller dogs that I had discarded before. I don’t like to use small dogs; they are put in cages, not runs, so I have to ask someone to move them into a run for my use. I also have a feeling (unscientifically) that small dogs react to stressful situations differently than large dogs do. But at this point I just wanted someone to enroll. One of the two turned out to be on meds which disqualified her. The other probably would have worked out — but his doctor had just gone home, so I couldn’t get permission to use him. (Surprising! People on a medicine rotation, as this resident was, are generally around much later than 5:45.) The lesson: if you think you might possibly want to use a dog, get the permission proactively. Don’t wait to see if someone better shows up later.

Then I went home and read more. I can’t quite get over the fact that I get to spend a year reading and writing. It’s great.

So that seems to be my routine for the early part of this semester. So far, so good. Soon I have to start doing things like doing the actual cortisol assay for the dogs I’ve enrolled, and build the results into a stress scale, but for now things are in a pretty solid rhythm.

Rats, dogs, foxes, and the SHRP

Working on my Master’s degree has made me yen for more letters after my name, so I’ve been doing some spare-time reading on subjects that might yield PhD-type projects. My putative interest is in development of the stress system in young dogs. The idea is that if a dog’s stress system develops poorly, whether through bad genetics or a bad early environment, then that dog is more likely to bite people when it grows up. The more we know about how their stress system develops, the more we can know about how to grow healthy dogs with good bite inhibition.

For several months I thrashed around in the literature, reading about development of the stress system in rodents (about whom we know quite a bit, because we are more willing to do experiments on them than on dogs), and reading about socialization periods in dogs. It was hard to find good direction, and I wasn’t quite sure where to start. Recently I have had a breakthrough, however.

First, some orientation. You are walking through the woods. You see a shape on the ground. Your brain interprets the shape: long, thin. Your amygdala (part of the limbic system of your brain) yells SCARY SHAPE SCARY SHAPE and you get a blast of adrenaline in your system. Half a second later your cortex (the thinking, conscious part of your brain) catches up: hey, that looks like a snake. Your hypothalamus (which deals with a lot of hormone regulation) sends a message to your pituitary (which releases a lot of your hormones), and the pituitary releases a hormone which travels down to your adrenals, near your kidneys. Your adrenals release our old friend cortisol, which gets into your blood and tells your body that you are having a stressful experience. Cortisol, you of course remember, is what I like to extract from the saliva of dogs to tell if they are unhappy about being stuck in a noisy hospital run. This whole system is what I’ve been referring to as the “stress system,” more properly called the HPA (hypothalamic-pituitary-adrenal) axis.

If you were a rat or mouse, instead of releasing cortisol, your adrenals would release corticosterone. It is a very similar hormone with similar effects. Dogs actually release equal parts cortisol and corticosterone, but we just study their cortisol levels. I still haven’t figured out why we chose cortisol to focus on in them; there are a lot of tools available for studying cortisol, since humans make it primarily, but also a lot for studying corticosterone, since we study rodents quite a bit.

Now, to get back to my recent reading, very young animals don’t get as frightened by scary things as slightly more mature animals or adults. This phenomenon has been studied intensively in the rat: rats younger than two weeks of age don’t show this corticosterone spike when exposed to something upsetting. This is called the “stress hyporesponsive period,” or SHRP.[1] There has been work on what part of the HPA system is responsible for this blunted response: the amygdala? The hypothalamus? The pituitary? Or are the adrenals themselves not responsive yet?

A good way to stress out an infant rat is to expose it to the odor of an adult male rat. Left to their own devices, adult males will happily eat infants, so the young rats are quite right to fear them. An infant rat, upon smelling a strange adult male, will become immobile. However, a neonatal rat younger than 14 days (in other words, one still in the SHRP) will not become immobile: it hasn’t yet developed the machinery to feel, or possibly just to express, fear. If you remove the infant’s adrenals, so that it is unable to make corticosterone, then even when it matures to older than 14 days it will still not properly become immobile when exposed to the scary smell. Moreover, if you inject corticosterone into one of these pre-14 day rats, it will be able to develop the immobility behavior at age 14 days, just like a normal rat. [2] This suggests that corticosterone is responsible for the immobility behavior. However, if you remove the adrenals of a rat which has already developed the immobility behavior (one which is older than 14 days), it will continue to become immobile in the presence of the scary smell. [3] And if you inject extra corticosterone into a rat too young to have developed the immobility behavior, it will develop it early. [4] This suggests that corticosterone is responsible just for the development of the behavior, not for allowing it to actually happen at specific times once it has initially appeared.

