Cryopreservation of mouse embryos by vitrification: A meta-analysis

A meta-analysis of cryopreservation studies vitrifying mouse embryos was undertaken to determine the treatment effect of vitrification. Treatment by vitrification decreased embryo viability compared with controls: the odds ratio was 9.02 (CI: 3.73-21.78; P < 0.001), a 24.90% (CI: 14.88-34.91; P < 0.001) reduction in risk was associated with embryos in the control group, and for every 4.00 (CI: 3.91-4.09) embryos treated by vitrification, one does not survive. A multiple regression analysis evaluated covariates of embryo survival. For each hour increase post-hCG treatment when embryos were cryopreserved, there was a decrease of 0.36% (SEM ± 0.01) in survival (P < 0.001). The number of embryos surviving vitrification decreased 0.25% (SEM ± 0.02) per day increase in age of the female mouse (P < 0.001), whereas there was no significant difference for control group embryos. For each 1 h increase post-hCG treatment after cryopreservation when blastocysts were assessed for viability, there was a decrease of 0.13% (SEM ± 0.01) in survival. The later interval post-hCG treatment when blastocysts were assessed, the less viable they were compared with earlier blastocysts, independent of the vitrification protocol. This effect was not observed for control embryos. A high percentage of variability in the treatment effect for vitrification was likely due to underlying heterogeneity among studies. A portion of the risk associated with vitrification could be attributed to the general effects of cryopreservation. Future research should identify effects in a cryopreservation protocol specific to vitrification that affect viability of mouse embryos.     

Cryopreservation of immature and in vitro matured porcine oocytes by solid surface vitrification

Cryopreservation of normal, lipid-containing porcine oocytes has had limited practical success. This study used solid surface vitrification (SSV) of immature germinal vesicle (GV) and mature meiosis II (MII) porcine oocytes and evaluated the effects of pretreatment with cytochalasin B, cryoprotectant type (dimethylsulfoxide (DMSO), ethylene glycol (EG), or both), and warming method (two-step versus single-step). Oocyte survival (post-thaw) was assessed by morphological appearance, staining (3',6'-diacetyl fluorescein), nuclear maturation, and developmental capacity (after in vitro fertilization). Both GV and MII oocytes were successfully vitrified; following cryopreservation in EG, more than 60% of GV and MII stage porcine oocytes remained intact (no significant improvement with cytochalasin B pretreatment). Oocytes (GV stage) vitrified in DMSO had lower (P<0.05) nuclear maturation rates (31%) than those vitrified in EG (51%) or EG+DMSO (53%). Survival was better with two-step versus single-step dilution. Despite high survival rates, rates of cleavage (20-26%) and blastocyst formation (3-9%) were significantly lower than for non-vitrified controls (60 and 20%). In conclusion, SSV was a very simple, rapid, procedure that allowed normal, lipid-containing, GV or MII porcine oocytes to be fertilized and develop to the blastocyst stage in vitro.     

Cryopreservation of ovine embryos: slow freezing and vitrification

Different methods for the cryopreservation of ovine embryos were evaluated in vitro (survival upon culture in vitro) and in vivo (pregnancy and lambing rates after transfer in field conditions). In the first 2 experiments, slow freezing conditions were evaluated. When glycerol and ethylene glycol were compared, no differences in the overall pregnancy rate were found (40.2 vs 51.3%), but better results were obtained with ethylene glycol than with glycerol in morulae (29.7 vs 59.4%, P < 0.05). In the second experiment, 2 methods of removing ethylene glycol were compared: a 1-step procedure using 0.5-M sucrose and a 3-step process for decreasing ethylene glycol concentration. There were no differences in the overall pregnancy rate (48.0 vs 48.0%) between the 2 methods. The last series of experiments were designed to compare 2 vitrification solutions: propylene glycol--glycerol (PG) and ethylene glycol--Ficoll 70--sucrose (EFS). There were no differences between the 2 vitrification solutions, based on the overall pregnancy rate (28.1 vs 40.0%). The vitrification technique and specially with EFS solution has resulted in good pregnancy rates. The EFS solution was particularly efficacious with morulae (55.5% pregnancy). These results demonstrate that vitrification with EFS can be used successfully for the cryopreservation of ovine embryos.     

