Partial funding from the USDA multi-state research project W-2171 "Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock" is gratefully acknowledged. The artistic talents of Ryan Callahan, who prepared the technical illustrations for the video portion of this article, are also gratefully acknowledged.

1. Evaluating Suitability of Embryos for Cryopreservation1
<ol>
<li>Specimens harvested from the reproductive tract of individual donor females should be placed into an isotonic embryo holding medium for 10 minutes prior to evaluation using a stereomicroscope. Be certain to keep embryos from each donor female separate from one another to enable identification of parentage of embryo transfer offspring.</li>
<li>Using low magnification (&le; 10X), specimens from an individual donor female should be segregated into separate groups consisting of unfertilized ova, degenerate embryos, or transferable quality embryos. Only quality grade 1 or 2 embryos at the compact morula through expanded blastocyst stages of development are suitable for cryopreservation, and all others should be discarded (unless synchronous recipient females are available for quality grade 3 embryos and a post-transfer pregnancy rate of approximately 25-30% is deemed acceptable).</li>
<li>Inspect quality grade 1 and 2 compact morula through expanded blastocyst stage embryos at a magnification &ge; 50X to ensure that the zona pellucida of the embryo is intact and that there is no material (e.g., cells, mucus) adhering to the zona pellucida. Any embryo with a cracked or missing zona pellucida or with a zona pellucida having adherent material should be segregated from other embryos and either be discarded or processed separately.</li>
</ol>
2. Washing Embryos to Reduce Likelihood of Disease Transmission2
<ol>
<li>Move zona pellucida-intact embryos in a minimal volume of isotonic embryo holding medium into the first well of embryo washing medium (e.g., phosphate buffered saline augmented with antibiotics and bovine serum albumin, newborn calf serum, or polyvinyl alcohol). Keep embryos from each donor female separate, and do not attempt to wash more than 10 embryos at one time. Move embryos in a minimal volume of medium into each successive wash to achieve a serial dilution &ge; 1:100, and be certain to use sterile embryo handling tips that are changed between each successive wash.</li>
<li>Move embryos through 4 more washes as described in 2.1.</li>
<li>If transmission of viral diseases is of concern, wash embryos two times in a 0.25% trypsin solution, for a total combined exposure time to trypsin of no more than 90 seconds.</li>
<li>After the two trypsin washes, wash embryos 5 times more in embryo washing medium as described in 2.1.</li>
<li>After the final embryo wash, place embryos into isotonic embryo holding medium.</li>
</ol>
3. Dehydrating Embryos Prior to Cryopreservation3
<ol>
<li>Embryos should be transferred in a minimal volume of isotonic embryo holding medium into a hypertonic solution (1.4-1.5 Molar concentration) of a cryoprotective agent (CPA) such as ethylene glycol or glycerol.</li> 
<li>Allow embryos to sit in freezing medium (hypertonic CPA solution) until they reach osmotic equilibrium. Because ethylene glycol permeates embryonic cells more rapidly than does glycerol, equilibration times are typically less for embryos in ethylene glycol (5 min) than in glycerol (10 min). Part of this equilibration time may be achieved during and after embryos are loaded into straws (described in next step).</li>
</ol>
4. Loading Embryos into a 0.25 ml Plastic Straw for Cryopreservation
<ol>
<li>Attach the cotton plug end of a 0.25 ml plastic straw (11.5 cm working length) to an embryo loading device, and aspirate approximately 1.3 cm of hypertonic CPA solution into a properly labeled straw. A variety of labeling procedures exists, including use of printed labels attached to either a straw sealing plug or to another straw connected with a straw adapter.</li>
<li>Remove the straw from the CPA solution, and aspirate approximately 0.15 cm of air to create a tiny air bubble within the straw.</li>
<li>Aspirate a single embryo in approximately 0.8 cm of hypertonic CPA solution into the straw.</li>
<li>Remove the straw from the CPA solution and aspirate approximately 0.15 cm of air to create a second tiny air bubble within the straw (air bubbles serve to physically isolate the embryo within the straw).</li>
<li>Aspirate approximately 9.1 cm of CPA solution to completely fill the straw, being certain to draw in a sufficient volume of CPA solution to moisten the polyvinylchloride (PVC) powder that exists within the cotton plug end of the straw. The CPA solution causes the PVC powder to gel and seal that end of the straw.</li>
<li>Seal the other end of the straw with PVC powder, plastic straw sealing plugs, or a heat sealer.</li>
</ol>
5. Placing Embryos into a Controlled Rate Embryo Freezing Machine
<ol>
<li>Either methanol bath or liquid nitrogen vapor freezing machines may be programmed for cryopreservation of preimplantation embryos.</li> 
<li>Load the straws containing the embryos into a freezing machine whose temperature has been cooled from ambient temperature to -6&deg;C. Load straws cotton plug end up (unless plastic straw sealing plugs or straw adapters are being used, in which case cotton plug end is placed down).</li>
<li>Allow embryos to sit at this temperature for at least 2 minutes before proceeding.</li>
</ol>
6. Seeding
<ol>
<li>Once embryos have cooled to -6&deg;C, use a pair of tongs supercooled in liquid nitrogen (or a cotton-tipped stick immersed in liquid nitrogen) to induce ice crystal formation in the CPA solution inside the straw by touching the tongs (or cotton tipped stick) to the column of solution either above or below the embryo.</li> 
<li>The water in the CPA solution will crystallize in the region exposed to liquid nitrogen, and ice crystals will spread to the column of CPA solution immediately surrounding the embryo.</li>
<li>Hold embryos at seeding temperature for 10 minutes before further cooling.</li>
</ol>
7. Continuing Dehydration of Embryos 
<ol>
<li>Cool embryos at a rate of 0.5&deg;C/min down to a temperature of -34&deg;C. This cooling rate is important to ensure continued dehydration of the embryo.</li>
<li>Hold embryos at -34&deg;C for 10 minutes before plunging embryos into liquid nitrogen (-196&deg;C).</li>
<li>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 dewar for short- or long-term storage.</li>
</ol>
8. Thawing Cryopreserved Embryos
<ol>
<li>Pull the canister into the neck of the liquid nitrogen dewar, ensuring that the canister remains below the frost line in the dewar.</li>
<li>Locate the cane containing the embryo to be thawed, and quickly and carefully remove the straw from the goblet, being certain not to warm other straws in the goblet.</li>
<li>Hold the straw in air for 3-5 seconds (to reduce the incidence of a cracked zona pellucida4), and then submerge into a 37&deg;C water bath for an additional 25-30 seconds.</li>
<li>Remove the straw from the water bath, wipe the straw (being careful not to smudge the embryo identification on the straw), use a straw cutting device to snip off the non-cotton plug end of the straw (if sealed using heat or PVC powder), or carefully remove the plastic sealing plug, and either:
<ol type="a">
<li>load the straw into an embryo transfer device and transfer as quickly as possible to a synchronous embryo recipient (but ONLY if the embryo was cryopreserved using ethylene glycol as the CPA), or</li>
<li>hold the open end of the straw over a dish containing a 1.0 Molar concentration of sucrose (a non-permeating compound), and use a pair of scissors to cut off the cotton plug end of the straw. 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 embryos to remain in sucrose solution for 10 minutes, and then transfer embryos into isotonic embryo holding medium for 10 minutes. Evaluate embryos post-thaw, load into a new straw, and transfer to suitable recipient females.</li>
</ol>
</li>
</ol>
9. Representative Results:
A variety of factors may influence the pregnancy and live birth rates resulting from the transfer of frozen-thawed preimplantation embryos to synchronous recipient females5. Embryos at developmental stages less advanced than compact morula or more advanced than expanded blastocyst often do not survive the cryopreservation process as well as embryos at the compact morula, early blastocyst, blastocyst, and expanded blastocyst stages of embryonic development. Embryos of quality grade 1 and 2 yield higher post-thaw pregnancy rates than do quality grade 3 embryos (which typically are not cryopreserved due to the low post-thaw pregnancy rates). Embryos mishandled during the embryo freezing or embryo thawing procedures are likely to exhibit reduced pregnancy rates. Embryos transferred to recipient females whose estrous cycles are not synchronized with that of the donor female typically produce lower pregnancy rates. Embryos produced in vitro or manipulated in some manner (e.g., biopsied or bisected) usually yield lower pregnancy rates. Under optimal conditions, pregnancy rates obtained after transfer of frozen-thawed in vivo derived embryos is typically 60-70% in cattle6,7, 65-75% in sheep8,9,10,11, and 60-70% in goats12,13,14,15. Similarly, pregnancy rates obtained after transfer of frozen-thawed in vitro produced embryos is typically 40-50% in cattle6,16, 25-35% in sheep17, and 30-40% in goats18.

