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A Breakthrough in Freezing Camelid
Embryos:
We are Paul and Sally Taylor, and we have been raising llamas for thirty years. We live in the US, near Bozeman, Montana, and about an hour away from Yellowstone Park. We began to be interested in embryo transfer in llamas about 1990. It was about that time that I first learned about some of the advantages of importation of embryos, something which was by then a routine practice in cattle and goat breeding. Importation of embryos was cheaper than importation of the live animals, and much safer from an animal health point of view, but the thing that captured my attention was that an imported embryo didn't increase the population in the importing country. Each imported embryo had to be carried by a reproductively-normal female, and she would likely be pregnant with some baby in any case. We hoped ET could compete with importation of the live animals before importation destroyed the market for llamas in the US. Very little was known about ET in the camelids at that time. A few ET babies had been produced by researchers in Scotland, but there was precious little practical knowledge about it. We were soon caught up in this exploration of uncharted territory, eventually building a small clinic and laboratory at our mountain ranch and purchasing a very good ultrasound machine. We learned about rectal palpation and the details of reproductive physiology in llamas, not by reading but by experimentation. Many thousands of hours of basic research went into this project in the years that followed, and I can remember Sally breaking down in tears when we saw our first ET pregnancy on the ultrasound screen. Since that time, late in 1993, we have produced over 300 live llama and alpaca babies by embryo transfer, both here in the US and in projects in Chile and Argentina. We were the first to get an alpaca baby carried by a llama, and we now have our names on several important scientific papers about early reproduction and embryo transfer in the South American camelids.
During all of this time, I was working to develop a practical method for
freezing these embryos, and this turned out to be the most difficult problem
of all. The embryos we could get from llamas and alpacas without resorting
to surgery were at a later stage of development than the embryos of cattle.
Camelid embryos start their development in the oviducts, just as bovine and
all other mammalian embryos do. The first few cell divisions after fertilization
happen inside the zona pellucida, the capsule surrounding the ovum. Eventually,
in a matter of a few days after fertilization, the embryo expands and breaks
out of this protective capsule in a process that is called hatching.
The difference in the camelids, and this is a huge difference, is that some mechanism retains the early embryo in the oviduct until after it hatches out of the zona pellucida. Early bovine embryos pass into the uterus about 4 days after conception, and they don't hatch until about 8 days, so there is a big window of time when they can be flushed non-surgically from the uterus with the zona still intact. Llama and alpaca embryos mature faster, hatching at about 6.5 days after the breeding, or only about 5 days after conception. Only then are they released into the uterus where they can be obtained by a simple flush. OK. Why is it so important to freeze embryos anyway, and why is it so important to get embryos still in the zona? Aside from the extra convenience and efficiency of being able to freeze embryos for transfer at a later time, which allows cattle breeders to flush embryos year round and transfer them all at a time when the resulting babies will be born in the spring, the fact is that only frozen embryos are eligible for international movement. This is because animal health authorities can't feel confident that an embryo is truly disease-free unless they can hold the embryo for a month or so after collection and then re-test the donor animals for the diseases of concern. A donor animal could have been exposed to FMD a few days or a few hours before the flush that produced the embryo. It takes at least several days after exposure to FMD before the animal's immune system reacts by producing antibodies to the disease, and it is these antibodies that the tests detect. It is not possible, with current technology, to hold a fresh embryo in culture for 30 days, so the only possibility for legal international movement of embryos is if they are frozen at the time of collection. Freezing of a bovine embryo with zona is a relatively simple and efficient matter. In fact, successful freezing of just about any mammalian embryo, while it is still in the zona, is routine. The freezing of hatched embryos, however, even hatched bovine embryos, has been all but impossible. The basic reason for this, I came to understand after 10 years of research, is the greater amount of water inside the hatched embryos and the larger size of these embryos. Size matters because: * freezing protocols depend on osmosis, the natural migration of molecules through a membrane, to get cryoprotectant molecules (ethylene glycol, glycerol, etc.) into the living cells where they can protect against damage from ice crystal formation *these osmotic movements proceed at a fairly constant rate per unit of surface *the ratio of volume to surface increases dramatically as the diameter of a sphere increases. So it just takes a lot longer for osmotic movement of cryoprotectant to result in an effective concentration throughout a larger embryo than it does for a smaller embryo. In fact, it takes 5 to 10 times as long to equilibrate (bring the concentration of cryoprotectant high enough in) a 7-day llama or alpaca embryo as it does to equilibrate a bovine embryo still in the zona *all known penetrating cryoprotectants are toxic to living cells if the exposure time is too great. A hatched blastocyst, the kind of embryo we get from the llamas and alpacas, looks like a little translucent soccer ball about half a millimeter in diameter. The living part is the spherical envelope of cells that surrounds a central volume of aqueous solution. This envelope is quite flimsy if you push against it, but it is very resistant to puncture. It's almost as if it were made of a fine nylon mesh.