Cryopreservation of flounder (Paralichthys olivaceus) embryos by vitrification

Conventional cryopreservation of complex teleost embryos has been unsuccessful, possibly because their large size (1-7 mm diameter), multi-compartmental structure and low water permeability lead to intracellular ice formation and chilling injury. To overcome these obstacles, we have developed a vitrification procedure for cryopreservation of flounder (Paralichthys olivaceus) embryos. In initial toxicity tests, propylene glycol (PG) and methanol (MeOH) were less toxic to embryos than dimethylformamide (DMF) or dimethyl sulfoxide (Me2SO), whereas ethylene glycol (EG) and glycerol (Gly) were toxic to all tested embryos. Embryos between four-somite and tail bud stages were more tolerant to vitrifying solutions than embryos in other developmental stages. Four vitrifying solutions (FVS1-FVS4) were prepared by combining a basic saline solution (BS2) and cryoprotectants PG and MeOH in different proportions (FVS1: 67, 20 and 13%; FVS2: 60, 24 and 16%; FVS3: 55, 27 and 18%; FVS4: 50, 30 and 20% of BS2, PG and MeOH, respectively). Their impact on flounder embryos was then compared. FVS1 produced the highest survival rate; whereas deformation rate was highest for FVS4. Five-step equilibration of embryos in FVS2 resulted in higher survival rates than equilibration in 4, 3, 2 or 1 steps. Flounder embryos varying from the 14-somite to the pre-hatching stage were cryopreserved in the four vitrifying solutions in liquid nitrogen for 1-7 h. From eight experiments, 20 viable thawed embryos were recovered from 292 cryopreserved embryos. Fourteen larvae with normal morphology hatched successfully from the 20 surviving frozen-thawed embryos from five experiments. Embryos at the tail bud stage exhibited greater tolerance to vitrification than embryos at other stages. These results establish that cryopreservation of flounder embryos by vitrification is possible. The technology has many potential applications in teleost germplasm resource conservation.     

不同群系小鼠胚胎玻璃化冷冻保存技术的研究

利用 EFS40 ,二步法对近交系 C5 7BL/6、DBA/2和远交群 ICR小鼠囊胚玻璃化冷冻保存 ,并对冷冻后胚胎体内、外发育效果进行比较。结果表明 ,相同条件下鲜胚经培养 ,近交系 C5 7BL /6小鼠的囊胚发育率 ( 93% )与 ICR( 1 0 0 % )相比差异不显著 ( P>0 .0 5 ) ;而两近交系的囊胚孵化率明显低于 ICR( P<0 .0 1 )。 C5 7BL/6、DBA/2小鼠囊胚冷冻后发育率 ( 93% ,96% )和孵化率 ( 5 2 % ,46% )与各自对照组 ( 1 0 0 % ,1 0 0 %和 61 % ,62 % )相比均无显著差异 ( P>0 .0 5 ) ;并且与 ICR冷冻组发育率和孵化率 ( 94% ,5 3% )之间也无显著差异 ( P>0 .0 5 )。两近交系冻胚移植妊娠与各自对照组和 ICR冷冻组比较均无显著差异 ( P>0 .0 5 )。C5 7BL/6胚胎移植产仔率 ( 35 % )与对照组 ( 5 1 % )之间差异显著 ( P<0 .0 1 ) ,而 DBA/2胚胎移植产仔率 ( 4 7% )与对照组和 ICR冷冻组 ( 39% ,5 8% )相比差异不显著 ( P>0 .0 5 )。     

Cryopreservation of mouse half-morulae and chimeric embryos by vitrification.

Mouse half-morulae were cryopreserved less than or equal to 1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2-propanediol as cryoprotectant. The developmental rates of the frozen-thawed half-embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty-one of the half-embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half-morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen-thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half-morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half-embryos of the components.     

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%).     

Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification

In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification.     