<img src="/files/ftp_upload/2764/2764table1.jpg" alt="Table 1" /> 
Table 1. Expected pregnancy rates following transfer of frozen-thawed embryos from domestic ruminant livestock species to synchronous recipient females.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+cryopreservation+in+domestic+mammalian+livestock+species.">Embryo cryopreservation in domestic mammalian livestock species.</a> CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. , (2010).</li><li>Rall, W. F., Meyer, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zona+fracture+damage+and+its+avoidance+during+the+cryopreservation+of+mammalian+embryos.">Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos.</a> Theriogenology. 31, 683-692 (1989).</li><li>Spell, A. R., Beal, W. E., Corah, L. R., Lamb, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+recipient+and+embryo+factors+that+affect+pregnancy+rates+of+embryo+transfer+in+beef+cattle.">Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle.</a> Theriogenology. 56, 287-297 (2001).</li><li>Hasler, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+status+of+oocyte+recovery,+in+vitro+embryo+production,+and+embryo+transfer+in+domestic+animals,+with+an+emphasis+on+the+bovine.">The current status of oocyte recovery, in vitro embryo production, and embryo transfer in domestic animals, with an emphasis on the bovine.</a> J. Anim. Sci. 76, 52-74 (1998).</li><li>Hasler, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+frozen+and+fresh+embryo+transfer+pregnancy+rates+in+cattle.">Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle.</a> Theriogenology. 56, 1401-1415 (2001).</li><li>Heyman, Y., Vincent, C., Garnier, V., Cognie, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+frozen-thawed+embryos+in+sheep.">Transfer of frozen-thawed embryos in sheep.</a> Veterinary Record. 120, 83-85 (1987).</li><li>McGinnis, L. K., Duplantis, S. C., Youngs, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+sheep+embryos+using+ethylene+glycol.">Cryopreservation of sheep embryos using ethylene glycol.</a> Anim. Reprod. 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S., Cogni&#233;, Y., Mermillod, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vitrification+method+allowing+direct+transfer+of+goat+embryos.">Improved vitrification method allowing direct transfer of goat embryos.</a> Theriogenology. 66, 1004-1011 (2006).</li><li>Nowshari, M. A., Holtz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+goat+morulae+to+blastocysts+before+freezing.">In vitro culture of goat morulae to blastocysts before freezing.</a> Theriogenology. 44, 983-988 (1995).</li><li>Machatkova, M., Hulinska, P., Reckova, Z., Hanzalova, K., Spanihelova, J., Pospisil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+production+of+embryos+from+high+performance+cows+and+the+development+of+frozen-thawed+embryos+after+transfer:+a+field+study.">In vitro production of embryos from high performance cows and the development of frozen-thawed embryos after transfer: a field study.</a> Veterinarni Medicina. 53, 358-364 (2008).</li><li>Mart&#237;nez, A. G., Valc&#225;rcel, A., Furnus, C. C., de Matos, D. G., Iorio, G., de las Heras, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+in+vitro-produced+ovine+embryos.">Cryopreservation of in vitro-produced ovine embryos.</a> Small Ruminant Research. 63, 288-296 (2006).</li><li>Han, Y., Meintjes, M., Graff, K., Denniston, R., Zhang, L., Ziomek, C., Godke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caprine+offspring+born+from+fresh+and+frozen-thawed+in+vitro-produced+embryos.">Caprine offspring born from fresh and frozen-thawed in vitro-produced embryos.</a> Veterinary Record. 149, 714-716 (2001).</li><li>Leibo, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one-step+method+for+direct+nonsurgical+transfer+of+frozen-thawed+bovine+embryos.">A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos.</a> Theriogenology. 21, 767-790 (1984).</li><li>Voelkel, S. A., Hu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+ethylene+glycol+as+a+cryoprotectant+for+bovine+embryos+allowing+direct+transfer+of+frozen-thawed+embryos+to+recipient+females.">Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females.</a> Theriogenology. 37, 687-697 (1992).</li><li>Rall, W. F., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-free+cryopreservation+of+mouse+embryos+at+-196&#176;C+by+vitrification.">Ice-free cryopreservation of mouse embryos at -196&#176;C by vitrification.</a> Nature. 313, 573-575 (1985).</li><li>Notter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+genetic+diversity+in+livestock+populations+of+the+future.">The importance of genetic diversity in livestock populations of the future.</a> J. Anim. 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No conflicts of interest declared.

