The overall goal of this procedure is to successfully cryopreserve pre-implantation embryos from cattle, sheep, and goats. This is accomplished by first examining specimens recovered from the donor female to identify those which are suitable for cryo-preservation.Quality. Grade one and two embryos are suitable for cryo-preservation.
However, unfertilized dova degenerate embryos and quality grade three embryos are not suitable for freezing embryos, less developed than a compact modular, or those which have begun or completed. The hatching process likewise are not suitable for freezing. The second step of the procedure is to substantially dehydrate the embryo by placing it into a hypertonic solution of a cryo protective agent, such as ethylene glycol.
Shown here is an embryo sitting in isotonic holding medium, where blue dots represent water molecules to begin the dehydration process. The embryo is placed into the hypertonic solution of ethylene glycol, a molecule represented by red dots. As the embryo sits in the ethylene glycol solution, several changes occur to the embryo.
Water rushes out of the cells of the embryo, causing the embryo to decrease in size. Concurrently ethylene glycol enters the cells of the embryo causing the embryo to increase in size until eventually the embryo reaches osmotic equilibrium. The third step of the procedure is to induce ice crystal formation in the solution surrounding the embryo.
A procedure known as seeding to enable further dehydration of the embryo as it is slowly cooled by touching the sides of the straw containing the embryo. With metal tong super cooled and liquid nitrogen ice crystals will begin to form in the solution surrounding the embryo. The ice crystals continue to spread through the solution until crystallization is complete.
The last step of the freezing procedure is to plunge the embryo into liquid nitrogen for short or long-term storage at minus 196 degrees Celsius. Ultimately, results can be obtained indicating that proper embryo dehydration prior to and during the cryo-preservation procedure can lead to the production of viable offspring after transfer of properly T thought embryos into a synchronous recipient female. Generally, individuals new to this method will struggle because of difficulties in discerning which specimens are suitable for cryo-preservation challenges.
For properly loading the embryos into the 0.25 ml straws for freezing, as well as inadvertent mistakes in handling of the embryos during the freezing and thawing procedures, Specimens harvested from the reproductive tract. Individual donor females should first be evaluated under a stereo microscope to determine their suitability for cryo-preservation. Place the embryos into an isotonic embryo holding medium for 10 minutes prior to evaluation.
Be certain to keep the embryos from each donor female separate from one another to enable identification of parentage of embryo transfer offspring using low magnification segregate specimens from an individual donor female into separate groups consisting of unfertilized dova to generate embryos or transferrable quality embryos. Embryos are graded based on a myriad of factors described previously by Lindner and Wright to determine whether or not they're of transferrable quality. Only compact Marla through expanded blasts stage embryos of quality grade one or two are suitable for cryo-preservation.
Grade three embryos while unsuitable for cryo-preservation, can be transferred to a recipient female and produce a pregnancy rate as high as 25 to 30%All degenerate embryos and unfertilized ova should be discarded embryos less developmentally advanced than a compact. More ora should be cultured or discarded. Inspect the grade one and two embryos at a magnification of greater than or equal to 50 x to ensure that the zona palita of the embryo is intact and that there is no material adhering to the zona palita.
Any embryo with a cracked or missing zoma palita or with a zoma palita having adherent material should be segregated from other embryos and be processed separately. After the selected embryos have been evaluated for the presence of an intact zop lucita, they're washed to reduce the likelihood of disease transmission. Keep embryos from each donor, female separate, and do not attempt to wash more than 10 embryos at one time.
To wash the embryos, transfer them in five microliters of isotonic embryo holding medium into 500 microliters of embryo washing medium. In the first well of a six. Well plate swirl the embryos around in the well to dilute any potential pathogens that may be present.
Change the sterile embryo handling tip before moving embryos to a new well for the next washing step. Then transfer the embryos into the second well, ensuring a one to 100 dilution with fresh embryo. Washing medium as in the first wash.
Swirl the embryos around in the well. Move the embryos through a total of five washes using a dilution of at least one to 100 with each successive wash and changing sterile embryo handling tips between each successive wash. Wash the embryos twice in 0.25%tryin solution for no more than 90 seconds total before the final set of five washes in embryo washing medium.
When washing is complete, place the embryos into isotonic embryo holding medium prior to cryo-preservation. Dehydrate the embryos by transferring them in a minimal volume of isotonic embryo, holding medium into a hypertonic solution of a cryo protective agent, such as ethylene glycol. After the embryos have undergone the initial dehydration, they need to be loaded into 0.25 ml straws for freezing.