鲈鱼胚胎的玻璃化冷冻保存

本文对鲈鱼(Lateolabrax japonicus)胚胎进行了玻璃化冷冻保存研究,筛选出了浓度较低、玻璃化程度较稳定的5种玻璃化液,冷冻时形成玻璃化的概率在48．1％～100％,在35～43℃的水浴中解冻时保持玻璃化的概率在44．4％～63．0％;玻璃化液VSD2在解冻时保持玻璃化的概率最高。对鲈鱼神经胚、20对肌节胚、尾芽胚、心跳胚、出膜前胚在玻璃化液VSD2中的适应能力及适合于玻璃化冷冻的胚胎时期进行了比较,结果显示：不同时期胚胎对玻璃化液的耐受能力不同,鲈鱼神经胚耐受能力最低,心跳胚耐受能力最强,出膜前期胚次之,心跳胚和出膜前胚适合于进行玻璃化冷冻。对0．5mol/L蔗糖的洗脱时间进行了选择,结果显示,洗脱10～20min效果较好。利用玻璃化程度较好的VSD2对鲈鱼不同时期胚胎进行超低温(-196℃)冷冻,获得了2．1％～27．9％的透明胚。将鲈鱼心跳胚冷冻解冻后获2粒复活胚,培养至出膜期,成活42～50h;出膜前期胚在冷冻解冻后有1粒胚复活,并且孵化出鱼苗[动物学报49(6)：843～850,2003]。     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

Cryopreservation of rat blastocysts by vitrification

Rat blastocysts equilibrated with vitrification solution (VS1), consisting of dimethyl sulfoxide, acetamide, propylene glycol, and polyethylene glycol were plunged directly into liquid nitrogen. The embryo suspension are solidified by an extreme elevation in viscosity of solution. The embryos are cryopreserved by vitrification without intra- and extracellular ice formation. The proportion of morphologically normal embryos after cooling and warming was 79% (117/149) and all (48/48) of the embryos cultured were developed to expanded or hatched blastocysts. Normal live young were obtained 41% of the time (28/69) after transfer of the cooled and warmed embryos to pseudopregnant recipients.     

Cryopreservation of rye protoplasts by vitrification

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20°C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0°C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN₂); and (d) recovery of the protoplasts from LN₂ and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN₂ if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3±2%. In contrast, survival of ACC protoplasts following recovery from LN₂ was 34 ± 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 ± 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.     

Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer

Apoptosis occurs during preimplantation development in both in vivo- and in vitro-produced embryos, and it may contribute to embryonic loss. The present study investigated the development of porcine nuclear transfer (NT) embryos reconstructed by using fetal fibroblasts as compared to embryos produced by in vitro fertilization (IVF). The onset and the frequency of apoptosis in NT and IVF embryos were examined via morphological and nuclear changes and TUNEL assay. The NT blastocysts had a similar number of nuclei as compared to IVF blastocysts and appeared to be morphologically similar. Relative to IVF embryos, the NT embryos had a lower cleavage rate (42.7% vs. 71.0%) and a lower developmental rate (11.1% vs. 28.6%) to the blastocyst stage. The earliest positive TUNEL signals were detected in the NT embryos on Day 5 of culture. The percentage of cells undergoing apoptosis in the NT embryos was higher than that of the IVF embryos and increased with time in vitro. Some of the abnormal morphological changes observed during early development related to apoptosis. Cytoplasmic fragmentation, developmental arrest, and nuclear condensation were typical characteristics of embryos undergoing apoptosis. Some mechanisms of the apoptotic pathway were triggered by changes in the NT embryos. The developmental rates of NT embryos might be improved by identifying specific apoptotic pathways and then intervening in these pathways to improve development.     

