Semen Collection & Freezing Journey

I found this post from nearly three years ago lurking in my “drafts” folder. I am publishing it in the stream-of-consciousness form as it was written at the time, and will add updates below.

I wanted to write this down, primarily for future myself… because nobody talks about it! But we have recently undertaken the journey towards collecting and freezing semen from Braam, so here I record our experiences and thoughts.

First – the decision.

Why collect at all? My thoughts:

  • Kooikers are a rare breed. They are widely spaced geographically and it is sometimes very difficult to thus arrange breedings. Also in a rare breed, it would be valuable to be able to resurrect certain lines through frozen semen if in a few generations they have been lost. These challenges make frozen semen a fairly natural choice in my eyes.
  • Braam is a virgin with no breeding plans on the horizon. He is “out of commission” while we wait on the fine folks at U of Utrecht to develop a genetic test for polymyositis. However, we may be waiting another ten years – and by that time, B will be far less likely to have viable sperm even if he receives a clear polymyositis result. So if we collect him at 3 years, we don’t have to worry about whether the test or his decline in fertility will come first.
  • I am personally interested, having worked on projects in Virginia and California on collection, cryopreservation, and AI in both cattle and critically endangered hoofstock species (primarily, dama gazelles). So I’m very curious to see how all this is run as a commercial venture. And, Braam was an AI baby himself! Cool, eh?

 

Preparation

There’s not a ton of information or specific advice or requirements listed online about this. So here’s what we did!

  • Brucella test. Some countries require a negative result to accompany any frozen semen shipped internationally, although the timing of negative result to collection appears to differ by country. Ours was done 10 days prior to collection. From my research, it appears a Brucella SNAP test is available through Zoetis and at least one other country, but I contacted three veterinary clinics (including two reproductive specialties) and none of them will use the SNAP. Instead, I had to get blood drawn and sent out to the lab. This ran me $45 as I had a wonderful coworker who drew blood for me and sent it out entirely at cost, but the clinics were quoting me around $100 for it.
  • Other testing? There is little talk about further testing of the male, but it appears there is some concern about herpesvirus and mycoplasma infections being spread through frozen semen. As Braam is a healthy, virgin dog, and I don’t find any solid recommendations to do these tests prior to collection, we skipped them.
  • Grooming? This was again something I invented. I cleaned up his pee-feathers to make everything more accessible and clean, and to reduce the possibility of sample contamination by hair, urine, bacteria or epithelial cells.

 

Extenders/Cryoprotectants?

Briefly… I want to address that it looks to me like science and the dog fancy hasn’t a clue what chemical goop is best to put dog semen in to freeze it. Why, I have no idea – we’ve had, what, 50 years since the discovery that glycerol keeps some spermatozoa from dying at -196C. Why then are we still using glycerol, when post-thaw sperm survival is so low? I did a bit of research and found papers claiming that ethylene glycol, milk, egg tolk (or Tris with egg yolk), methyl-formamide, coconut milk, and sucrose all work better than glycerol. None of them, however, appear to have been tested against each other, and the research methods and limited number of study dogs makes me doubt the results of most of these studies anyway. In my past work, I saw with my own eyes how different buffers/extenders/cryoprotectants in different concentrations can help or hurt frozen sperm samples. Each individual’s sperm responds differently to these conditions, so male X might have better post-thaw motility in egg yolk, for instance, while male Y does better in just glycerol. Of course this makes me want to experiment on my own boys and determine their best medium for freezing.. but I don’t have access to expensive lab equipment anymore!

Some of this is a little cringey to read now but it is still something that is not discussed, so here is what happened!

We ended up driving 1000 miles in January 2018 to California to have both boys collected and frozen by Sirius Canine Fertility in Grass Valley. I had their brucellosis bloodtests done beforehand, and we had worked on desensitizing to touch. (And, I’ve since learned there’s no reason to test for herpes or mycoplasma.) The staff at Sirius was great about getting the boys comfortable and letting me see the results on the screen. We ended up getting 4 straws frozen from Braam and 6 frozen from Casper, with decent post-thaw motility. We receive an invoice from them every January for the year’s storage costs.

Although I did buy a microscope and extenders (Zoetis Fresh Express and Minitube/MOFA’s products appear to be the gold standard) I also discovered that repro vets don’t particularly like it when you worry about extenders or cryoprotectants! They each have their preferred product that will be used almost certainly, unless post-thaw or simulated shipment parameters drop significantly during testing, which is really unlikely to be investigated in your first attempt at collection and/or freezing. I could write a whole separate post about how to collect and ship for a chilled-semen breeding… perhaps in the future. Until then, feel free to contact me for questions or guidance.

I’ve also accumulated a list of dietary supplements that are reported, either anecdotally with enough frequency to trigger my interest, or in literature, to improve sperm quality:

  • DHA (omegas)
  • Vit E a/o C
  • Selenium?
  • Silica – DE?
  • Linoleic, linolenic, oleic acid
  • Glycoflex, etc (green lipped muscle)
  • Antioxidants
  • Carnitine (L-Carnitine?)

I’ve tried all these except selenium supplements and the boys’ samples are consistently fine, although I don’t have a “control” or any sort of scientific method going on.