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1.
The present study was conducted to evaluate the possibility of using in vitro‐produced parthenogenetic (PA) embryos for co‐transfer with morulae that had been collected in vivo and cryopreserved. The proportion of PA blastocysts (20.5%) was higher than that of their in vitro fertilization (IVF) counterparts (16.6%). Although there were no differences in morphology or diameter between the two groups, the number of cells in early PA blastocysts after in vitro culture for 6 days was lower than for IVF blastocysts (25.7 and 30.4 cells, respectively), and the number in recovered PA blastocysts was also smaller than that in recovered IVF blastocysts (37.4 and 50.2 cells, respectively). When 10 morulae warmed after vitrification were co‐transferred with 10 PA blastocysts (total 20 embryos) to the uterus of five recipients, the rates of pregnancy and farrowing did not differ, but the average period until spontaneous abortion tended to be longer relative to the control (when 20 morulae were transferred). These data suggest that in vitro‐produced PA embryos offer the possibility of assisted pregnancy for cryopreserved embryos; further experiments will be needed to confirm the beneficial effect of this approach on piglet production.  相似文献   

2.
The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.  相似文献   

3.
This study was undertaken to examine pre- and postimplantation developmental potency of cryopreserved embryos that had undergone in vitro growth (IVG), maturation (IVM) and fertilization (IVF) of oocytes from the preantral follicle stage. An oocyte culture system for IVG and IVM was used in oocyte-granulosa cell complexes (OGCs) derived from preantral follicles in 12-day-old mice. The rate of oocyte maturation was improved by the addition of gonadotropins (FSH/LH) and cytokines (IGF-I/SCF) to culture medium for IVG. During culture for IVG, estradiol-17β and progesterone concentrations increased progressively to the latter period of culture. This culture system enabled IVG, IVM, IVF and pre- and postimplantation development. From 90 cryopreserved 2-cell stage embryos transferred into recipients after warming, 10 live pups were produced. Cryopreservation of embryos by vitrification at the 2-cell stage showed no harmful effect on development to the blastocyst stage or on the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). This study demonstrated that embryos derived from oocytes grown in vitro have tolerance for vitrification and competence to develop to term after warming. This IVG-IVM-IVF technology combined with embryo cryopreservation might be useful for assisted reproduction in mice.  相似文献   

4.
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   

5.
We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.  相似文献   

6.
This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.  相似文献   

7.
Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%. Here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggyBac system. In embryos in which hyPBase mRNA and pPB-CAG-TagRFP DNA were co-injected into the cytoplasm, TagRFP fluorescence was observed after the 2-cell stage; when 30 ng/µl pPB-CAG-TagRFP DNA and 30 ng/µl hyPBase mRNA were co-injected, 94.4% of blastocysts were TagRFP positive. Furthermore, a high concentration of hyPBase mRNA resulted in creation of mosaic embryos in which the TagRFP signals partially disappeared. However, suitable concentrations of injected DNA and hyPBase mRNA produced embryos in which almost all blastomeres were TagRFP positive. Thus, the hyperactive piggyBac transposon system is an easy-to-implement and highly effective method that can contribute to production of transgenic mice.  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.
In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At 10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until 168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic development.  相似文献   

10.
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11.
The aim of this study was to determine the optimum conditions for vitrifying in vitro produced day 7 porcine embryos using different vitrification devices and blastocoele collapse methods. Firstly embryos were collapsed by micro-pipetting, needle puncture and sucrose with and without conducting vitrification. In the next experiment, non-collapsed embryos were vitrified in an open device using either superfine open-pulled straws (SOPS) or the CryoLoopTM system, or vitrified in a closed device using either the CryoTipTM or Cryo BioTM’s high security vitrification system (HSV). The post-thaw survival of embryos vitrified in the open devices did not differ significantly (SOPS: 37.3%; CryoLoopTM: 37.3%) nor did the post-thaw survival of embryos vitrified in the closed devices (CryoTip™: 38.5%; HSV: 42.5%). The re-expansion rate of embryos that were collapsed via micro-pipetting (76.0%) did not differ from those that were punctured (75.0%) or collapsed via sucrose (79.6%) when vitrification was not performed. However, embryos collapsed via sucrose solutions (24.5%) and needle puncture (16.0%) prior to vitrification were significantly less likely to survive vitrification than the control (non-collapsed) embryos (53.6%, P < 0.05). The findings show that both open and closed vitrification devices were equally effective for the vitrification of porcine blastocysts. Collapsing blastocysts prior to vitrification did not improve survival, which is inconsistent with the findings of studies in other species. This may be due to the extremely sensitive nature of porcine embryos, and/or the invasiveness of the collapsing procedures.  相似文献   

12.
This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.  相似文献   

13.
The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.  相似文献   

14.
Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP3R1), the channel responsible for Ca2+ release during IVF in porcine oocytes. Localization of IP3R1 close to the plasma membrane and total expression level of IP3R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP3R1 by the vitrification procedure.  相似文献   

15.
To completely avoid ice crystal formation and thus get a higher survival rate, vitrification methods have been commonly used for cryopreservation of oocytes and embryos. However, currently used vitrification methods for oocytes and embryos are not suitable for the cryopreservation of preantral follicles (PFs). In the present study, stainless steel mesh was fabricated into mini mesh cups to vitrify isolated PFs. Moreover, isolated follicles were encapsulated and then subjected to vitreous cryopreservation to facilitate in vitro culture/maturation of follicles after warming. The results showed that the percentages of viable follicles did not differ significantly between the vitrification group and fresh group soon after warming (81.25% vs. 85.29%, P>0.05) and after a 7-day culture period (77.78% vs. 83.33%, P>0.05). No difference in mean follicular diameter was observed between cryopreserved and fresh follicles when cultured in vitro. Transmission electron microscopic analysis revealed that vitreous cryopreservation could maintain the ultrastructure of follicles in alginate beads. In conclusion, the present vitrification method could efficiently cryopreserve isolated human ovarian follicles encapsulated by calcium alginate, which could be put into immediate use (in vitro culture/ maturation) after warming. However, more follicles and some detailed biochemical analyses are required to further investigate the effects of vitrification on the long-term growth of human encapsulated PFs.  相似文献   

16.
Field trials were carried out on the use of swamp buffaloes (2n = 48) as recipients of in vitro-derived vitrified river buffalo (2n = 50) embryos. River buffalo embryos were produced through a total in vitro production system using frozen semen from a top progeny-tested bull and slaughter-house-derived ovaries and cryopreserved by a vitrification technique. Recipient swamp buffaloes were Philippine carabaos selected on body condition, parity, and reproductive status and owned by village farmers. Eighty post-warmed riverine embryos were transferred non-surgically to 40 swamp buffalo recipients, of which three healthy riverine calves were born by normal delivery and one stillbirth, representing a 5.0% (4/80) full-term development and 10% (4/40) calving rate with 75% (3/4) normal and 25% (1/4) stillbirth. The results demonstrate that, under field conditions and despite the difference in chromosome number between the embryo and recipient, swamp buffaloes are potential surrogate mothers of imported, genetically superior river buffalo frozen embryos. Thus, embryo IVP-vitrification and transfer technology is a potential tool in the propagation of genetically superior buffaloes. Precautionary measures, however, are recommended to avoid dystocia and stillbirth.  相似文献   

17.
The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.  相似文献   

18.
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   

19.
Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the 8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.  相似文献   

20.
This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.  相似文献   

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