Cryogenics in Embryo Transfer
By Meyla Bianco Johnston with Guidance from Dr. Curtis Youngs
During the course of researching “Embryo Transfer in Alpacas: Leaders in Research and Practice Explain,” from Alpaca Culture’s June 2013 issue, I became extremely curious about exactly why alpaca embryos were so difficult to freeze. The wool was pulled from my eyes during the “Cryopreservation of Alpaca Embryos” seminar by Dr. Curtis Youngs at the Pacific Crest Accoyo America Auction in Hillsboro, Oregon on June 29. Reproductive Biologist Dr. Youngs is a professor at the Department of Animal Science at Iowa State University. His research is centered on embryo transfer and specifically on cryopreservation; he lectures regularly on topics such as embryo transfer and laboratory courses he has developed. He has worked with many species including goats, sheep, cattle, and pigs. Youngs was a member of the team that produced the first live goat kids in the United States from frozen-thawed embryos. In cattle, Dr. Youngs says, “Nearly one million embryo transfers are done per year, and more than half of those obtained from living animals have been cryopreserved prior to their transfer.” In all of his research, Dr. Youngs emphasizes that one thing is clear: don’t try to fit one species’ protocol for cryopreserving embryos on another. For example, the standard protocol widely utilized to freeze cattle embryos scarcely works with pig embryos, which contain a very high lipid content. Even embryos from Jersey dairy cattle, which also contain many lipids, produce a 10-15% lower pregnancy rate than embryos from other cattle breeds. Alpaca embryos contain a lot of lipids, but no one has yet reported in the scientific literature the production of live cria after the transfer of cryopreserved alpaca embryos. Youngs hopes to change that.
Obstacles to Cryopreservation of Embryos in Alpacas
In alpacas, freezing embryos is particularly difficult because the embryo has already hatched from its protective outer shell (the zona pellucida) by the time it reaches the uterus. As a result, the alpaca embryo possesses a 18 | Alpaca Culture • September 2013
much larger number of cells compared to embryos from other species. Developmentally advanced alpaca embryos also form themselves into a doughnut shape that is unusual and hard to manipulate. Dr. Youngs explained why else freezing embryos is so difficult: water is the main biological ingredient of embryos. Water forms ice at 32 degrees Fahrenheit (0 degrees Celsius), and when that happens, extremely jagged and damaging ice crystals form. These crystals often come in contact with cell organelles and cell membranes, causing severe cell damage and subsequent embryo death. There are two main approaches used to successfully freeze embryos: an equilibrium and a non-equilibrium approach. They are performed differently, but both depend on dehydrating the embryos prior to freezing. All of this cryopreservation work takes place after embryos have been successfully flushed non-surgically from the uterus of a mated female alpaca.
Osmosis is a key component to embryo cryopreservation, so it is important to have an understanding of how it works. It can be defined as the movement of compounds across a semi-permeable membrane to create equilibrium (or an equal concentration of solutes on both sides of the cell membrane). See illustration at right. The idea with this method is to remove as much water as possible from inside the cells comprising the embryo. The water in the embryo is replaced with something else that is less damaging than water when it freezes. This “something else” is called a cryoprotective agent, and commonly used cryoprotective agents include ethylene glycol and glycerol. The more cells that are present in an embryo, the more difficult the embryo is to dehydrate because some of the cells are deep inside the ball of cells that make up the embryo. As the embryo is introduced to a cryoprotective agent, there is an initial decrease in embryo cell volume, followed by a later increase as the cells attempt to achieve equilibrium. After the embryo is successfully dehydrated in this initial step, it is loaded into a ¼-ml straw and is physically isolated between two small air bubbles. The straw (containing the embryo) is put into an embryo-freezing machine at -21 degrees Fahrenheit (or -6 degrees Centigrade). After two minutes, seeding is initiated. Seeding Dr. Youngs defines seeding as the purposeful induction of ice crystal formation in the cryoprotective solution OUTSIDE the embryonic cells. This is accomplished by touching the straw with very cold tongs. Ice crystals then form in the solution outside of the embryo,
Preparing an Embryo For Freezing
Cryoprotective agent moves in
Embryo at equilibrium in simple salt solution containing H20.
H20 moves out
Embryo is introduced into a cryoprotective solution. This solution contains a cryoprotective agent that does not exist in the embryo. The embryo wants to maintain osmotic equilibrium, so it begins to move water out of the cells and cryoprotective agent into the cells through osmosis.
The individual cells of the embryo have sent water out, causing the embryo to shrink in size.
