Monday, March 17, 2014

Learn about the genetics of dog behavior with me

As promised, my big announcement: I will be teaching two online courses for the Association of Professional Dog Trainers this summer.

DNA, courtesy of Wikipedia


 The courses are open to APDT members and non-members, so anyone can take them. I'm working hard to make them fun, and I would love to see some blog readers or Twitter followers in the class!

The first class is just a basic grounding in genetics, nothing really special about dogs (although I use dogs for many of the examples). So if you have a solid or even just passable genetics background, you don't need to take that. If you're not sure you could explain what a gene is or what a chromosome is, though, you would probably be a bit overwhelmed in the second course without having taken the first one.

The APDT is an organization that I've respected for quite a while, so I'm thrilled to be working with them. If these classes are well-received, they will repeat periodically, so if you can't take one or both this summer, you should get another chance later.

Saturday, March 15, 2014

The state of the Zombieverse: spring 2014

No matter how many times I resolve that I will post at least once a week, life always seems to get in the way. So I figured I’d make lemonade from the lemons and tell you guys about what I’ve been up to. I know a lot of you enjoyed the posts about life as a shelter medicine intern, but I’ve been suspicious that life in the lab would be less interesting. (“Today I found the perfect set of pipetters! It was awesome!”) Still, it seems possible that there are people out there who wonder what first year PhD students spend their time doing, so here it is.
Me escaping from lab for Scio!

I went to ScienceOnline Together 2014, which was a great deal of fun. The head of my lab smiled tolerantly and let me go — she doesn’t do much layperson-level science communication herself, but she knows I love that stuff and figures there’s probably a good reason for me to get better at it. I went to a discussion about engaging undergraduates in science (run by two undergraduates), where I learned that Facebook is the Thing right now but Twitter might be catching up; a brainstorming session about providing explanations of various scientific concepts to people at a variety of levels, elementary school through expert; and an intergalactic gala, where I met Malcolm Reynolds, the Doctor, and an inflatable Dalek, and ate carbonated ice cream which was made before my eyes.

Back home but still on the science communication track, I have been kicking around ideas with another science blogger here about improving science communication education at UIUC. Let me tell you a story about how much the world needs help training aspiring scientists about science communication! I was in class with some undergrads, and they were talking about our recent midterm. One said: “I didn’t really understand that question where we were supposed to explain the findings as if to a scientist, and then as if to a non-scientist. What did that mean?” The other replied: “I don’t know, so I just wrote the same thing twice.” Ouch.

So yeah, I had some midterms. The one mentioned above was for an excellent class on genes and behavior. I really dig the structure of this class: every week we read some articles, then discuss them on the message board. We come to class already understanding the articles fairly well, and we discuss them more in person, both in small groups and as a full class with the professor. It’s a great design, though it does suffer around midterm time when the students are too tired to muster the energy to do paper analysis. Still, it is always better to read articles than a text book!

I also had a project for a statistics class. Statistics can be mind-bogglingly boring, but it is really essential to understand it if you want to be able to analyze your data well. In my experience doing my Master’s work, asking a statistician for help will lead to some terrifyingly complicated analyses that you will only understand during the moments that he is explaining them, and will immediately become completely opaque when you are trying to explain them to your advisor the next day. Anyways, this stats class was designed for grad students, not undergrads, and this project was to do some analysis of our own data. I am a first year student, so what do I not have yet? Data. I have a bunch of RNA which had been sitting in the sequencing machine for weeks. My hope was that sequencing would finish up in time for me to analyze that data for the class. Nope. So, with the teacher’s permission, I made up the data. It was kind of fun. Not enough significant results? Let me just change those numbers... This is apparently not something we are supposed to do in real life, unfortunately.

