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>
	<li>Lindner, G. M., Wright, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+embryo+morphology+and+evaluation.">Bovine embryo morphology and evaluation.</a> Theriogenology. 20, 407-416 (1983).</li><li>Stringfellow, D. A., Stringfellow, D. A., Givens, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+sanitary+handling+of+in+vivo+derived+embryos.">Recommendations for the sanitary handling of in vivo derived embryos.</a> Manual of the International Embryo Transfer Society. , 65-68 (2010).</li><li>Youngs, C. R., Leibo, S. P., Godke, R. 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. Sci. 30, 273-280 (1993).</li><li>Shelton, J. N. <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+viability+of+fresh+and+frozen-thawed+sheep+demi-embryos.">Factors affecting viability of fresh and frozen-thawed sheep demi-embryos.</a> Theriogenology. 37, 713-721 (1992).</li><li>Bettencourt, E. M., Bettencourt, C. M., Silva, J. C. E., Ferreira, P., Matos, C. P., Ram&#227;o, R. J., Rocha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+rates+following+the+transfer+of+ovine+embryos+cryopreserved+using+three+protocols.">Fertility rates following the transfer of ovine embryos cryopreserved using three protocols.</a> Small Ruminant Research. 82, 112-116 (2009).</li><li>Chemineau, P., Procureur, R., Cogni&#233;, Y., Lef&#232;vre, P. C., Locatelli, A., Chupin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+freezing+and+transfer+of+embryos+from+a+bluetongue-infected+goat+herd+without+bluetongue+transmission.+Theriogenology.">Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue transmission. Theriogenology.</a> 26, 279-290 (1986).</li><li>Baril, B., Casamitjana, P., Perrin, J., Vallet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+production+freezing+and+transfer+in+Angora,+Alpine+and+Saanen+goats.">Embryo production freezing and transfer in Angora, Alpine and Saanen goats.</a> Zuchthyg. 24, 101-115 (1989).</li><li>Guignot, F., Bouttier, A., Baril, G., Salvetti, P., Pignon, P., Beckers, J. F., Touz&#233;, J. L., Cogni&#233;, J., Traldi, A. 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. Sci. 77, 61-69 (1999).</li><li>Thibier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+retrieval+committee+statistics+of+embryo+transfer-+year+2008.+The+worldwide+statistics+of+embryo+transfers+in+farm+animals.">Data retrieval committee statistics of embryo transfer- year 2008. The worldwide statistics of embryo transfers in farm animals.</a> Embryo Transfer Newsletter. 27, 13-19 (2009).</li></ol>

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 border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalog number</th>
 <th>Comments (optional)</th>
 </tr>
 <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>ViGro
thaw 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>&nbsp;</td>
 </tr>

 <tr>
 	<td>SYNGRO
Holding 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&#x2019;s phosphate buffered saline</td>
 </tr>

 <tr>
 	<td>BioLife
BSA Fraction V
</td>
 <td>Agtech, Inc.</td>
 <td>B09</td>
 <td>lyophilized bovine serum albumin, fraction V</td>
 </tr>

 <tr>
 	<td>BioLife
antibiotic-antimycotic
</td>
 <td>Agtech, Inc.</td>
 <td>B01</td>
 <td>amphotericin-B, penicillin G, streptomycin sulfate</td>
 </tr>

 <tr>
 	<td>BioLife
trypsin kit
</td>
 <td>Agtech, Inc.</td>
 <td>C22A</td>
 <td>25 g lyophilized trypsin, sterile water, sterile D-PBS </td>
 </tr> 
 </tbody>
</table>
<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the equipment</th>
 <th>Company</th>
 <th>Catalog number</th>
 <th>Comments (optional)</th>
 </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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Nanyang Technological University

Research

JoVE Journal

Biology

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

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

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 ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

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Chapters in this video

Dissection of 6.5 dpc Mouse Embryos

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

Double Whole Mount in situ Hybridization of Early Chick Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Upright Imaging of Drosophila Embryos

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Isolation and Purification of Kinesin from Drosophila Embryos