Ask John: Question on freezer malfunction

Categories: Evidence-Based ET
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Published on: September 13, 2016

By John F.Hasler

A few years ago I was asked to change my newsletter column from “Ask John” to “Evidence Based ET”.  However, for this Sept. 2016 edition we are back to some questions submitted to me for which there are not really any hard data available.

For our younger members, some background on my experience in freezing embryos follows. In 1977, my first year out in commercial ET practice after finishing my post-doc at CSU, I flew to Cambridge, England and met with Dr. Steen Willadsen. One year earlier in 1976, Steen had published a paper that described freezing sheep embryos followed by the production of pregnancies after the embryos were thawed and transferred. This followed the first report of successful freezing of mammal embryos (mice) in 1972 by Whittingham, Leibo and Mazur. During my visit to Cambridge, Steen showed me how to make ampules from glass tubing and how to make freezing medium containing DMSO as the cryoprotectant.  I came home enthusiastic and optimistic, but spent a couple of very frustrating years with ampules blowing up not infrequently upon at thawing and very few embryos appearing to survive. We finally gave up on slowing lowering the ampules into the neck of a liquid nitrogen tank and Alan McCauley and I paid $12,000 (equivalent to about $30,000 in today’s dollars) for a Planer freezer.  However, we still had to make our own freezing media and the recommended freezing program was over 3 hours long.  Lastly, the biggest mistake we made for several more years was to transfer the very best embryos into available recipients and freeze what was left, often late at night after we returned to home base.  That old Planer freezer was much too big and unwieldy to haul around from farm to farm.

Life improved markedly after we hired Dr. Techan Takeda, who had just completed a post-doc with George Seidel at CSU. Techan brought us his experience with freezing embryos in 0.5ml straws with glycerol as the cryoprotectant.  We also bought an alcohol bath freezer for use in the lab and two portable Hoxan freezers for on-farm freezing.  A switch to 0.25 ml straws followed, and quite reliable freezing in a much faster program was used by the industry for about 10 years.

The use of ethylene glycol as the cryoprotectant in a freezing system now known direct transfer (DT) was introduced to our industry in 1992 following its development by Steve Voelkel at Granada Genetics.  We are continuing to use DT in the ET industry almost exclusively 25 years later.

Freezing, thawing, handling and transfer of ET embryos has really changed very little.  However, I am not infrequently asked about how to handle malfunctions of programmable freezers. The question that came in recently involved “what should one do when the power goes out and the embryos have not yet reached plunge temperature?”  I don’t think that there are any really good data on exactly what temperature must be achieved before plunging cattle embryos.  A certain amount of dehydration must be achieved and that is just not likely to be sufficient at say -15 or 20°C.  My advice would be to go ahead and plunge the embryos if they have reached -25°C or lower.  If they have only reached -10 or 12, I would warm them up and start over.  Between -10 and 25 is kind of a never-never land and there may just not be any way to salvage the situation.  Even in a lab with two freezers it is unlikely that another freezer would be up and running and, depending on the brand and nature of the freezer that failed, it is unlikely that a second freezer could be brought on line quickly enough.

Anecdotal data, unless it is based on 1 embryo, can be useful and I encourage any of you who has a freezer malfunction, but then goes ahead and tries to salvage the situation, to please let me know the outcome when the embryos are thawed and frozen.

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