Role of equilibration before rapid freezing of mouse embryos

The time requirements for permeation by glycerol and dehydration by sucrose before rapid freezing of Day-3 mouse embryos by direct transfer to -180 degrees C were studied. When the embryos were equilibrated in 2.0, 3.0, or 4.0 M-glycerol + 0.25 M-sucrose for 2.5 to 40 min, the post-thaw viability increased (P less than 0.001) with the length of equilibration period at 4 degrees C. At 20 degrees C the volume of embryos increased with the duration of equilibration up to 20 min (P less than 0.001), but the post-thaw viability was not affected. The effect of equilibration in glycerol-sucrose was determined at 20 degrees C for embryos which were previously permeated by glycerol, dehydrated by sucrose or left in PBS + 5% FCS. The survival of previously permeated embryos was not affected by equilibration for 1-16 min in glycerol-sucrose. The maximum survival rate was attained after shorter equilibration in glycerol-sucrose for embryos without pretreatment (4 min) than for those previously dehydrated (8 min). It is concluded that increases in the intracellular glycerol level are beneficial for the viability of rapidly frozen mouse embryos and previous or concomitant exposure to sucrose unfavourably affects glycerol permeation.     

Survival of mouse embryos after being frozen in glycerol-sucrose mixture

Random bred female albino mice (6-8 weeks old) were used as a source of embryos. 8- to 16 cell embryos were dehydrated in glycerol-sucrose mixture in 0.25 ml straws at room temperature. Straws were cooled at the rate of 5 degrees C/min to -7 degrees C. Seeding was induced by touching the out side of the straw at -7 degrees C. Straws were further cooled at 0.5 degree C/min down to -35 degrees C and then plunged into liquid N2. Thawing of straws was done by direct transfer into water at 35 degrees C. Frozen-thawed embryos were cultured in a CO2 incubator maintained at 39 degrees C. Out 190 embryos (8-16 cell) initially frozen, 169 (88.94%) were recovered on thawing. 158 (93.5%) out of 169 were apparently normal and used for culture. 75 (47.46%) developed to morulae/early blastocysts and 72 (45.56%) to expanded blastocysts on 24 and 48 hr culture respectively. In conclusion, the incorporation of sucrose in the freezing medium at a concentration of 0.25 M has led us to propose a freezing, thawing and transfer method without dilution of glycerol. The technique being quite simple is worth trying in farm animals where importance of this technique in non-surgical transfer of frozen-thawed embryos will be a boon.     

Liquid nitrogen vapour freezing of mouse embryos

Mouse morulae were frozen rapidly to -196 degrees C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7 degrees C after seeding into liquid nitrogen vapour at -170 to -180 degrees C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M-glycerol, 2 M-propylene glycol, 2 M-ethylene glycol; 5-30 min equilibration time at 0 degrees C; 3-60 min holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. Morulae frozen in this fashion, cultured and transferred to recipients developed into normal young.     

Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour

The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20 degrees C, exposure to less than or equal to 1.0 M-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36 degrees C, however, greater than or equal to 1.0 M-sucrose significantly reduced the developmental potential (P less than 0.001). In the freezing experiments the embryos were placed in 0.5 ml straws containing 40 microliters freezing medium separated by an air bubble from 440 microliters sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20 degrees C than at 36 degrees C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P less than 0.001). The best survival was obtained when the freezing medium contained 3.0 M-glycerol + 0.25 M-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20 degrees C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0.5 M, 69%; 1.0 M, 73%; 1.5 M, 64%). The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.     

Factors affecting the survival of one- and two-cell rabbit embryos cryopreserved by vitrification

The effects of equilibration time, glycerol (GLY), and 1,2-propanediol (PROH) concentration, and of vitrification and sucrose solution on the viability of 1- and 2-cell rabbit embryos were investigated. After collection, the embryos were equilibrated for 5 or 10 minutes in phosphate buffered saline (PBS) containing 10% GLY-20% PROH and were exposed for 30 seconds at 4 degrees C or were exposed and vitrified in one of two vitrification solutions 35% GLY-35% PROH or 20% GLY-50% PROH. The in vitro survival rates of 1-cell embryos equilibrated for both 5 and 10 minutes were lower (34.0 and 48.0%, respectively) than those of 2-cell embryos (78.8 and 68.5%, respectively; P<0.01). No differences were noted in the viability of embryos exposed to the 2 vitrification solutions. Following vitrification in a mixture of 35% GLY-35% PROH, the survival rates of 1- and 2-cell embryos were 18.3 and 13.7% and 19.6 and 10.4% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1- and 2-cell embryos vitrified in a solution of 20% GLY-50% PROH were 25.7 and 35.4% and 26.2 and 21.3% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1-and 2-cell embryos stored in 1M sucrose solution were 63.8 and 84.0%, respectively. In conclusion, the viability of vitrified 1- and 2-cell rabbit embryos was reduced as a consequence of their equilibration before vitrification, the exposure to vitrification solution and the dilution in a sucrose solution rather than of the vitrification process itself.     