Did you hear me mention RNA in the sequencer? Yes, I am also doing research! Last semester was a lot of time at the bench, doing ridiculously finicky extraction work to get RNA out of tissue samples. RNA, you will remember from high school biology or some such, is a single-stranded copy of DNA. The cell makes these single stranded copies for use in making proteins, so RNA is part of the whole translation mechanism whereby DNA turns into an organism. Because RNA is single stranded, it isn’t as stable as DNA (which is double stranded), so handling RNA is a really annoying process involving gloves and this magic spray bottle which kills the evil RNA-eating demons which apparently live in the air, your hands, on counters, and on the mobile phones that undergraduates like to leave in your work area.

But once you get the RNA extracted into teeeeny little vials, you can send it off to be sequenced. What I will get back (what I got back a few days ago, but which involves a lot of processing before I can extract useful information from it) is information about which genes are expressed at different levels in particular tissues. I am using tissues from foxes selected for tameness versus foxes selected for aggression. (Remember, I work on the tame fox project.) Why is that interesting? Well, it seems likely that a lot of the differences that we see in tame foxes aren’t due to changes in their actual genes, but changes in how those genes are regulated. So maybe tame foxes express more of a particular gene rather than a different form of that gene. If I know which genes are expressed at higher or lower levels in tame foxes, I can start to guess at the functions of those genes, in foxes and, eventually, in dogs and humans.

What else? Helping the head of my lab teach her class in domestic animal behavior. I have been working behind the scenes to find good papers for students to read and writing questions to assess the students’ understanding of those papers. It’s the perfect job for me. I get to read a lot of papers about domestic animal behavior, and I get paid to do it.

Finally, I’ve been working on another project which is days (DAYS, I tell you) away from being ready to be announced here. I think it’s safe to say I find it more exciting than you will, but I still think you should check this blog daily in breathless anticipation!

Saturday, February 22, 2014

The spectrum of hoarding

There was almost no furniture in the entire house. This was unusual for hoarder houses, which are usually packed full of stuff or just of trash. But she was a somewhat unusual hoarder, with only 26 cats and one dog. We stood in the living room and talked to her about her animals. We could see into the kitchen, which was swarming with cats, on the counters, poking out of empty cabinets, sleeping in the sink. The woman we were talking to told us how she loved them but how her son had called us in because he was concerned about them and about her.

While we talked, her dog urinated in the middle of the empty floor in front of us. She didn't notice. The room contained a couch and an entertainment center and absolutely nothing else: the entertainment center had a few knicknacks but was mostly empty. On it sat the box of flea preventative that our team had given her months ago. It was unopened.

And everywhere was the smell of urine. I could barely stand to be in the room, but I could do so without a mask, which made it one of the cleaner hoarder homes. All I could think of was getting outside and getting a breath of air that was not thick with ammonia, so thick that my eyes watered and I could not fill my lungs. But this woman lived here. She could no longer smell it. She no longer noticed or cared when her dog urinated in the middle of the floor. And I remembered another time I had smelled this smell: at a cat shelter in another part of the country.

That was in rural New England. The two women running the place called it a shelter, but I knew it was really a hoarding house. They did not know how many cats they had. They would find cats unexpectedly dead, because no one had noticed for days or weeks when they were sick. And everywhere the smell. Not as bad as in the hoarder house in the big city, but bad enough to scare away adopters. Yes, they opened their doors to adopters. They advertised their cats for adoption. But adopters who brought these cats home risked bringing a sick animal into their lives, or contracting ringworm from their new pet, a story I heard unfold at least once from this shelter. The women running the shelter grilled every potential adopter and turned many away for not being good enough for their cats. And they continued taking in more cats — if they turned them away, they said, what would happen to them? But could another fate have been worse than dying trapped in that place, where adopters did not want to come, and were turned away when they did? Was this place a shelter or a hoarding house or some weird combination of the two?

But the combination isn’t weird. It happens all the time. I saw it again at a shelter in the deep South. This shelter proclaimed that they would kill no animals, and they did not, even the dogs who had been there for seven or eight years, that had gone insane and were unadoptable. These dogs were loving with the shelter staff, but when I came within a dozen yards of their enclosures they would erupt in terrifying, violent barking. I had no doubt that if I entered their enclosures I would be badly bitten. One dog was not aggressive, but hardly seemed to see the rest of the world any longer: he spun in circles around his enclosure, up on the roof of his dog house, down on the ground again, paws hitting exactly the same point every time. Over and over and over.