4 H2O Cryoprotective Agent
As the cryoprotective agent enters the cells of the embryo, equilibrium is once again achieved between the embryo and the surrounding solution. Water molecules inside the cells have been replaced to an extent by cryoprotective agent. Technical assistance from Dr. Curtis Youngs
and crystals progress through the straw evenly, without creating too much destruction to the embryo. After ice crystals form outside the embryo, the embryo boots more water out in its effort to achieve osmotic balance. After seeding, the embryos are slow cooled at a rate of 1.8 degrees F (1 degree Celsius) per minute down to -29 degrees F (-34 degrees Celsius). During this process, the embryo becomes almost completely dehydrated. Embryos are then transferred into liquid nitrogen (-320 degrees F, -196 C) where they can be stored indefinitely. Today’s equilibrium method of cryopreservation takes approximately one hour to complete, whereas it used to take as long as three hours, so progress is definitely being made, Dr. Youngs emphasizes.
the Alpaca Research Foundation (ARF) because animals produced via embryo transfer (ET) cannot be registered in North America. However, the Morris Animal Foundation and Bioniche Animal Health provided funding for Dr. Youngs’ study which was conducted in Perú with the aid of other collaborators (Mallkini Alpaca Ranch [the breeding facility of the Michell Group, with around 3,000 animals], Dr. Willie Vivanco [Vivanco Enterprises] Dr. Gustavo Gutierrez [La Molina University in Lima, Perú], and Ethel Huaman and Silvia Leon [CIETE – Embryo Transfer Training and Research Center]). During a trip to Perú in 2007, Dr. Youngs observed firsthand the hardship subsistence farmers face every day. He was moved to begin working with alpacas to help that community and others make improvement Non-Equilibrium Method to the genetic quality of their alpacas. Because Unlike the equilibrium method of emvitrify | vi-truh-fy | transporting live animals is so expensive and bryo cryopreservation that involves slow Verb. Convert (something) transporting a cryopreserved embryo in a cooling, the non-equilibrium method into glass or a glasslike plastic straw is cheap, the potential of ET relies upon an ultra-rapid rate of cooling. substance, here by exposure to to assist those in dire financial circumThe underlying idea of this method is extreme cold. stances in Perú is obvious. that the water is cooled so quickly that Another reason Dr. Youngs felt comit doesn’t have time to form damaging hypertonic | hy-per-ton-ik| pelled to research alpaca embryo cryoice crystals. Vitrification is the term Adjective. Having increased prespreservation was that there was hardly commonly used to describe the ultra sure or tone, in particular: any research being conducted in that area fast cooling of embryos. The entire Biology having a higher osmotic and he wanted to add to the scientific non-equilibrium embryo cryopreservapressure than a particular fluid, body of information. tion process takes place in less than ten typically a body fluid or intraSuccesses and Room to Grow minutes. cellular fluid. Dr. Youngs’ study in South America The non-equilibrium method begins yielded many results – a couple of triumphs and much like the equilibrium method in terms of far more disappointments. The team performed several exposing embryos to a hypertonic solution of a cryoptodozen embryo transfers in one week. After transfers, retective agent. However, the vitrification method uses a cipient females were exposed to males to determine their much higher concentration of cryoprotective agents and receptivity to breeding. After two periods of male exdoes not allow the embryo to reach initial equilibrium posure, 9% of the equilibrium method embryo transfers before it is placed into a super-concentrated solution appeared to have resulted in pregnancies, although disapof cryoprotective agents.The embryo is loaded into the pointingly, 0 percent of the vitrified embryos appeared to straw, the straw is sealed and then plunged into liquid be successful. Dr. Youngs plans to return to his research nitrogen or liquid nitrogen vapor at -220 degrees F (-140 during next year’s breeding season but with some degrees C). Dr. Youngs characterizes the method as “not changes to the protocols. For example, he will ensure as user-friendly” during the warming process but points optimal management of recipient females, and begin to out that it also makes an embryo freezer unnecessary. cryopreserve embryos more quickly after recovery from However, a smaller diameter straw with a thinner wall donor females. Perhaps next year, Alpaca Culture will be must be used so the embryo is in the smallest amount of able to report ongoing pregnancies from the transfer of liquid possible when it is plunged into liquid nitrogen. It cryopreserved alpaca embryos. is important to note here that the embryos can’t stay in the super-concentrated solution too long or they die. SOURCES: Field Research Dr. Youngs explained that he had presented a proposal to fund his alpaca embryo cryopreservation research but his proposal was not selected for funding by
20 | Alpaca Culture • September 2013
• Youngs, Curt. “Cryopreservation of Alpaca.” Pacific Crest Accoyo America Auction. Pacific Crest Accoyo America and Little Creek Farm Alpacas. Pacific Crest Accoyo America Norte, Hillsboro. 29 June 2013. Lecture. • Personal interviews with Dr. Youngs.
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Cryogenics in Embryo Transfer