Cryopreservation of mouse embryos at -196 degrees C by vitrification

Embryos (8-16 cell) were obtained from random bred albino mice (6-8 weeks old) that were induced to superovulate by injections of 5 I.U. PMSG and 5 I.U. hCG given 48 hr apart. Embryos were exposed to intracellular cryoprotecting medium (glycerol 10%, 1-2 propanediol 20% in PBS) for 10 min and then transferred to extracellular vitrification medium (25% glycerol, 25% 1-2 propanediol in PBS). Vitrification medium containing embryos, and diluent (1 M sucrose) were loaded in a straw and immediately plunged into liquid N2. After thawing at 20 degrees C, the contents of the straw were mixed by shaking (1 step dilution) and emptied in a petri dish. After 3 washings in culture medium the embryos were kept in CO2 incubator for further development. In 3-step dilution procedure the dilution of cryoprotectants was done in 0.5 and 0.25 M sucrose before culture. Embryos in 3-step dilution of cryoprotectants exhibited high survival as compared to 1-step dilution (20.23% vs 6.55%).     

Factors affecting survival of deep frozen bovine embryos in vitro: The effect of freezing container and method of removing cryoprotectant

The effect of freezing container and method of glycerol removal on in vitro survival of frozen-thawed Day 7 bovine embryos was investigated. Two hundred and fifteen embryos were frozen in ampules or straws, in either vertical or horizontal position and at a cooling rate of 0.3 degrees C/minute from -7 degrees C to -35 degrees C, before being plunged into liquid nitrogen. Samples were thawed in a water bath at +35 degrees C and glycerol was removed by either step-wise dilution (increments 0.25 M) or by exposure to 1.0 M sucrose for 10 minutes. A total of 197 embryos was recovered post-thaw (91%) with an overall survival after 1, 3, 6 and 24 hours in culture of 87, 81, 71, and 23%, respectively. Embryonic quality and percent survival, as assessed morphologically, did not change significantly between 1 and 3 hours but decreased significantly between 6 and 24 hours in culture (p < 0.05). Survival at 24 hours was significantly higher after removal of the cryoprotectant with sucrose when compared to the step-wise glycerol dilution (p < 0.05). Overall, embryonic survival in straws equaled that in ampules; freezing orientation of straws did not affect results. Further, glycerol removal with sucrose tended to yield survival superior to that provided by a step-wise dilution technique.     

A comparative study of mouse embryo freeze-preservation including the examination of a thermoelectric freezing device

In Study 1 over 2000 4- to 8-cell mouse embryos were randomly pooled and assigned to 1 of 12 treatment groups. A 2 X 2 X 3 factorial design was used to analyze two types of cryoprotectant/post-thaw (PT) dilutions (dimethyl sulfoxide [Me2SO]/stepwise dilution versus glycerol/sucrose dilution), two storage containers (glass ampoules versus plastic straws), and three cooling treatments. Two commercial, controlled-rate freezing machines were examined, employing either nitrogen gas (Planer) or thermoelectric (Glacier) cooling. Embryos were cooled slowly (0.5 degrees C/min) to -35 or -80 degrees C and then cooled rapidly by transfer into liquid nitrogen (LN2). Thawed embryos were cultured for 24 hr after which developmental stage, post-thaw survival (PTS), embryo degeneration rate (EDR), quality grade (QG), and fluorescein diacetate viability grade (VG) were assessed. Overall, PTS and EDR were similar (P greater than 0.05) among the three freezing unit/plunge temperature treatments. Cumulative results of container and cryoprotectant/PT dilution treatments consistently demonstrated greater PTS, QG, and VG ratings and lower EDR values when embryos were frozen in ampoules using glycerol/sucrose dilution. Embryos treated with Me2SO/stepwise dilution were particularly sensitive to freezing damage when stored in plastic straws and plunged into LN2 at -35 degrees C. Study 2 was directed at determining whether Study 1 methods for diluting Me2SO-protected embryos markedly affected PTS rates. Post-thaw culture percentages were no different (P greater than 0.05) for four- to eight-cell Me2SO-treated embryos frozen in ampoules (using the forced-LN2 device), thawed, and diluted either conventionally in reduced concentrations of Me2SO or in the sucrose treatment normally accorded glycerolated embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.     

