In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol

The aim of the present study was to compare the survival rates of goat morulae and blastocysts after different freezing procedures. The viability of frozen-thawed embryos was assessed both in vivo and in vitro. Two cryoprotectants, ethylene glycol and glycerol, were used and three cryoprotectant removal procedures were compared: progressive dilution in 1.0, 0.5, 0.3 and 0 M of cryoprotectant in PBS; a similar progressive dilution with cryoprotectant in PBS plus 0.25 M of sucrose; or one-step transfer in PBS containing 0.25 M of sucrose. In vitro development of frozen-thawed blastocysts was always higher than that of frozen morulae irrespective of the cryoprotectant (52 129 = 40.3% vs 23 161 = 14.3% ; P< 0.001). In vivo, however, frozen-thawed morulae developed equally as well as blastocysts after an identical freezing-thawing protocol. Development both in vivo and in vitro showed ethylene glycol to be a better cryoprotectant than glycerol for goat embryos at both developmental stages (23 vs 0%, 45 vs 35% in vitro; 34.5 vs 21%, 35 vs 23% in vivo for morulae and blastocysts, respectively).     

High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant

A method for obtaining a high survival rate of frozen-thawed mouse embryos is presented. Eight-cell mouse embryos were frozen inside small plastic straws in the presence of 1-2 propanediol and stored at -196 C. After thawing, the embryos were diluted for only 5 min in a 1.0 M sucrose solution to remove the 1-2 propanediol from the cells. At high rate of thawing (is equivalent to 2500 C/min) more than 88% of the embryos survived in vitro to the blastocyst stage provided that the dilution of propanediol was performed rapidly during thawing. At a lower rate of thawing (is equivalent to 300 C/min), survival tended to be higher (94.7%) when dilution was done 5 min after thawing. When the frozen-thawed embryos were transferred to the oviducts of day 1 pseudopregnant recipients either directly after the dilution of 1-2 propanediol or after 24 or 48 hr of culture, a high proportion of them (65.9%) develop normally to viable fetuses.     

应用乙二醇冷冻小鼠胚胎：优化和简化程序的探索

提高解冻胚胎的发育能力和简化冷冻解冻程序是胚胎冷冻研究的两大永恒的主题。尽管乙二醇（EG）广泛用于家畜胚胎冷冻,但很少用于冷冻小鼠和人胚胎。为数很少的以EG慢冻小鼠或人胚胎的研究均采用较为复杂的人胚冷冻程序,未见简化程序和用EG冷冻小鼠桑椹胚的报道。采用简单的牛胚胎冷冻程序研究了发育时期、EG浓度、平衡方法、添加蔗糖以及解冻后脱除EG等对小鼠胚胎冻后发育能力的影响。结果显示：（1）致密晚期桑椹胚冻后体外培养囊胚发育率（81.92%±2.24%）和孵出率（68.56％±2.43%）显著（P＜0.05)高于4-细胞、8-细胞胚胎和致密早期桑椹胚胎;（2）1.8mol/L EG冷冻小鼠致密晚期桑椹胚的囊胚发育和孵出率显著高于其它浓度;（3）在EG中平衡10min的冻后囊胚发育显著好于平衡5、20或30min;（4）两步平衡冷冻胚胎的囊胚发育率和孵出率显著高于一步平衡;（5）用EG冷冻小鼠胚胎无需添加蔗糖;（6）解冻后可不脱除EG;（7）冻后发育的早期囊胚和囊胚细胞数明显少于体内发育胚胎。因此,用EG冷冻小鼠胚胎的最佳方案为：致密晚期桑椹胚用1.8mol/L EG不添加蔗糖、两步平衡15min、以简单的牛胚胎冷冻程序冷冻解冻、解冻后不脱除EG直接培养或移植。     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

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.     

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).     