Because embryos consist predominantly of water, it is crucial that preimplantation embryos be adequately dehydrated and slowly cooled in accordance with the protocol described herein to avoid intracellular ice crystal formation. Once the cooling process has begun, it is important to maintain unidirectional temperature change and to avoid temperature fluctuations. Initiation of ice crystal formation ("seeding") at an appropriate temperature for the specific CPA solution is critical. 
Modifications to this slow cooling/rapid thawing method for preimplantation embryo cryopreservation include the one-step method (where the last column of medium loaded into the straw is sucrose instead of CPA solution)19 and the direct transfer method (where embryos frozen in ethylene glycol are thawed and transferred directly to the uterus of a recipient female without first removing the ethylene glycol from the embryo)20. To reduce the volume of ethylene glycol deposited into the uterus of a direct transfer (DT) recipient female, it is advisable to fill the 0.25 ml straw with 6 cm holding medium before continuing as described previously. It should be noted that a voluntary standard exists within the commercial embryo transfer industry that preimplantation embryos cryopreserved for DT should be frozen in yellow-colored straws and stored in yellow-colored goblets in the liquid nitrogen dewar. The commercial industry also has specific labeling requirements for all cryopreserved embryos that should be followed.
One alternative to this method is vitrification21, a non-equilibrium method of cryopreservation where a more highly concentrated (6-8 M) CPA solution is cooled ultra-rapidly causing the CPA solution to change from liquid to a "glassy" state without ice crystal formation. Vitrification is likely better suited for cryopreservation of embryos that are less developmentally advanced than compact morula due to their increased temperature sensitivity.
This technique has application for preservation of unique germplasm resources22, as well as to the international commercial embryo transfer industry where more than 55% of approximately 500,000 bovine preimplantation embryos transferred in calendar year 2008 were cryopreserved.23