Because this is the most technically demanding aspect of the procedure, it's important for technicians to practice loading the straws using only medium and not embryos until they gain proficiency. With the technique, the location of the embryo within the straw as well as the size and location of the air bubbles are crucial to successful embryo cryopreservation. To begin loading an embryo into a 0.25 milliliter plastic straw for cryo-preservation, attach the cotton plugin of a properly labeled straw to an embryo loading device, which in this demonstration is a shortened tomcat catheter attached to a one cc all plastic syringe asate, approximately 0.05 milliliters of hypertonic CPA solution into the straw.
Remove the straw from the CPA solution and asate a small volume of air to create a tiny air bubble within the straw. Next asate a single embryo surrounded by approximately 0.025 milliliters of hypertonic CPA solution into the straw. Remove the straw from the CPA solution and aspirate a small volume of air to create a second tiny air bubble within the straw.
The two air bubbles serve to physically isolate the embryo within the straw aspirate CPA solution to completely fill the straw being certain. To draw in a sufficient volume of CPA solution to moisten the poly vinyl chloride powder that exists within the cotton plug end of the straw. The CPA solution will cause the PVC powder to gel and seal that end of the straw.
Finally seal the other end of the straw with the heat sealer. The controlled rate freezing machine used here for cryo-preservation of pre-implantation embryos is a liquid nitrogen vapor freezing machine whose temperature has been cooled from ambient temperature to minus six degrees Celsius. To begin this procedure, load the straws containing the embryos cotton plug and up into the freezing machine.
Allow embryos to sit at this temperature for at least two minutes before proceeding to seating. Once the embryos have cooled to minus six degrees Celsius, use a pair of tongs, super cooled and liquid nitrogen to induce ice crystal formation in the CPA solution inside the straw by touching the tongs to the column of solution either above or below the embryo. Since embryos must not be kept outside the freezing machine any longer than necessary, for the purposes of this video, we'll use a straw without an embryo to demonstrate what happens in the straw during seeding.
When the supercooled tongs touch the straw region containing the CPA solution, the water in the CPA solution will crystallize in the region exposed to cool temperatures caused by the liquid nitrogen cooled tongs and ice crystals will spread to the column of CPA solution immediately surrounding the embryo After 10 minutes at minus six degrees celsius, cool the embryos at a rate of 0.5 degrees Celsius per minute, down to a temperature of minus 34 degrees Celsius. Hold the embryos at minus 34 degrees Celsius for 10 minutes. Finally, place cryopreserved embryos into an appropriately labeled goblet filled with liquid nitrogen attached to an appropriately labeled cane, and place the cane into a canister of a liquid nitrogen diar for short or long-term storage at minus 196 degrees Celsius to thaw cryopreserved embryos.
First, pull the canister into the neck of the liquid nitrogen D uar, ensuring that the canister remains below the frost line. In the doer, locate the cane containing the embryo to be thawed and quickly and carefully remove the straw from the goblet. Be careful not to warm other straws in the goblet.
Hold the straw in the air for three to five seconds to reduce the incidence of a cracked Z of lucita, and then submerge the straw into a 37 degree Celsius water bath. For an additional 25 to 30 seconds, remove the straw from the water bath and wipe the straw being careful not to smudge the embryo identification on the straw. Then use a straw cutting device to snip off the non-con plug end of the straw.
Although embryos, cryopreserved de ethylene glycol may be directly transferred to the uterus of a synchronized recipient female immediately after thawing, technicians who wish to perform a post thaw visual assessment of the embryo prior to transfer must first remove the ethylene glycol from the embryo. To accomplish this, hold the open end of the straw over a dish containing a one molar concentration of the non permeating compound sucrose, and use a pair of scissors to cut off the cotton plug end of the straw. The contents of the straw should freely flow into the sucrose solution, but pushing a small column of air through the straw may be necessary.
If any residual medium exists inside the straw, allow the embryos to remain in sucrose solution for 10 minutes and then transfer the embryos into isotonic embryo holding medium for 10 minutes. After 10 minutes, an isotonic embryo holding medium, the embryos can be evaluated under the microscope. This table shows the expected pregnancy rate following transfer of frozen thought embryos from domestic ruminant livestock species.
After watching this video, you should have a good idea of how to cryopreserve pre-implantation embryos from cattle, sheep, and goats. This procedure can be a valuable addition to your laboratory's repertoire of skills and can be highly useful in preserving unique genetic lines.
