Chapter 77 Kenneth Bondioli School of Animal Sciences, Louisiana State University, Baton Rouge, Louisiana, USA The utilization of embryo cryopreservation has become an integral part of the technique of embryo transfer, particularly the commercial application in the bovine embryo transfer industry. This is demonstrated by the industry statistics compiled by the International Embryo Transfer Society (IETS). Of the 572 342 in vivo-derived bovine embryos transferred worldwide in 2011, 324 149 or 57% were cryopreserved.1 Embryo cryopreservation in conjunction with embryo transfer allows for efficient utilization of recipients, reduces the need to move cattle, allows an efficient means for marketing of genetics, and greatly facilitates the international movement of bovine genetics. There are generally two types of procedures used in the cryopreservation of bovine embryos: (i) slow-rate or “conventional” cryopreservation and (ii) vitrification. Vitrification refers to the solidification of water as a glass-like structure without the formation of ice crystals. Slow-rate freezing employs a low concentration of cryoprotectant, induction of ice crystal formation outside of embryonic cells, and slow reduction of temperature so that these ice crystals grow and draw water from the embryonic cells without ice crystal formation inside the cells. This process is continued until the concentration of cryoprotectant within the cells becomes high enough to allow the formation of a glass-like structure. Vitrification on the other hand employs high concentrations of cryoprotectants and very rapid cooling rates so that a glass-like structure is immediately formed within the cells. This brief description of the two methods indicates that the terminology is not technically accurate since both methods ultimately result in vitrification. The terminology of “slow-rate freezing” and “vitrification” used to distinguish the different methods of arriving at this state is well accepted and commonly used. Regardless of the cryopreservation method used, the selection of embryos to be cryopreserved is always a major factor influencing the success of the procedure. The stage of embryonic development can be a factor influencing or affecting the efficiency of cryopreservation. Oocytes and early-stage (pre-compaction morula) embryos generally survive the freeze–thaw cycle of cryopreservation less efficiently than later stages. Fortunately, the compacted morula to blastocyst stage bovine embryo (normally recovered 7 days after behavioral estrus), which is the stage utilized for nonsurgical bovine embryo transfer, survives cryopreservation with a high degree of success. The “quality” or embryo grade is a factor that varies considerably and has a major influence on the efficiency of cryopreservation. Embryo quality or embryo grading is an attempt of estimate the probability that a given embryo will result in full-term development if transferred to a suitable recipient. The morphological characteristics of bovine embryos that are the basis of embryo grading were described by Linder and Wright2 and have become reasonably standardized within the commercial embryo transfer industry. These criteria for embryo grading, as well as the criteria for classification of embryonic stage, have been described in the IETS Manual of Embryo Transfer.3 This manual has been widely accepted as a standard for procedures employed for embryo transfer throughout the world. (Editor’s note: see Chapter 79 for complete coverage of embryo evaluation and grading.) It is important to realize that many of the differences in morphological characteristics that constitute the embryo grading criteria are amplified by the freeze–thaw process. Many of these differences that result in embryos being assigned lower quality grades are a direct or indirect reflection of the number of viable cells within the embryo. The freeze–thaw cycle can only be expected to reduce the number of viable cells, so these characteristics that result in embryos being assigned lower grades are amplified by the freeze–thaw cycle. Thus it is common practice to limit the application of cryopreservation to embryos with higher-quality grades (grades 1 or 2). While stage of development is a factor in embryo cryopreservation for some species (human, pig, and horse for example), embryonic stage is not a major factor for cryopreservation of bovine embryos within the context of normal embryo transfer. Differences in the ability to survive cryopreservation based on embryonic stage have been observed for bovine embryos,4 although all stages normally recovered from a day 7 (day 0 being observed or expected estrus) nonsurgical embryo collection survive cryopreservation at an equal rate. A possible exception to this exists for hatched blastocysts, which have been observed to not survive cryopreservation well. Hatched blastocysts are not normally recovered at day 7 from cattle but are sometimes encountered when collection is delayed until day 8. A cryoprotectant is defined as any substance that aids in cell survival during freezing and thawing. Cryoprotectant compounds can be divided into two general classes, penetrating and nonpenetrating. Penetrating cryoprotectants are those which cross the cell membrane and act intracellularly. These compounds have a small molecular weight and a polar molecular structure that can mimic that of water. For slow-rate or conventional cryopreservation of bovine embryos one of two penetrating cryoprotectants are commonly used, glycerol or ethylene glycol (EG). For vitrification, penetrating cryoprotectant molecules frequently used include glycerol and EG as well as dimethyl sulfoxide (DMSO) and isopropyl alcohol. Nonpenetrating cryoprotectant compounds do not cross the cell membrane and work extracellularly. These compounds affect the osmolarity of a freezing solution and provide membrane-stabilizing properties. These compounds are generally sugars and include sucrose, galactose, and trehalose. These cryoprotectant compounds are not normally included in the freezing solutions utilized in slow-rate freezing but are frequently used to control the movement of water across the cell membrane after thawing (discussed below). Nonpenetrating cryoprotectant compounds are commonly included in vitrification solutions. Freezing solutions used in slow-rate freezing commonly include only one cryoprotectant compound, while those used for vitrification commonly include multiple cryoprotectant compounds. Freezing solutions are prepared by addition of a cryoprotectant to a buffer solution. Traditionally, this buffer has been phosphate-buffered saline supplemented with either serum or bovine serum albumin (BSA). More recently, freezing solutions have become available from commercial sources. These solutions often utilize a zwitterionic buffer such as MOPS or HEPES instead of the phosphate buffer and a synthetic macromolecule such as polyvinyl alcohol instead of serum or BSA. This latter substitution is extremely important for the international movement of cryopreserved embryos. Penetrating cryoprotectants are normally added at 1.4 mol/L for glycerol or 1.5 mol/L for EG. The method of cryopreservation commonly referred to as slow-rate freezing entails a reduction in temperature at a slow or controlled rate to allow extracellular ice crystals to grow and draw water from the cells without ice crystals forming within the cells. As mentioned previously the terminology is somewhat of a misnomer since the slow rate of cooling only occurs for a short period of time followed by very rapid cooling and vitrification of the remaining water. The rate of cooling must be controlled by some manner and this is typically performed by some version of an electronically controlled freezing apparatus. The freezing solutions described above are clearly hypertonic and will cause cells to shrink on initial exposure and then slowly expand to their original size as the cryoprotectants enter the cells. Initially, it was believed that this type of osmotic stress would reduce embryo viability and embryos were exposed to the cryoprotectant solutions in a stepwise manner. Empirical studies demonstrated that stepwise addition of cryoprotectant was not necessary. This is particularly true for EG. Embryos are placed in the freezing solution and allowed to equilibrate with the cryoprotectant. Since the cryoprotectants at this concentration are not very toxic, equilibration times are not critical and equilibration will continue while the embryos are “loaded” into the appropriate “package” and during the first part of the freezing process. Embryos placed in a freezing solution will float because of the hypertonic nature of the solution and slowly sink as cryoprotectants enter the cells. Normal practice is to start loading embryos as soon as they have settled to the bottom of dish, with a minimum of 10 min of equilibration before starting the freezing process.
Cryopreservation of Bovine Embryos
Introduction
Cryopreservation methods
Selection of embryos for cryopreservation
Cryoprotectants
Slow-rate freezing
Addition of cryoprotectants
Packaging of embryos for cryopreservation
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