Cryopreservation of bovine blastocysts obtained by intracytoplasmic sperm injection

The freezability and survivability of zona-intact and zona-free (hatched) bovine blastocysts obtained by intracytoplasmic sperm injection (ICSI) were assessed. Day 7 or 8 blastocysts were cryopreserved by slow freezing using 1.5 M glycerol and 0.2 M sucrose. Embryos were exposed to solutions in a 2-step procedure at room temperature and frozen in a programmed cell freezer. Blastocysts that re-expanded within 6 h of post-thaw culture were considered viable. The cleavage, morula and blastocyst development rates after ICSI were 52.4 (131/250), 39.7 (52/131), and 24.4% (32/131), respectively. Blastocyst stage embryos were randomly divided into 2 groups. The first group of embryos was frozen with their zonae intact, while the second group was allowed to hatch from their zonae during the additional 18 h culture, after which they were frozen. The data showed that more Group 2 blastocysts (14/16, 87.5%) than Group 1 (12/16; 75.0%; P<0.05) survived, and more zona-free bovine blastocysts frozen with glycerol as the cryoprotective agent (CPA) than zona-intact blastocysts after slow freezing retained their viability.     

Quick freezing of rat embryos

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).     

Sucrose dilution of frozen mouse embryos: interaction of glycerol and sucrose concentrations

Several concentrations of glycerol for cryoprotection and several concentrations of sucrose for cryoprotectant dilution were examined with frozen, thawed and cultured mouse embryos. Four hundred and eighty late morulae to early blastocyst stage embryos were collected from 35 superovulated mice (B6D2 x Swiss Webster crosses back-crossed to Swiss Webster males) 3-1/2 days after breeding. The embryos were transferred through increasing concentrations of glycerol in modified Dulbecco(1)s phosphate buffered saline (MDPBS) to reach three final concentrations of 1.0 M, 1.4 M and 1.8 M. The embryos were loaded in 0.5-ml French straws appropriately filled with the cryoprotectant and sucrose solutions for each treatment. The straws were cooled with a standard fast-freezing program to -35 degrees C, then plunged into liquid nitrogen. After 58 days of storage at -196 degrees C the straws were thawed in a 37 degrees C water bath. Cryoprotectant dilution was accomplished with a standard step-wise procedure or in the straw with one of three concentrations of sucrose solution (0.25 M, 0.5 M, 1.0 M) in MDPBS. The embryos were then washed twice in MDPBS, twice in Whitten's media for embryo culture and then placed in microdrops of Whitten's media under paraffin oil in a water saturated 5% CO(2) in air atmosphere at 37 degrees C. Embryos were observed 24 hours later for development to the expanded blastocyst stage. The proportion of embryos developing in vitro from the three glycerol concentrations were not significantly different with standard step-wise dilution procedures for glycerol removal. After step-wise cryoprotectant removal, blastocyst expansion occurred in 49%, 44% and 52% of embryos frozen in 1.0 M, 1.4 M and 1.8 M glycerol, respectively. The 1.0 M sucrose dilution of 1.0 M glycerol showed the highest development (60.5%) in vitro but was not significantly different from any of these three step-wise diluted glycerol concentrations. The step-wise dilution of the three glycerol concentrations and dilution of the 1.0 M glycerol and 1.0 M sucrose were all superior (P < 0.01) to any other dilution procedure examined.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

Status of cryopreservation of embryos from domestic animals.