And the dogs who were not yet insane were not moving out fast enough. I could see their fates. This shelter was so overwhelmed with the number of dogs they were managing that they did not have the energy to keep these dogs mentally healthy, or to do the extra legwork it takes to adopt out a large dog in an area like the South which is so overpopulated with them. These dogs needed transfers to different shelters, they needed adoption events, they needed foster care to get a break from the shelter. They got none of those things. And as soon as a cage opened, it was filled with a new dog. This shelter actually transferred dogs in from outside their community. If they had not done so, they told us, what would have happened to those dogs? Where else could they have gone?

When I started to look, I started to see it everywhere. How many shelters provided enough space for their cats? Even the shelters that have big condo-style cages for cats in their adoption areas, with enough space to move around and a separate area for the litter box, even these shelters still have cats in tiny three by three foot cages in the back, in the sick rooms, in the intake and holding rooms: not enough room for a cat to stretch out, not enough room to get away from the litter box to eat. Even these shelters tell me of course they can’t use that antibiotic, because it must be given twice a day, and they don’t have time to visit so many sick animals twice a day, so they must use the one that doesn’t work as well but can be given less often. Even these shelters say, Of course we would love to have dog play groups, but we don’t have enough trained staff to manage them.

So what is sheltering and what is hoarding? A good shelter provides a needed service: a brief place for an animal to stop on its road to a new home, some medical care, some help finding that home. Keeping that stay brief is the hard part. It is, in fact, a very hard part, balancing keeping the animal healthy (it’s hard and potentially unethical to adopt out a sick pet), finding the right adopter (for that pit bull type dog that looks like row upon row of others in your shelter, for that orange cat that doesn’t come to the front of his cage to meet adopters), keeping them mentally healthy while you’re at it (play groups, training, just plain time out of the cage and time with humans).

And keeping large animals like dogs takes space. In fact, keeping a small animal like a cat takes space, much more space than we as a sheltering community realized until recently. Sheltering can so easily start to slip down the spectrum. It is a spectrum! Many shelters, real shelters, shelters that have legal non-profit status with the government, shelters that don’t smell like urine and have lots of volunteers and get grants and have spay-neuter services, yes these shelters too can fail to provide minimally acceptable care for their animals. Simply because they have too many.

What’s the answer? In one sense, it is simple. Call in someone from the outside, who can see your facility with unbiased eyes. Someone who doesn’t have to answer all the calls for pets that need homes. Someone who can look at the pets that you have and tell you how you are keeping them. This person must know how to assess humane capacity, so it can’t be just any animal lover. They should be able to look at your facility, know how much space is appropriate for animals of different sizes, and calculate how much space you have for each animal. Then they must look at your staffing, and calculate how much staff time you have for each animal. Not just to feed and clean them, but to spend time with them. To notice when they are sick: to look at them every day, carefully, and think about what they need and how to get it to them. To not be running ragged putting out fires, but to be able to plan adoption events, and to notice that dog that’s been here for months and needs special work to get out.

When this person tells you how many animals you can care for humanely, not just how many you can fit in your facility, you will be shocked. You will deny it. You will say that’s half what you can handle. You will point to your records: you’ve had many more than that for years! But they will insist: yes, you’ve had more. But you haven’t had them humanely. You have had a toe on the road to the hoarder end of the spectrum.

So in another sense, it is not at all simple. Because embracing humane capacity means accepting that you have not done as well in the past for your animals as you could. It means not beating yourself up over this, not feeling guilty, not indulging in denial. It means moving forward: it means promising yourself and the animals in your care that you will do better in the future.

And even harder, it means saying no. No to the animals that need a place. No to the people who just can’t find a home for their pit bull type dog but have to move, and the new place does not take animals. No to the mom whose dog bit her child but is such a loving dog with adults. No to the stray cat who is so sweet. Surely you can find homes for these animals? If you don’t, who will?

