What’s in a bit of dog spit?

No, really, what’s in dog spit? I feel like I know a fair bit about dogs, I have two (one of them, Meg, is gracing your screen above as I type)- I’m also a fourth-year zoology student. I know that dogs were domesticated around 27,000 years ago1, I know that this caused a series of behavioural and physiological changes in both humans and dogs2. Humans carried on domesticating other species enabling the development of livestock farming and dogs were molded into whatever role humans wanted them to fill- hunter, guarder, companion, the list goes on.

Dogs-to-Breathless-Bulldogs
The various uses of the domestic dog through coevolution with humans13

 Human spit is quite interesting from an evolutionary standpoint, as we advanced technologically and began manufacturing food extensively, the proportion of starch in our diet increased. High levels of undigested starch lead to obesity3. To combat this, we needed to produce more of an enzyme called Amylase, coded for by the AMY gene. We’d always produced this in our stomach and small intestine but evolutionary pressure from diet changes led to copy number increases of the AMY gene, so more amylase was produced and also, importantly, a whole gene duplication4. Humans now possessed two copies of the AMY gene, AMY1, and AMY2, that could evolve separately. This second AMY gene (strangely called AMY1) was expressed in the salivary glands meaning amylase could be produced in the mouth, triggering starch digestion earlier. Isolated communities with low starch diets that suddenly transition to a western diet often have lower AMY copy numbers and experience high levels of obesity and obesity-related diseases when their starch content increases5. So, as far as bodily substances go, spit seems to be quite evolutionarily important for humans.

But what about dogs? Their diets are significantly higher in starch than their prehistoric counterparts’. Surely, they must also have evolved salivary amylase. When I looked on Web of Science (a desperate student’s best friend) I found conflicting evidence (a desperate student’s worst nightmare). There is an abundance of research into pancreatic amylase expansion in dogs post domestication, they followed a similar route to us in terms of expansive copy number increases that vary individual to individual6. Spit scientists are clearly cat lovers; however, my searches for “canine” and “salivary amylase” yielded 10 results; 10 results I might add that ranged from those denying that it exists, to those that just test the levels of salivary amylase as part of a larger study as if there is no debate on it

Papers published in the early 1900s through 1990s found no evidence of salivary amylase or AMY gene expression within the salivary glands7,8. I’m generally of the belief that time makes things better, cheese, wine, men, and scientific methods are no different; the enzyme assay kits we use in 2019 are a lot more sensitive than those used in 1990. Science doesn’t just stop when you don’t get a positive result- you try again, a different kit, different time, different temperature because there is always more to learn. That is just what 4 sets of scientists have attempted to do in the past 2 years- I would like to list you all of their names but that would mean me typing out 23 names of variable difficulty and I don’t fancy that sorry.  All four papers used modern enzyme assay kits and three of them found significant levels of amylase in their canine subjects9,10,11, the fourth found either no amylase or negligible amylase though this was part of a larger proteomic study meaning amylase assaying was not the primary focus12. Only the study with negative results gave the breeds of their subjects in the main paper (Beagle and Labrador retriever).

I decided that it would be informative to run amylase assays on the saliva both before and after the presentation of a treat item, food is a proven trigger for salivary amylase in humans, on a large number of dogs from the same group. I successfully arranged to use dogs training as assistance dogs in my study- I have access to a large pool of Golden Retrievers (like Meg), Labrador Retrievers and crosses of these breeds. Other than the benefit of a large, stable sample pool, working with trained assistance means I’m much less likely to get my hand bitten off when I stick a cotton swab in a specimen’s mouth!

Talking about cotton swabs in the mouth, here is my 10 step guide to getting a dog’s saliva:

k.png
10 steps to taking a canine saliva sample

Step 1: Find yourself a willing dog

Step 2: Introduce your dog to the salivette

Step 3: Remove cotton swab from outer casing (Meg got suspicious at this point)

Step 4: Inset the swab in one side of the mouth, soaking with saliva and scraping the inside of the cheek to obtain DNA- place sample back in the tube

Step 5: Tempt dog with a tasty treat

Step 6: Test the patience of your dog

Step 7: Repeat step 3 on the opposite side

Step 8: Congratulations, you have two samples

Step 9: Realise that the marker rubs off so use masking tape instead (we get marks for ingenuity right?)