It was 2 years ago that I finally accepted the fact that it would never be possible to freeze these hatched embryos unless I could reduce the total volume of aqueous solution inside the envelope of cells and control the concentration of cryoprotectant in that volume. Understanding that and actually doing something about it were two very different things. I wanted to inject cryoprotectant into this central volume so it didn't have to migrate through the cells I was trying to protect in order to get inside, but all my efforts to puncture the envelope, even with a very sharp glass micropipette, were in vain.
The standard approach used for microinjection through the zona, the technique used for injection of a single sperm or for cloning purposes, involves the use of a holding pipette to suck the surface of the zona and hold it tight against the opening at the tip. This holding pipette is presented from one side of the zona while an injection pipette is poked at it from the opposite side. This just didn't work in the case of a hatched blastocyst. The envelope just invaginated ahead of the sharp tip of the injection pipette. It became obvious that it would take a new approach, creation of a new device, for injection into and aspiration from the central volume of a hatched embryo. I tried several different approaches to this problem before finally settling on a combination pipette, a holding pipette with an injection pipette inside it. This device, which I was able to cobble together from items I had around the lab and could buy at the local hobby shop, I called the co-axial micro injection system (CMIS) for purposes of scientific writing about it. In conversations about it with other researchers, however, it soon got the nickname "the Dracula Pipette" because of its suck-and-puncture function. Before we could inject into these embryos, hardly any of them survived any of our freeze and thaw experiments. As soon as we got the Dracula Pipette up and running it became obvious that almost all survived, regardless of the specific protocol used. No matter what type of cryoprotectant or its concentration (within a wide range) almost all the frozen-thawed embryos survived. In fact, only the third embryo processed using the Dracula system resulted in our first-ever pregnancy from a frozen embryo! This breakthrough device allowed us to inject cryoprotectant directly into the central volume of a hatched blastocyst and then to remove almost all of the resulting fluid from inside the envelope of living cells. This shortened the total time of exposure to cryoprotectant for these cells and resulted in a higher intracellular concentration of cryoprotectant at the moment of the freeze. Then, immediately after the thaw, we could again grasp the deflated envelope and reinflate it with a culture solution to allow the cryoprotectant inside the cells to migrate out in both directions, toward the central volume as well as toward the outside. All that remained was to find the very best combination of solutions and timing to freeze these embryos of the camelids for commercial uses. We and other camelid researchers around the world are working on that fine-tuning right now. Because almost every embryo survives the freeze to tell us its story, we are finally closing in on a protocol that will give us virtually the same survival and pregnancy rate for frozen embryos as for fresh embryos. At the same time, llama and alpaca breeders in almost every country where these camelids exist are applying for government permits to allow importation of frozen embryos. In the US, UK, EEU, Chile, Australia and New Zealand, regulators are finding formal requests for importation of frozen llama and alpaca embryos on their desks. Within a year or two, it should be possible to move frozen hatched blastocysts of llamas and alpacas to just about anywhere from just about anywhere. As one longtime alpaca breeder in the US put it, "This will really change the landscape." Because we have had experience with moving live llamas from South America to North America, we understand two other, less obvious, advantages of moving embryos rather than live animals. The first is animal welfare. No matter how good the intentions or what precautions are taken, pre and post embarkation quarantines, testing and transport are very stressful for the animals involved. Almost always some die, and the others often suffer negative physical and psychological effects for years afterward. Embryo collection and movement will makes these stresses just a bad memory, a relic of the past. Also, movement of live animals results in the loss of their genetic potential for the exporting country. In Peru, for example, buyers from Australia and the US skimmed the cream of the alpaca breeding stock and shipped them out of the country, forever damaging the ability of Peruvian breeders to produce the fine fiber that was a national heritage. By contrast, if only frozen embryos had been exported from Peru, all of the pre-existing genetic potential could have been preserved. In the future, there can be several centers in each country where fine alpacas or llamas exist or are wanted, to flush their embryos for export or to thaw and transfer high-quality imported embryos. International trade in camelid genetics will become routine, and a robust world market will open up for even the smallest producers of quality genetics, no matter where they live. I believe that alpaca and llama breeders everywhere will give new consideration to the possibility of doing embryo transfer work themselves rather than depending on high-priced professionals. As one famous vet who had told us that ET in llamas was impossible said when I told him we had our first ET pregnancy ten years ago, "Well, it's not rocket science, is it?" In fact, all it takes is a small hand, some sensitivity of the fingertips and some sensitivity to the animals. The supplies necessary are cheap and easy to get, thanks to the popularity of bovine ET work. A few years back I did a demonstration program of practical llama ET in Argentina, in the open under rough conditions. This resulted in a 70% pregnancy rate of the transferred embryos. ET requires rectal palpation, and at first Sally and I were petrified that we eventually would rupture the rectum of one of our llamas. This hasn't happened, and we now have over 10,000 palpations behind us. The danger is still there and we are still very careful, but the record proves that this work can be done with very little risk to the donor and recipient females. Serious breeders of alpacas and llamas can put this valuable tool, embryo transfer, which holds the promise of a tenfold increase in the production of select females, to work for themselves. Thanks to the breakthrough in freezing of embryos they can look forward to being able to buy great new genetics from anywhere on earth and to being able to sell their best genetic production to other serious breeders no matter where they are. |
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