But that is the central point: you can’t do it humanely, but you try anyway, you will add to the problem. Sheltering is where it is because shelters try to do too much, and if a shelter’s doors are open, animals will pass through them. Shelters which have put waiting lists into place have found that many people do manage to find homes for their animals, if they have to keep trying. Some don’t, and they wait until a place opens. Will these people abandon their animals on the street? Will they take them to a quiet spot and shoot them between the eyes? Very, very rarely does this happen: when it does, it is big news. But you know what? Doing that is illegal. And that is where we need to prevent it: with laws, and enforcement of them. Not by saying, Okay, I will take over your responsibility for you.

That is what we as a society must embrace: responsibility. Not taking in animals that we can’t care for appropriately. And not accepting someone else’s responsibility. Being strong, and doing only what we can, and no more. Because by doing more, we actually do less.

Tuesday, February 11, 2014

Shelter dogs, movement, and stress

The always-awesome group of researchers at the Center for Shelter Dogs (associated with the Animal Rescue League of Boston, MA) has just published a new paper. They took a bucketful of different kinds of stress measurements of dogs in their shelter and looked to see if there were correlations between the different kinds. They are working on the same problem that I tackled in my Master’s work: it is awfully hard to tell which dogs in a population are stressed; can we use some kind of easy marker (like observing behavior) to do it? They got similar results to mine: yes, it’s super hard! There is no silver bullet answer. But they provide some interesting insight into how to move forward in the quest to improve stress detection methods.

Jones S., Dowling-Guyer S., Patronek G.J., Marder A.R., Segurson D’Arpino S. & McCobb E. (2014). Use of Accelerometers to Measure Stress Levels in Shelter Dogs, Journal of Applied Animal Welfare Science, 17 (1) 18-28. DOI:

The biggest contribution this paper makes, I think, is the use of an accelerometer to test activity levels in shelter dogs. They attached this device to the collars of dogs when they first came in to the shelter, and got a report of how much the dogs moved around over the course of 24 hours. Because cortisol is such a difficult measurement of stress to interpret, it’s important to supplement cortisol measurements with other measurements, to sort of triangulate your answer. Movement in the kennel is one that I haven’t seen measured before in shelter dogs, and is something that might provide some interesting answers: do dogs move around a lot when they are stressed (pacing) or very little (depression)? I’m really happy to see this new measurement entering the shelter dog literature.

The researchers also attempted to provide a stress score of the dogs by watching them and subjectively assessing stress levels based on a minute’s worth of behavior. This is the holy grail of stress studies: can we look at a dog and tell how stressed it is? If so, we wouldn't need to do all this correlation with cortisol levels. I think we all want to say that someone who really understands dog body language can tell if a dog is stressed by looking at it. I know that I believe that I can estimate the average dog's stress level by looking at it. If you see a dog with low body posture, refusing to meet your eyes, maybe shaking, it’s stressed, right? How hard is that?

Well, if you try to compare your observations (particularly over a very short period of time — in my work, I had more luck with 20 minutes than with 2), the answer is, it’s extremely hard. In this study, they tried to assess an average level of stress in the dogs by taking multiple cortisol measurements, both salivary and urinary. Salivary cortisol changes so very fast that I wouldn’t expect it to correlate well to a day’s worth of exercise, or even to behavioral observations unless they were taken within just a few minutes of the saliva sample, but as this study involved multiple saliva samples over time, I was interested to see if a better average measure was obtained. The researchers also compared salivary cortisol samples with urinary cortisol samples, and since urine builds up in the bladder over time, I’d expect urinary samples to produce a better average as well.

So what did they find? To the question of movement (measured by the accelerometer) vs cortisol (salivary and urinary):
  • Maximum activity level correlated with salivary cortisol (p = 0.025)
  • Maximum activity level did not correlate with urinary cortisol
  • Mean (average) activity level correlated with mean urinary cortisol (p = 0.028)
  • Mean activity level did not correlate with salivary cortisol
To the question of the behavioral scoring: no correlation with cortisol of either kind.