The protective action of polyvinyl alcohol during rapid-freezing of mouse embryos

Biological products like serum and BSA are routinely used in embryo freezing solutions. These products are undefined and can potentially expose the embryos to infectious agents. Therefore, this experiment was designed to evaluate in vitro and in vivo survival of mouse embryos frozen in solutions supplemented with a chemically defined macromolecule, polyvinyl alcohol (PVA). Morula-stage embryos from superovulated mice were collected, frozen by a rapid freezing procedure, and cryoprotectant diluted out (after thawing) in media supplemented with either 10% fetal calf serum (FCS), 0.1 mg/mL PVA, or a combination of 10% FCS and 0.1 mg/mL PVA. Frozen-thawed (good to excellent quality) and nonfrozen (control, collected in FCS supplemented medium) embryos were cultured in medium M16 (32) supplemented with either 4 mg/mL BSA or 0.1 mg/mL PVA for 72 h. Embryos frozen in solutions supplemented with FCS or PVA and nonfrozen embryos were transferred to pseudopregnant recipients. Recipients were humanly killed on Day 15 after transfer, and the rate of implantation and percentage of live fetuses were recorded. The supplementation of collection, freezing and cryoprotectant dilution solutions with FCS, PVA or FCS plus PVA did not influence (P > 0.05) the rate of survival and in vitro development of embryos to hatched/hatching blastocyst-stage. However, a higher (P < 0.01) in vitro development rate to hatching/hatched-stage was recorded when frozen-thawed embryos were cultured in medium supplemented with BSA than with PVA. There was no difference (P > 0.05) in the rate of implantation (68 vs 72%) or percentage of live fetuses (62 vs 60%) between pregnant recipients with embryos frozen in medium with FCS or PVA. The rate of implantation and development of embryos frozen in medium supplemented with PVA or FCS was comparable (P > 0.05) to that of nonfrozen embryos. It may be concluded that PVA can be substituted for FCS in medium for freezing mouse embryos; however, it can not be completely substituted for BSA in the in vitro culture of embryos to the hatched blastocyst stage.     

Deep freezing of mouse one-cell embryos and oocytes using different cryoprotectants

The objective of this study was to compare iso-osmolar concentrations (1.5 M) of 1,2-propanediol, glycerol, dimethylsulphoxide and a combination of 1 M propanediol + 0.5M glycerol (PDGLY) as cryoprotectants for murine ovulated oocytes and one-cell embryos. A higher (P < 0.01) percentage of one-cell embryos developed to the two-cell stage when frozen-thawed with 1,2-propanediol (83%) as compared with glycerol (43%), dimethylsulfoxide (51%) or PDGLY (7%). Data recalculated on the basis of two-cell embryos/number of normal one-cell embryos after thawing indicated no differences among single cryoprotectant groups. More (P < 0.01) frozen-thawed, in-vitro fertilized oocytes developed to the two-cell stage when 1,2-propanediol (35%) was used as cryoprotectant as compared with glycerol (15%). Freezing-thawing resulted in a reduced number of two-cell embryos after oocytes were fertilized in-vitro as compared with fresh oocytes. 1,2-propanediol was a better cryoprotectant than glycerol, dimethylsulphoxide or PDGLY for deep freezing of murine oocytes or one-cell embryos.     

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.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

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.     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

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.     

Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation

The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts.     

Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose

The present study was designed to examine the suitability of ethylene glycol as a cryoprotectant for equine embryos. Blastocysts recovered nonsurgically from Day 6 donor mares were cryopreserved by conventional 2-step freezing in the presence of 10% ethylene glycol (EG), 10% glycerol (Gly), or 10% ethylene glycol + 0.1M sucrose (EG + Suc). After thawing, the EG and Gly were removed by a 6-step manner, and the EG + Suc was diluted to one fourth in the freezing straw. The postthaw blastocysts were transferred nonsurgically into the uteri of recipient mares on Days 4 to 7 after ovulation. Pregnancy rates, based on Day 15 ultrasonography, were 25.0% (2/8) and 37.5% (3/8) for the blastocysts frozen in EG and Gly, respectively. Direct transfer following thawing and in-straw dilution of blastocysts frozen in EG + Suc resulted in a pregnancy rate of 63.6% (7/11). In fresh Day 6 blastocysts (control group), the pregnancy rate was 70.0% (7/10). These results indicate that the combined use of ethylene glycol and sucrose in a 2-step freezing regimen allows for the direct transfer of frozen-thawed blastocysts into recipient mares, with an acceptable pregnancy rate.     

Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol

Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n = 5,525) were utilized to study survival following exposure to cryoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survival. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 8. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo B2 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol.     

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)     

Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours (38.5 degrees C, 5% CO(2)) in modified TCM-199 medium supplemented with 5% superovulated cow serum (SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M 1, 3-butylene glycol (BG) solutions. They were then loaded into 0.25-ml straws, placed into an alcohol bath freezer at 0 degrees C, cooled from 0 degrees C to -6 degrees C at -1 degrees C/minute, seeded, held for 10 minutes, and cooled again at -0.3 degrees C or -0.5 degrees C/minute to -30 degrees C. Straws were then plunged and stored in liquid nitrogen. After thawing in 30 degrees C water, the embryos were rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred nonsurgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at -0.3 degrees C vs -0.5 degrees C/minute was not significantly different (P>0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of -0.3 degrees C/minute (64.6%, 31 48 ) than at -0.5 degrees C/minute (22.6%, 12 53 ). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows: MC (48%, 10 21 ); DEG (30%, 3 10 ); EG (74%, 20 27 ); and PG (40%, 4 10 ). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF bovine embryos.     

A method for one-step freezing of mouse embryos

A simple one-step method of freezing mouse embryos directly in liquid nitrogen is described. The main objective of this study was to assess post-thaw survival following predehydration in various mixtures of glycerol and sucrose. Also investigated was pretreatment with glycerol prior to dehydration and effects of embryo stage. When sucrose was held constant (0.25 M) and glycerol concentration varied (1.0-4.0 M), post-thaw survival was best (67%) in 2.0 M glycerol. Pretreatment in glycerol provided no advantage over no pretreatment. When glycerol was held constant (2.0 M) and sucrose concentration varied (0-1.0 M), optimum post-thaw survival (81%) was found in 0.5 M sucrose. Morulae survived better than blastocysts (86% vs 72%, respectively). Transfer of thawed embryos frozen by the optimum treatment (2.0 M glycerol + 0.5 M sucrose) resulted in a birthrate of 41%, compared to 54% for fresh controls. This technique could find application in freezing and thawing of livestock embryos on the farm.     

Factors affecting survival of mouse blastocysts vitrified by a mixture of ethylene glycol and dimethyl sulfoxide

The present study was conducted to determine suitable conditions for mouse blastocysts vitrified by a solution containing 25 % v/v (4.5M) ethylene glycol and 25% v/v (3.4M) dimethyl sulfoxide (VSi). In Experiment 1, blastocysts were exposed to 50% diluted VSi (50% VSi) for 10 minutes then to VSi for 0.5 minutes at room temperature (22 approximately 24 degrees C) or at 4 degrees C, followed by vitrification. The survival rates of these embryos exposed at each temperature were not significantly different. In Experiment 2, embryos were exposed directly to VSi for various time periods at room temperature and diluted in mPBS with 0.5 M sucrose without vitrification. The viability of embryos decreased after more than a 3 minute exposure. When the embryos were exposed to VSi for 0.5, 1, 1.5 and 2 minutes followed by vitrification, the survival rates were 78, 80, 76 and 50%, respectively. In Experiment 3, embryos were vitrified after exposure to 50% VSi for various time periods and then to VSi for 0.5 minutes at room temperature. One minute exposure to 50% VSi resulted in the highest survival rate. In Experiment 4 and 5, the cooling rate (from approximately 70 to approximately 2500 degrees C/minute), sucrose concentration (0, 0.5 and 1 M) of dilution solution, and dilution time (1 or 5 minutes) did not affect the viability of the vitrified embryos. Following exposure to 50% VSi for 1 minute and to VSi for 0.5 minutes at room temperature, embryos were cooled 1) at approximately 2500 degrees C/minute and diluted in 0.5 M sucrose in mPBS after warming or 2) at approximately 200 degrees C/minute and diluted in mPBS. In vivo development rates after the transfer to recipients were 38 and 42%, respectively. These values were similar to that of fresh control embryos.