<table><tbody><tr><td>BioLife freeze medium</td><td>Agtech, Inc.</td><td>C18, C18A</td><td>1.5 M ethylene glycol</td></tr><tr><td>Emcare ethylene glycol freeze solution</td><td>ICPBio Reproduction</td><td>IC8, IC10</td><td>1.5 M ethylene glycol</td></tr><tr><td>ViGro ethylene glycol freeze plus</td><td>Bioniche Animal Health</td><td>EVM835</td><td>1.5 M ethylene glycol</td></tr><tr><td>Emcare 10% glycerol freeze solution</td><td>ICPBio Reproduction</td><td>IC12</td><td>1.34 M glycerol</td></tr><tr><td>Emcare 1 M sucrose thaw</td><td>ICPBio Reproduction</td><td>IC16</td><td>1.0 M sucrose</td></tr><tr><td>ViGro one-step thaw</td><td>Bioniche Animal Health</td><td>EVM247, EVM847</td><td>1.0 M sucrose</td></tr><tr><td>ViGrothaw 1-2-3 plus</td><td>Bioniche Animal Health</td><td>EVM248, EVM848</td><td>5%, 2.5%, 0% glycerol with 0.5, 0.5, 0.6% sucrose</td></tr><tr><td>Emcare CSU thawing kit</td><td>ICPBio Reproduction</td><td>IC20</td><td>6%, 3%, 0% glycerol with 10.3% sucrose</td></tr><tr><td>BioLife holding and transfer medium</td><td>Agtech, Inc.</td><td>C15, C15A</td><td>modified PBS with 0.4% BSA (bovine serum albumin)</td></tr><tr><td>Emcare holding solution</td><td>ICPBio Reproduction</td><td>IC2, IC4, IC6</td><td>0.4% BSA (bovine serum albumin), MOPS buffer (inert zwitterions)</td></tr><tr><td>ViGro holding plus</td><td>Bioniche Animal Health</td><td>EVM024, EVM824</td><td></td></tr><tr><td>SYNGROHolding medium</td><td>Bioniche Animal Health</td><td>ESM024, ESM224, ESM828</td><td>contains hyaluronan; no products of animal origin</td></tr><tr><td>BioLife modified D- PBS</td><td>Agtech, Inc.</td><td>C06</td><td>modified Dulbecco&amp;#39;s phosphate buffered saline</td></tr><tr><td>BioLifeBSA Fraction V</td><td>Agtech, Inc.</td><td>B09</td><td>lyophilized bovine serum albumin, fraction V</td></tr><tr><td>BioLifeantibiotic-antimycotic</td><td>Agtech, Inc.</td><td>B01</td><td>amphotericin-B, penicillin G, streptomycin sulfate</td></tr><tr><td>BioLifetrypsin kit</td><td>Agtech, Inc.</td><td>C22A</td><td>25 g lyophilized trypsin, sterile water, sterile D-PBS </td></tr><tr><td>Biol-Cool controlled rate freezer</td><td>FTS Systems</td><td>BC-IV -40</td><td>Methanol bath freezer</td></tr><tr><td>Freeze Control controlled rate freezer</td><td>Cryologic</td><td>CL2200</td><td>Liquid nitrogen freezer</td></tr></tbody></table>

cryopreservation of preimplantation embryos of cattle, sheep, and goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

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Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

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30.6K Views.  Iowa State University. Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Video: Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Genetics

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Developmental Biology

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Rabbit Embryo Thawing: A Warming Procedure to Recover Cryopreserved Rabbit Embryos for Embryo Transfer

Source: Garcia-Dominguez, X. et al., <a href="https://www.jove.com/v/58055">Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for thawing vitrified rabbit embryos and the precautionary measurement steps for optimal embryo recovery.

Rozmrażanie zarodków królików: procedura rozgrzewająca w celu odzyskania kriokonserwowanych zarodków królików do transferu zarodków

Source: Garcia-Dominguez, X. et al., Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model. J. Vis. Exp. ...