The discovery of glycerol as an effective cryoprotectant for spermatozoa led to research on cryopreservation of embryos. The first successful offspring from frozen-thawed embryos were reported in the mouse and later in other laboratory animals. Subsequently, these techniques were applied to domestic animals. Research in cryopreservation techniques have included studies concerning the type and concentration of cryoprotectant, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal. To date, successful results based on pregnancy rates have been obtained with cryopreserved cow, sheep, goat, and horse embryos but no success has been reported in swine. Post-thaw embryo survival has been shown to be dependent on the initial embryo quality, developmental stage, and species. The freezing techniques most frequently used in research and by commercial companies are identified as "equilibrium" cryopreservation. In this technique the embryos are placed in a concentrated glycerol solution (1.4 M in PBS supplemented with BSA) at room temperature and the glycerol is allowed to equilibrate for a 20-min period. During the cooling process the straws are seeded (-4 to -7 degrees C) and cooling is continued at a rate of 0.3 to 0.5 degree C/min to -30 degrees C when bovine embryos may be plunged into LN2. Sheep embryos are successfully frozen with ethylene glycol (1.5 M) or DMSO (1.5 M) rather than with glycerol. Horse embryos have been frozen in 0.5 rather than 0.25 cc straws but with cooling rates and seeding and plunging temperatures similar to those used with bovine embryos. Swine embryos have shown a high sensitivity to temperature and cryoprotectants probably due to their high lipid content and a temperature decrease to 15 or 10 degrees C causes a dramatic increase in the percentage of degenerated embryos. However, a recent study has shown that hatched pig blastocysts survived exposure below 15 degrees C. Recent research has shown that embryos may also be frozen by a "nonequilibrium" method. This rapid freezing by vitrification consists of dehydration of the embryo at room temperature by a very highly concentrated vitrification media (3.5 to 4.0 M) and a very rapid freeze that avoids the formation of ice allowing the solution to change from a liquid to a glassy state. Vitrification solutions consist of combinations of sucrose, glycerol, and propylene glycol. With this technique, 50% pregnancy rates have been reported with the bovine blastocyst.     

Vitrification of pronuclear-stage mouse embryos on solid surface (SSV) versus in cryotube: comparison of the effect of equilibration time and different sugars in the vitrification solution

The cryopreservation of pronuclear-stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear-stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene-glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this "equilibration" solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15-25 embryos were transferred into a 1.8 ml cryotube containing 30 microl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN(2)) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40 degrees C water bath and then immediately diluted with 300 microl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15-20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2-microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. Vitrified droplets were stored in cryovials in LN(2). Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37 degrees C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high-survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear-stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.     

Use of sucrose during removal of cryoprotectants after thawing eight-cell mouse embryos

Eight-cell mouse embryos were frozen in 0.5-ml plastic straws in modified Dulbecco's phosphate buffered saline (PBS) plus 5% steer serum plus either 1.32 M dimethyl sulfoxide (DMSO) or 1.32 M glycerol. Upon thawing, embryos were diluted 1:4 with 0.0, 0.2, 0.6, or 1.0 M sucrose solutions within the straws. Thawing was either in air at ambient temperature or in 8 degrees C or 38 degrees C water. After 48 h of culture, more embryos frozen in DMSO and thawed in 8 degrees C and 37 degrees C water developed to blastocysts (87 and 93%, respectively) than embryos thawed in air (75%; P < 0.05). No significant differences in development were noted among the three thawing regimens when embryos were frozen with glycerol. There was no significant effect of concentration of sucrose during dilution on development of embryos postthaw. With glycerol as the cryoprotectant, damage to zonae pellucidae increased as thawing rates increased, whereas the opposite was observed with DMSO as the cryoprotectant (P < 0.05).     

Effect of reduced concentrations of glycerol and various macromolecules on the cryopreservation of mouse and cattle embryos

The effect of different macromolecules [bovine serum albumin (BSA), Pluronic F-68, (ET surfactant), or sodium hyaluronate (SH)] on postthaw survival of mouse morulae and in vivo- and in vitro-derived bovine blastocysts frozen in 10, 5, or 1% glycerol solutions was investigated. Embryos were equilibrated with cryoprotectant solution at 25 degrees C for 10 min, seeded at -5 degrees C, cooled at 0.5 degrees C/min to -35 degrees C, and plunged into liquid nitrogen. Embryos were thawed in a 35 degrees C water bath, glycerol was removed with 0.6 M sucrose at 25 degrees C for 5 min, and postthaw viability was evaluated after 1, 24, and 48 h in culture. The addition of BSA supplementation improved postthaw survival of mouse morulae frozen in 5% glycerol, but not in 10% glycerol. All three macromolecular supplements were effective in increasing survival of mouse morulae in 5% glycerol but only BSA and SH were effective in increasing postthaw survival of in vivo- and in vitro-derived bovine blastocysts. None of the macromolecular supplements improved postthaw survival of embryos frozen in 1% glycerol.     

Cryopreservation of oyster (Crassostrea gigas) embryos

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.     

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.