So how we interpret all this?

First off, this study ended up enrolling only 13 dogs, taking quite a few measurements of each dog (I haven’t actually covered all of their findings here), and trying to draw conclusions. On the one hand, I want to emphasize that this is how stress studies in dogs are done. I did exactly the same thing in my stress study of hospitalized dogs. It is just extremely hard to enroll enough dogs. If you read the paper itself you get a feel for what the primary researcher went through, as she lists the reasons she had to exclude dogs from the study. It reminded me of my intense frustration during my Master’s work as I had to give up on dog after dog for a variety of reasons. This is par for the course in all these studies: it is almost impossible to get the time and funding to enroll enough subjects to have solid statistical findings. So I am not in any way criticizing these researchers, who did a great job introducing some interesting findings.

But on the other hand, it’s important to recognize that with so many questions and such a small sample set, it is almost impossible to trust the statistical significance of the results. If you do a study in which you ask 100 questions, and you set your p value at 0.05 (which is usual), then you are saying that you expect 5 of your answers to look significant even though they are not. That's what a p value is: setting the bar at which you accept a few false positives. One way around this is to have more subjects, which will lower your p values. Then you can say you'll only accept p < 0.01 or something even more stringent. This makes your findings a bit more trustworthy.

For the number of questions this study asked and the size of their sample set, I would take their findings with a grain of salt. Does activity level correlate with cortisol level? I think it's likely that it does. But I also think that what this study tells us is “this is an interesting area which is worth more study,” not “you should trust that these findings are absolutely true.”

Moreover, what are high cortisol levels telling us about dogs who move around a lot? Exercise itself can increase cortisol levels. This can be a good stressor. So are these dogs distressed or not? This is a problem which is going to be very hard to pick apart. I think a lot of the dogs in a shelter who pace incessantly are indeed very distressed, but some, as this paper points out, are coping with their distress by means of that exercise and are doing better than the dogs who don’t move. The paper also asks the question about how to interpret movement in small versus large dogs. Small dogs simply have relatively more room to move in a little shelter kennel. So how does that change the equation?

As for the lack of correlation between the behavioral stress score and cortisol levels: I feel pretty confident interpreting that one despite my comments about statistics above, because this is a question that has been asked before, and is always answered the same way. We can’t tell what a dog’s cortisol level will be by looking at its behavior. Why not? Is it because we don’t know what the dog's inner experience is (we don’t know if the dog is feeling stress)? Or is it the cortisol level that is lying to us, doing a terrible job of telling us about stress levels, and we’re interpreting the behavior just fine? We don't know, but we do keep trying to find out. Hopefully one day we will.

In the end, I enjoyed this paper. Kudos to the researchers for exploring stress levels in a variety of ways, instead of just one or two. This study was well designed, in my opinion; it just needed a lot more dogs. Hopefully this group or another one will be able to pull together a bigger study going forward.

Tuesday, February 4, 2014

The thing about shelter consulting

I’ve been doing a lot of thinking about shelter consulting over the last few weeks. The thing is, shelters need help. There is a massive amount of work to do to make things better for the animals who are unlucky enough to end up in the American shelter system. Some shelters do things pretty well, some not so much, but they can all do better, and they can all benefit from the work being done by shelter medicine specialists. We have learned so much in the past decade about how to get animals out of shelters faster and how to keep them healthy while they are in there, and that information is percolating into the shelter system at an upsettingly low rate.

But how can the specialists work with the shelters to get these changes implemented? I have seen specialists offer to help shelters for free, because they are paid by animal welfare organizations and by universities to offer free consulting and extension work. The shelters accept in theory, but when the time comes for the director or the board to commit to real change, they push back. No, we’ve always done it this way. No, what you’re saying doesn't make sense to us. No, we don't think this is the right thing to do.