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 m sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 m glycerol and 0.4 m sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 m sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids. Received: 19 September 1996 / Revision received: 3 January 1997 / Accepted: 24 February 1997     

Ultrastructure of porcine embryos following development in vitro versus in vivo

The present study examined the ultrastructural appearance of porcine embryos from the four-cell stage to the blastocyst grown either in vivo or in vitro. Embryos were collected at slaughter from superovulated gilts and were fixed for transmission electron microscopy either immediately or after various periods of in vitro culture. In general, the morphology of in vivo and in vitro grown embryos was similar. In vivo grown four-cell stages contained dense fibrillar nucleoli. At the eight-cell stage the nucleoli possessed increasing amounts of chromatin and granules. In both stages the mitochondria were spherical or ovoid in shape and had only few cristae. In morulae and blastocysts the nucleoli were mainly of the fibrillogranular type, and the mitochondria were filamentous and possessed more cristae, of which many were tubular. Two major ultrastructural deviations were observed in about half of the in vitro cultured embryos. First, nucleolus-like structures were found outside the nuclei in the cytoplasm of blastomeres. These structures were spherical and composed of chromatin-like material containing characteristically a single large and several small vacuoles. The structures were frequently associated with profiles of smooth endoplasmic reticulum (SER). A second type of deviation was aggregates of SER appearing as spiral coils or multiangular complexes. Some embryos displayed both types of deviations. The physiological significance of these deviations remains speculative. They may be involved in the considerably reduced capability of porcine embryos to develop to piglets following in vitro culture.     

Cryopreservation of rat hippocampal slices by vitrification

Although much interest has attended the cryopreservation of immature neurons for subsequent therapeutic intracerebral transplantation, there are no reports on the cryopreservation of organized adult cerebral tissue slices of potential interest for pharmaceutical drug development. We report here the first experiments on cryopreservation of mature rat transverse hippocampal slices. Freezing at 1.2 degrees C/min to -20 degrees C or below using 10 or 30% v/v glycerol or 20% v/v dimethyl sulfoxide yielded extremely poor results. Hippocampal slices were also rapidly inactivated by simple exposure to a temperature of 0 degree C in artificial cerebrospinal fluid (aCSF). This effect was mitigated somewhat by 0.8 mM vitamin C, the use of a more "intracellular" version of aCSF having reduced sodium and calcium levels and higher potassium levels, and the presence of a 25% w/v mixture of dimethyl sulfoxide, formamide, and ethylene glycol ("V(EG) solutes"; Cryobiology 48, pp. 22-35, 2004). It was not mitigated by glycerol, aspirin, indomethacin, or mannitol addition to aCSF. When RPS-2 (Cryobiology 21, pp. 260-273, 1984) was used as a carrier solution for up to 50% w/v V(EG) solutes, 0 degree C was more protective than 10 degrees C. Raising V(EG) concentration to 53% w/v allowed slice vitrification without injury from vitrification and rewarming per se, but was much more damaging than exposure to 50% w/v V(EG). This problem was overcome by using the analogous 61% w/v VM3 vitrification solution (Cryobiology 48, pp. 157-178, 2004) containing polyvinylpyrrolidone and two extracellular "ice blockers." With VM3, it was possible to attain a tissue K(+)/Na(+) ratio after vitrification ranging from 91 to 108% of that obtained with untreated control slices. Microscopic examination showed severe damage in frozen-thawed slices, but generally good to excellent ultrastructural and histological preservation after vitrification. Our results provide the first demonstration that both the viability and the structure of mature organized, complex neural networks can be well preserved by vitrification. These results may assist neuropsychiatric drug evaluation and development and the transplantation of integrated brain regions to correct brain disease or injury.     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Optimal developmental stage for vitrification of parthenogenetically activated porcine embryos

The objective of this experiment was to determine the optimal developmental stage to vitrify in vitro cultured porcine parthenogenetically activated (PA) embryos. Embryos were vitrified by Cryotop on Day 4, 5 or 6 after oocyte activation (Day 0), and immediately after warming they were either time-lapse monitored for 24h or analyzed by differential staining. After warming, the embryos had to be cultured for at least 8h before their survival rates were stabilized. Both the survival rate at 8h and the hatching rate at 24h of Day 4 embryos were significant higher than those vitrified on Day 5 or 6 (P<0.05), no matter if they were morulae or blastocysts. These results demonstrate that porcine PA embryos can survive successfully after vitrification/warming, that the optimal time for vitrification was Day 4 for both morulae and blastocysts, and that 8h after warming was the time needed to make an early evaluation of porcine PA embryo survival.