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1.
The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN2 for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.  相似文献   

2.
Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5–15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.  相似文献   

3.
Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.  相似文献   

4.
The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.  相似文献   

5.
The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring.  相似文献   

6.
The chemical toxicity of cryoprotectants to porcine embryos was examined by the evaluation of survival and DNA damage after exposure to cryoprotectants. Porcine blastocysts were exposed to 10% of ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GLY) for 1 h at room temperature (23-25 degrees C) and then cultured in vitro for 24 h. The survival rates of blastocysts exposed to PD and GLY were significantly lower than those of control blastocysts in which the embryos were exposed to carrier solution without cryoprotectants. Significantly more DNA-fragmented nuclei occurred in the cryoprotectant-exposed blastocysts, compared with the control blastocysts. Moreover, the indices of DNA-fragmented nuclei in the blastocysts without blastocoele re-formation after culture were significantly higher than those with blastocoele re-formation, irrespective of the exposure treatment. These results indicate that the exposure of porcine blastocysts to cryoprotectant decreases the survival rates and increases the DNA-fragmented nuclei in embryos.  相似文献   

7.
This study examined the effects of different vitrification medium compositions and exposure times (2, 4 and 6min) on the post-thaw development of buffalo embryos produced in vitro (IVP). The compositions were (1) 40% ethylene glycol (EG); (2) 25% glycerol (G)+25% EG, and (3) 25% EG+25% dimethylsulfoxide (DMSO). The base medium was 25mM Hepes-buffered TCM-199+10% steer serum +50microg/mL gentamycin. The IVP embryos were cryopreserved by a two-step vitrification method at 24 degrees C. After warming, the embryos were cultured in vitro for 72h. The vitrification of morulae and blastocysts in 25% EG+25% DMSO with an exposure time of 2 and 4min, respectively, resulted in a better hatching rate than other combinations. The hatching rate of morulae vitrified in 25% EG+25% G, 25% EG+25% DMSO, and blastocysts vitrified in 40% EG, 25% EG+25% DMSO were negatively correlated with exposure time. However, the hatching rate of blastocysts vitrified in 25% EG+25% G was positively correlated with exposure time. The study demonstrated that the post-thaw in vitro development of IVP buffalo embryos was affected by the vitrification medium composition and exposure time.  相似文献   

8.
My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.  相似文献   

9.
The purpose of this study was to develop a practical cryopreservation method for in vitro-produced (IVP) and sex-predetermined bovine blastocysts that will be applicable to direct transfer of the post-thaw embryos. Blastocysts were harvested 7 days after IVF and allocated to either an intact or biopsy group. The cryoprotective solution contained 0.7 M glycerol and 0, 0.05 or 0.1 M sucrose. Slow cooling at a rate of -0.5 C/min was terminated at -25, -30, or -35 C, and rapid cooling in liquid nitrogen was followed. After one-step thawing and dilution, the IVP blastocysts were cultured for 3 days to assess their survival. The post-thaw survival rate of intact blastocysts after termination of slow cooling at -30 C in 0.7 M glycerol plus 0.1 M sucrose (96.2%) was significantly higher than that at -25 C in 0.7 M glycerol alone (44.4%). The post-thaw survival rate of biopsied bovine blastocysts after termination of slow cooling at -25 C in 0.7 M glycerol alone (53.8%) tended to be lower than that at -25 C in 0.7 M glycerol plus 0.05 M sucrose (91.3%) or -30 C in 0.7 M glycerol plus 0.1 M sucrose (92.3%). Thus, addition of a small amount of sucrose to 0.7 M glycerol cryoprotective solution shortened the process of slow cooling for both the intact and biopsied bovine embryos. Judged from the survival levels in vitro after thawing and one-step dilution of embryos (>80%), this is an improved method of cryopreservation for subsequent direct transfer of IVP and biopsied bovine blastocysts.  相似文献   

10.
This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN2). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN2 for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN2.  相似文献   

11.
影响玻璃化冷冻兔胚胎效果的一些因素   总被引：4，自引：0，他引：4  
试验对影响玻璃化冷冻兔胚胎效果的一些因素进行探讨，以找出理想的玻璃化冷冻方法。在测试的５种玻璃化溶液中，含３５％乙二醇（ＥＧ）和１．０ｍｏｌ／Ｌ蔗糖的溶液（ＶＳ１）对胚胎的毒性最小。用ＶＳ１冷冻桑椹胚和囊胚的理想程序是：在室温下使胚胎分别在２０％ＥＧ和３５％ＥＧ中平衡２、３分钟后，移入ＶＳ１中，０．５分钟内（囊胚也可在２分钟后）投入液氮中冷冻。桑椹胚的存活率为９１．７％（３３／３６），囊胚的存活率为９７．１％（３３／３４）～９７．３％（３６／３７）。８～１６细胞胚胎的理想冷冻程序为：在室温下使胚胎在２０％ＥＧ、３５％ＥＧ中平衡２、３分钟，移入４℃的３７％ＥＧ＋１．０ｍｏｌ／Ｌ蔗糖溶液中平衡２分或１０分钟后冷冻，胚胎存活率分别为１００％（３７／３７）、８６．１％（３１／３６）。  相似文献   

12.
The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post‐warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post‐warm survival rate of blastocysts vitrified in medium containing 1,2‐propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post‐warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non‐penetrating cryoprotectant did not differ. There was also no significant difference in post‐warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post‐warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post‐warm survival of IVP porcine blastocysts. The improved post‐warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.  相似文献   

13.
In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 mul of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO(2) in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.  相似文献   

14.
The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO2 incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 µl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the non-vitrified control. However, the hatching rate of cyto-b-treated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.  相似文献   

15.
The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.  相似文献   

16.
This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG + 2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG + 16.5% DMSO + 0.5M sucrose. After warming, embryos were cultured for 48 hr. Re‐expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane‐damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p < .05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN‐t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.  相似文献   

17.
小鼠2-细胞胚胎细管法和OPS法玻璃化冷冻保存技术的研究   总被引：8，自引：0，他引：8  
本试验在室温 (2 0℃和 2 5℃ )条件下 ,利用不同浓度的玻璃化溶液 (EFS和EDFS) ,对小鼠 2 细胞胚胎进行细管法和OPS法玻璃化冷冻保存。在 2 0℃室温条件下 ,用EFS4 0平衡 1min细管一步法冷冻 ,解冻后囊胚发育率仅为35 .0 % ,和新鲜 2 细胞体外培养的对照组 (6 5 .0 % )的差异极显著 (P <0 .0 1)。当 2 细胞胚胎在 10 %EG +10 %D溶液中预处理 5min ,再移入EDFS中平衡 30s二步法冷冻保存 ,解冻后囊胚发育率达 4 7.8%～ 4 8.8% ;当室温升至2 5℃时 ,二步法冷冻保存后 2 细胞的囊胚发育率达到 5 2 .2 % ,与对照组无显著差异 (P >0 .0 5 )。改用OPS二步法EFS30冷冻组保存后的 2 细胞胚胎的囊胚发育率高达 6 2 .2 % ,为试验中的最佳组。用最佳细管法和OPS法冷冻组解冻后培养至囊胚移植给受体母鼠均获得产仔  相似文献   

18.

Background

At present, vitrification has been widely applied to humans, mice and farm animals. To improve the efficiency of vitrification in straw, bovine oocytes were used to test a new two-step vitrification method in this study.

Results

When in vitro matured oocytes were exposed to 20% ethylene glycol (EG20) for 5 min and 40% ethylene glycol (EG40) for 30 s, followed by treatment with 30% glycerol (Gly30), Gly40 or Gly50, a volume expansion was observed in Gly30 and Gly40 but not Gly50. This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40 (approximately 5.6 mol/L) and Gly50 (approximately 7.0 mol/L). Since oocytes are in EG40 just for only a short period of time (30 s) and at a lower temperature (4°C), we hypothesize that the main function of this step in to induce dehydration. Based on these results, we omitted the EG40 step, before oocytes were pretreated in EG20 for 5 min, exposed to pre-cooled (4°C) Gly50, for 30 s, and then dipped into liquid nitrogen. After warming, 81.1% of the oocytes survived, and the surviving oocytes developed into cleavage stage embryos (63.5%) or blastocysts (20.0%) after parthenogenetic activation.

Conclusions

These results demonstrate that in a two-step vitrification procedure, the permeability effect in the second step is not necessary. It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.  相似文献   

19.
绵羊玻璃化冷冻胚胎直接移植试验研究   总被引：1，自引：0，他引：1  
应用EFS40玻璃化液对6.5～7日龄的绵羊胚胎进行玻璃化冷冻及解冻后直接移植试验.结果:桑椹胚、囊胚冷冻解冻后移植的妊娠率分别为37.50%(3/8)和54.55%(6/11),胚胎存活率分别为33.33%(3/9)和50.00%(6/12),差异均不显著(P＞0.05);胚胎解冻后用0.5 mol/L蔗糖脱防冻剂与直接用胚胎存放液脱除防冻剂的妊娠率分别为44.44%(4/9)和50.00%(5/10),胚胎存活率分别为40.00%(4/10)、45.45%(5/11)差异不显著(P＞0.05);10枚解冻后的胚胎细管内脱防冻剂后,直接装管移植给8只受体,妊娠率为50.00%(4/8),胚胎成活率为40.00%(4/10),与同期常规冷冻解冻组相比无显著差异(P＞0.05).  相似文献   

20.
This study was conducted to examine the utility of vitrification for bovine embryos with low‐quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one‐step in‐straw method were compared with those of fresh control embryos in Experiment 3. Frozen‐thawed or vitrified‐warmed blastocysts were cultured with TCM‐199 supplemented with 100 μmol/L beta‐mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO2 in air, their survival after 24 hr were compared. The development to term of fair quality in vivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen‐thawed embryos were lower (p < .05) than that of vitrified‐warmed ones. When vitrified embryos were warmed in 0.3 mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified‐warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one‐step cryoprotectants dilution is utilized to preserve low‐quality bovine embryos.  相似文献   

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