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)
•