Step 10: Place your samples into your highly fashionable white polystyrene cool box full of ice blocks

Now you know how to take a saliva sample you could just do my project for me right? What, no takers? Anyway, once I have collected my samples from the dogs at the assistance dog kennel and training centre, I’ll have to dash back to the lab in Nottingham pretty sharpish so I can centrifuge my salivettes, separating the saliva in the bottom of the tube from the DNA held on the swab. Once they’re spun down I can just leave them at -80°c until I’m ready to do my amylase assay- it’s like hitting the pause button on a DVD, the enzyme and DNA won’t degrade so long as they stay on ice…. or so I hope.

I expect to find amylase in my samples based on the four most recent papers on the subject, if I do, I can then play around with some really interesting stats to find out what drives any potential variation between and within breeds. I’m recording sex, age and breed as factors I can test so I hope I get a chance to produce some interesting stats. I can feel you all glaring at me now, I like stats, I’m weird okay!? There’s no point in lab or field work if you can’t analyse your results is there?

Given the limited research on the subject, my work could contribute to our understanding of canine evolution in a tangible way. I’m not going to tell you that planning this project has been a ball, I’m not working in my supervisor’s field so there are times when we’re both as clueless as the other, and working with live animals is bound to throw up some problems. We’re continually making progress though, and when I’ve got my hands on my first samples tomorrow, I’ll be one step closer to finding out what’s in my dog’s spit.

 

Link to sources:

  1. http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC3494089&blobtype=pdf
  2. http://wwwdata.forestry.oregonstate.edu/orb/BiotechClass/2004%20materials/2A-AG%20HIST/DiamondDomestication-2002-Nature01019.pdf
  3. https://www.cambridge.org/core/services/aop-cambridge-core/content/view/6DB071DD5989F7D0096DD70B0EEDC77B/S1368980004000175a.pdf/div-class-title-diet-nutrition-and-the-prevention-of-excess-weight-gain-and-obesity-div.pdf
  4. http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC2377015&blobtype=pdf
  5. https://watermark.silverchair.com/ddv098.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAjswggI3BgkqhkiG9w0BBwagggIoMIICJAIBADCCAh0GCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQM4V3CyVeRtCtm_MaGAgEQgIIB7g03pjN—O2XBvoJreYXSiWEcZo2JBcP84Il8hXrthO-CUHogMNZJTH9u6pui25h9am0dH0FjVfmgcJh_Dft9lF5s7D8-JaMRR1enQnlNTs7XcGzP6rdpUFqlPxmMX3IPLog3fD-2JCFSSRDlEfanyP4tKliSjo7c5LYoHj7HZg_AFtsHNf3FVw7HWFuSEgGQ9Ot06GVxmY2Gl6-OFYDu37ISnFmaa5T9Dn8y5aLaGI0Mgar2ek_LKAodwBtpvBlWTc1sQlVkTAoseyGxaxdPqPlLKsNCtTv6KnRTIXuHpz-qhDAEX1SkatjBdfyUjfqrzsNCstU83nv3SX9VWJt7J2xzsm1Skpnw7joMQ-5qfWdDnzvzdWuS3HeJqIOSFeS4MR4j0uFl4jKwBye7g2EBie_GvbkLG3W7nB8DC9TEmlhLBNjCpzudAzGH1Lybf3hihVA_-EclAIZRjLVqR-9MUdmpQhs1hdT364bBFhcw6WImF35MazUBhRXhs3AoORleMosCT7OlXcKCKnWyp0FTESiL5A2I2UggBl2EHKI1t9-_EnNf9i-P77ffqZ6TGymPJsUy5BeSo9zYyZvclqYOelbrhmvhCzTeHCrdVWmGXpQIfGSD1Nsq22K7GKu-nHWLOZTqRnv7Z5ZQGace3p
  6. https://www.nature.com/articles/nature11837
  7. http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC330360&blobtype=pdf
  8. https://link.springer.com/article/10.1007/BF02214725
  9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681786/
  10. https://www.ncbi.nlm.nih.gov/pubmed/28830550
  11. https://www.ncbi.nlm.nih.gov/pubmed/29871742
  12. https://www.ncbi.nlm.nih.gov/pubmed/29327448
  13. https://www.ancient-origins.net/history/mighty-bear-dogs-breathless-bulldogs-how-human-manipulation-has-changed-shape-canines-021341

 

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