And why shouldn’t they? The consultants have years of expertise, but it’s hard to believe that someone who’s giving you advice really knows what they’re talking about unless you’ve seen them in action. And the staff at these shelters don’t get to see the consultants successfully helping other shelters. They just see the consultants coming in from the outside, not having worked at their shelter, not understanding their specific history, with no close personal ties to shelter staff. (Some of the shelters where these stories have played out are in small communities where everyone knows everyone and always has.) They often don’t even understand the consultants’ credentials. This one is a “doctor” but apparently not a veterinarian — what does someone with a PhD know about shelters? This one is just a "consultant" and not a veterinarian — what’s her actual experience? This one is a veterinarian, but veterinarians aren’t shelter staff, and in the staff's past experience, most veterinarians don’t know much about how shelters operate. She says she has advanced training in shelter medicine, but what does that really mean? How is it relevant to this shelter?

The consultants almost inevitably get impatient. It is hard to take the time to get to know a shelter, build ties with the staff, and introduce change at the rate at which staff can accept it. It’s hard to do that with one shelter when there are dozens more that need your help too, and you just want this one fixed so you can move on to the next one. It’s hard to take the time and accept the slow rate of change when you see animals being neglected and even abused in a shelter, with the staff not understanding that the level of animal care is unacceptable. When you truly feel that the situation in a shelter is an emergency, how can you take the time to do things right? How can you stop yourself from barging in and just getting it done? But if you do those things, you will lose the trust of the shelter staff. When is that worth losing?

Sometimes shelters actually go out of their way to invite big consultations with multiple specialists. I have seen this in universities. A shelter sends a request, and often pays for the service. A dozen or so veterinary shelter medicine specialists spend a week crawling all over the shelter. They often antagonize the staff, simply by watching and photographing them at work. The staff feel that they are being graded and judged — and they are; there's really not a way around it with this "learn everything about the shelter in one week" approach. At the end, the consultants give their findings. I have seen staff cry at the final presentation. I have seen consultants deliver massive documents, hundreds of pages long, with recommendations. No one should be surprised that these recommendations are not followed. The experience isn’t pleasant. But is there a better way?

Shelter consultants are actively seeking better ways, and while I was at a shelter medicine program, I saw the consultation procedure evolve rapidly. But I think the essential dilemma remains: how can consultants give a single shelter the time that it needs to digest change, when their job is to help many shelters? Is the process simply too labor-intensive?

One shelter director told me recently that she didn’t feel the need to work with consultants because there is so much information available directly to her now, online at Maddie's Fund and ASPCAPro and at shelter conferences like Expo. Why should she let outsiders tell her what to do when she can learn herself? There is a lot to be said for this approach. I am absolutely a fan of education, and of providing new findings to everyone who’s interested. I think that if shelter staff really own their own education, they will accept new procedures much more quickly. Personally, I’d always rather make my own decisions after understanding the problem space, rather than being told what to do by someone else. (Flashbacks to being a teenager!)

But I think that if all these available resources lead shelter staff to feel that they can do it all themselves without consultants, they are missing a valuable resource. Consultants are experts. They are immersed in this stuff. And, perhaps even more importantly, they can take a step outside of the experience of one shelter, and draw on experiences from multiple shelters, something that can be extremely difficult for staff to do. They can focus staff on their primary problem -- something staff may not even be able to recognize, due to having lived with it for so long. I visited one shelter with dogs who had been on site for years. The shelter was full of behavioral emergencies, dogs who were engaged in full blown stereotypical behavior, dogs who were terrified constantly, dogs who were aggressive to all strangers. The shelter staff couldn’t see it. The dogs were great with them.

So, does it ever work? I am going to close this pessimistic post with a video about one time when it did work. Here, the two leaders of the Maddie's Shelter Medicine Program at the University of Florida accept recognition from the city council of a nearby community for their help with that community's municipal shelter. Watching this video made my heart warm. The councillor presenting the award listed accomplishments I never would have expected a politician to recognize as important. Maybe it is all possible. May we all find a way to work with shelters, not against them. And may shelters find a way to work with us.


Sunday, January 19, 2014

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!

Monday, January 6, 2014

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

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

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: