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1.
Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.  相似文献   

2.
The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.  相似文献   

3.
为提高绵羊体细胞核移植效率,以绵羊卵丘细胞为核供体,在融合后的核质互作期间加入咖啡因,在激活后对重构胚进行培养的过程中,加入组蛋白去乙酰化酶抑制剂Scriptaid。结果表明:咖啡因浓度以2.5 mmol/L或5 mmol/L为宜,尽管卵裂率、桑椹胚率以及囊胚发育率与对照组差异不显著(P>0.05),但囊胚细胞数显著高于对照组(P<0.05);以0.2μmol/L Scriptaid处理组囊胚发育率最高,达24.31%,显著高于对照组和0.8μmol/L Scriptaid处理组(P<0.05),但与0.4μmol/L Scriptaid处理组相比差异不显著(P>0.05);囊胚细胞总数以0.2μmol/L Scriptaid处理组最高,显著高于0.8μmol/L Scriptaid处理组(P<0.05),但是与对照组和0.4μmol/L Scriptaid处理组差异不显著(P>0.05)。以上结果表明,咖啡因处理对绵羊核移植重构胚的囊胚发育率无显著影响,但可以显著提高囊胚细胞总数;Scriptaid可以显著提高绵羊核移植重构胚的囊胚发育率。  相似文献   

4.
本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40% EG(ethylene glycol,EG)、25% EG＋25% DMSO(dimethylsulphoxide,DMSO) 和20% EG＋20% DMSO＋0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率。结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8%)和微滴系统(8.3%)(P<0.01)。采用20% EG＋20% DMSO＋0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40% EG(P<0.01);HMC重组胚的融合率和囊胚率均高于传统核移植法(P<0.05;P<0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异。以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20% EG＋20% DMSO＋0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果。  相似文献   

5.
Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non‐domestic animal species. However, problems arise during the development of these embryos, which may be related to species‐specific differences in nuclear–cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria‐related genes NRF1, MT‐CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34–33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45–12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80–87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT‐CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.  相似文献   

6.
The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.  相似文献   

7.
Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.  相似文献   

8.
This study was conducted to evaluate whether refining mineral oil and the addition of hemoglobin and/or glucose to a serum-free medium could improve in vitro-development of embryos cultured in a chemically semi-defined microdroplet culture system. Block strain, outbred (ICR) mouse 1- or 2-cell embryos were cultured in 5 microl droplets of Chatot, Ziomek and Bavister medium overlaid with mineral oil of different types, and preimplantation development to the blastocyst stage was subsequently monitored. In the experiment 1, either Sigma (M-8410) or BDH (GPR) mineral oil with or without washing was used for embryo culture and, distilled water (DW) or culture medium was used as a washing agent. As results, better (P<0.0001) development of 1-cell embryos was found in the Sigma than in the BDH; more blastocysts developed in Sigma oil washed with culture medium than in the others (37% vs. 0%). Subsequently, 1- (experiment 2) or 2-cell (experiment 3) embryos were cultured in the droplets overlaid with medium-washed Sigma oil, to which 0.001 mg/ml hemoglobin and/or 5.6 mM glucose were supplemented at the 1-cell and the 4-cell stages, respectively. Regardless of embryo stages, blastocyst formation was significantly improved by the addition of hemoglobin (54 to 48% vs. 42 to 31% in 1-cell and 83 to 78% vs. 65 to 68% in 2-cell embryos) and this effect was independent of glucose addition. In conclusion, the selection and washing of mineral oil, and the addition of hemoglobin is beneficial for improving the efficacy of a drop embryo culture system using a serum-free medium.  相似文献   

9.
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10.
The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects.  相似文献   

11.
The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.  相似文献   

12.
This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.  相似文献   

13.
Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible.  相似文献   

14.
Nowadays, the use of foetal calf serum (FCS) during in vitro embryo culture is very controversial. Whilst some authors have encouraged its use, others reject it because of its harmful effects. Although in vitro embryo production in red deer is a promising assisted reproductive technique, it is still in its infancy and a great effort is needed to update the protocols used. The aim of this study was to assess whether FCS supplementation in red deer embryo culture medium is necessary to produce blastocyst and, if so, when is the best time to add it in terms of blastocyst production and quality. In vitro blastocysts were cultured with FCS added at 24, 48 or 96 hours post‐insemination (hpi). In addition, a treatment without FCS was used as control. Six hundred and ninety‐four cumulus–oocyte complexes were collected for in vitro fertilization. Cleavage rate was examined at 48 hpi, and blastocyst yield was recorded on days 6, 7 and 8. FCS had no influence on cleavage and blastocyst rate for any of the treatments studied. However, the number of cells was higher (p = .025) in those blastocysts cultured with FCS from 48 hpi compared with FCS‐free culture media (93.88 ± 7.76 vs. 54.11 ± 8.36). In conclusion, the addition of FCS to the embryo culture medium at 48 hpi improves the quality of red deer blastocyst, although it does not affect the percentage of embryos obtained.  相似文献   

15.
Studies to date have shown that bison embryo development in vitro is compromised with few embryos developing to the blastocyst stage. The aim of this study was to use bison-cattle hybrid embryos, an interspecific cross that is known to result in live offspring in vivo, as a model for assessing species-specific differences in embryo development in vitro. Cattle oocytes fertilized with cattle, plains bison and wood bison sperm were assessed for various developmental parameters associated with embryo quality, including cell number, apoptosis and ATP content. Decreased development to the blastocyst stage was observed in hybrid wood bison embryos compared with the other treatment groups. Although both wood bison and plains bison hybrid blastocysts had significantly lower cell numbers than cattle blastocysts, only wood bison hybrid blastocysts had a greater incidence of apoptosis than cattle blastocysts. Among the treatment groups, ATP levels and expression profiles of NRF1, TFAM, MT-CYB, BAX and BCL2 were not significantly different in both 8- to 16-cell stage and blastocyst stage embryos. These data provide evidence of decreased developmental competence in the wood bison hybrid embryos, owing to inadequate culture conditions that have increased apoptotic events.  相似文献   

16.
This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.  相似文献   

17.
The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.  相似文献   

18.
以CZB为基础培养液,培养小鼠4、8-细胞胚胎单卵裂球,研究葡萄糖、牛磺酸和猪输卵管上皮细胞共培养在其体外发育中的作用。结果表明:牛磺酸添加与否对4、8-细胞胚胎单卵裂球的囊胚发育率分别为29%、30%和14%、14%,无显著性差异(P>0.05)。添加葡萄糖后,4、8-细胞胚胎单卵裂球的囊胚发育率分别为36%和19%,有显著提高(P<0.05)。含有葡萄糖而牛磺酸的添加与否对4、8-细胞胚胎单卵裂球的囊胚发育率分别为36%、38%和19%、20%,无显著性差异(P>0.05)。各组内4、8-细胞胚胎单卵裂球形成的囊胚细胞数分别为(10.44±1.24～(12.43±1.18)和(7.57±0.97)～(8.48±1.16),均无显著性差异(P>0.05)。4、8-细胞胚胎单卵裂球与猪输卵管上皮细胞共培养,其囊胚发育率分别为47%和26%,囊胚细胞数分别为(17.57±1.13)和(11.43±0.92),均高于单一培养(P<0.05)。  相似文献   

19.
This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp‐70) were also examined. Day 7 and 8 bovine in vitro‐produced blastocysts were submitted to an HHP treatment (60 MPa, at 32°C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post‐warming) and hatching (48 h post‐warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP‐treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32°C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP‐treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp‐70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post‐warming.  相似文献   

20.
In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.  相似文献   

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