What’s going on up in the brain while all this is happening? When infant rats are too young to express (or possibly feel) fear, are their amygdalas just failing to activate? When neurons in a particular brain region have been recently active, they contain a protein called c-fos. You can check a brain region for the prescence of extra c-fos to see if it has been doing anything in the recent past. This was done with young rats. Rats too young to have developed the fear response did not have amygdala activity (no extra amygdala c-fos) after exposure to the scary smell; if they were injected with corticosterone to cause them to develop the fear response early, then they did have amygdala activity; rats old enough to have developed the fear response did have amygdala activity; and rats whose adrenals were removed prior to developing the fear response did not have amygdala activity. [4] Unfortunately, this study does not appear to have looked at whether rats which were allowed to normally develop the fear response (intact adrenals), but then had their adrenals removed after initial development of the response, still showed amygdala activation. Perhaps that question has been answered elsewhere.

So what does all this mean for dogs? Do dogs have an SHRP? I found one unreferenced assertion that they do, but I have not yet found a study actually examining the canine SHRP. The SHRP does exist in various species, and it seems likely to me that it exists in the dog. Puppies start out fearless, and develop fear later. I suspect that a canine SHRP will prove to be an important part of socialization: the time that puppies don’t yet feel fear may be an important one for introducing them to lots of different kinds of people, so that they can learn that these people are a normal part of puppy life and are not to be feared later on.

The development of the HPA system has been studied in domesticated silver foxes — foxes selectively bred to not fear humans. (These foxes show surprising physical similarities to other domesticated animals in body shape and color, despite not having been bred for these features, leading to speculation that there is some general mechanism of domestication. That general mechanism of domestication is actually what I’d like to get at in a PhD project.) Researchers took two groups of foxes: domesticated foxes, and foxes bred for increased aggressiveness to humans. They tested them for behavioral reactions to humans and cortisol level increases after exposure to humans, at ages 30 days, 45 days, and 60 days. The aggressive foxes did not show aggressive behavior or cortisol spikes at 30 days, but they did show it at 45 and 60 days. The domesticated foxes, on the other hand, did not show aggressive behavior until 60 days, and their behavior at that time was described more as “defensive” than “aggressive.” They never showed the cortisol spike. [5]

Is this the same thing as a silver fox SHRP? I’m not sure that this study exactly gets at that, but it seems suggestive. Questions I’d like to ask about the SHRP in dogs are: Does the SHRP definitely exist in dogs? Is the SHRP length different in dogs and wolves? Does the length of the SHRP affect the socialization of the dog? Is the SHRP length different in different dog breeds? And, most important but most difficult to get at, does length of SHRP have anything to do with a dog’s fearfulness as an adult?


[1] Walker Claire-Dominique, Perrin Marilyn, Vale Wylie, Rivier Catherine. Ontogeny of the Stress Response in the Rat: Role of the Pituitary and the Hypothalamus. Endocrinology. 1986;118:1445-1451.

[2] Takahashi L. K., Rubin W. W. Corticosteroid induction of threat-induced behavioral inhibition in preweanling rats. Behavioral neuroscience. 1993;107:860-866.

[3] Takahashi L. Organizing action of corticosterone on the development of behavioral inhibition in the preweanling rat. Developmental Brain Research. 1994;81:121-127.

[4] Moriceau S. Corticosterone controls the developmental emergence of fear and amygdala function to predator odors in infant rat pups. International Journal of Developmental Neuroscience. 2004;22:415-422. [Free full text.]

[5] Plyusnina I., Oskina I., Trut L. An analysis of fear and aggression during early development of behaviour in silver foxes. Applied Animal Behaviour Science. 1991;32:253-268.