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1.
Parthenogenetic activation is an important factor in successful production of cloned mammals. Because it has been reported that aged oocytes are more sensitive to parthenogenetic activation than young oocytes, the present study examined the effects of oocyte aging on the in vitro and in vivo developmental potential of nuclear-transferred (NT) mouse oocytes receiving cumulus cells. The potentials of young NT oocytes (14 h after human chorionic gonadotrophin [hCG] injection) to develop into blastocysts was, however, significantly higher than that of aged oocytes (20 h after hCG injection; 16% vs 6%). When the nuclei of NT oocytes at the 2-cell stage were fused with enucleated fertilized 2-cell embryos, the potentials of the serial NT embryos to develop into blastocysts were no different for both young and aged oocytes (74% vs 74%). Live young, however, were obtained only after transfer of serial NT blastocysts developed from young NT oocytes (2%). In contrast to a report using embryonic nuclei as the nuclear donors, the results of the present study indicate that young oocytes are superior to aged oocytes as a source of recipient cytoplasm for mouse somatic cell cloning.  相似文献   

2.
Nuclear transfer preserves the nuclear genome of freeze-dried mouse cells   总被引：1，自引：0，他引：1  
Mouse spermatozoa can be freeze dried without losing genetic integrity and reproductive potential. However, it is not known if freeze-dried mouse cells similarly maintain their genetic integrity and developmental potential following nuclear transfer. Here, we investigated the developmental capacity and embryonic stem (ES) cell derivation of reconstructed oocytes by nuclear transfer using freeze-dried cumulus or ES cells. Cumulus and ES cells were lyophilized overnight and stored at 4 C for up to 1 week. After rehydration, all cells showed membrane damage and were unviable. However, following nuclear transfer, 1-4% of the reconstructed oocytes developed to the blastocyst stage. A total of five nuclear transfer ES (ntES) cell lines were generated from blastocysts and morulae. All ntES cell lines had normal karyotypes and were positive for the ES-cell-specific markers (alkaline phosphatase, Oct3/4 and Nanog). After aggregation of ntES cells with fertilized embryos, chimeric mice with a high level of coat color chimerism were generated. Our findings show that the genomic integrity of cells can be maintained after freeze-drying and that it is possible to produce offspring from the cells using nuclear transfer techniques.  相似文献   

3.
Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.  相似文献   

4.
The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.  相似文献   

5.
In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.  相似文献   

6.
Among the factors that affect in vitro embryo development, oxygen atmosphere is considered to be of great influence. In this study, we evaluated the influence of two different oxygen atmospheres during in vitro fertilization (IVF) of ovine oocytes on their developmental capacity and quality assessed by cryotolerance. Cumulus oocyte complexes derived from ovaries of slaughtered sheep were matured in vitro and subsequently fertilized under low (5%) or high (20%) oxygen atmospheres, and cultured in SOF + aa + 0.4% BSA in 5% CO2 and 5% O2 up to blastocyst stage. The cleavage rates obtained in the fertilization system at 20% O2 were significantly higher than those obtained in the 5% O2 fertilization system (61.2% vs 50.8%; p < 0.01). The distribution of cleaved oocytes at 22, 26 and 40 h of culture intervals was not different in the low or high O2 atmosphere (31.4%, 26.4% and 42.1% vs 28.0%, 29.3% and 42.7% respectively). Blastocysts output on the 6th day post-fertilization (dpf) was significantly higher when oocytes were fertilized under 5% O2 concentration (63.04% in 5% O2 vs 47.36% in 20% O2), while on the 7th dpf the higher number of blastocysts was obtained in the 20% O2 system (35.10%.in 20% O2 vs 26.09% in 5% O2). After vitrification no differences were observed between low or high oxygen atmosphere in the viability rates of blastocysts obtained on day 6 (93.6% vs 96.5%), on day 7 (46.3% vs 41.7%) and on day 8 (11.1% vs 6.6%). After differential staining, no significant differences were observed in the total cell number and inner cell mass and trophoblastic cells ratio of blastocysts produced on 6 dpf (189.6 +/- 51.3 and 0.260 +/- 0.07 vs 223.3 +/- 45.6 and 0.277 +/- 0.09), on 7 dpf (168.3 +/- 25.1 and 0.316 +/- 0.06 vs 172.1 +/- 33,6 and 0.320 +/- 0.06) and on 8 dpf (121.2 +/- 23,8 and 0.302 +/- 0.03 vs 117.0 +/- 35.1 and 0.313 +/- 0.04) under low or high oxygen atmosphere respectively). In conclusion, our data suggest that low oxygen atmosphere during IVF affects positively the production of high quality ovine blastocysts.  相似文献   

7.
In the present study, we investigated the effect of activation treatments on the actin filament distribution and in vitro development of somatic cell nuclear transfer (SCNT) embryos in miniature pigs. We combined three activation methods, ionomycin (ION), electrical stimulation (ES), and cycloheximide treatment (CH), to prepare seven activation treatments (ION, ES, CH, ION + CH, ION + ES, ES + CH and ION + ES + CH). First, we investigated the activation rate of oocytes and in vitro development of parthenotes. The activation rates of the oocytes in the ION, ES, CH, ION + CH, ION + ES, ES + CH, and ION + ES + CH groups were 42.9, 51.3, 0.0, 82.1, 80.6, 78.1 and 78.6%, respectively, showing that the rates of the combined treatment groups were significantly higher (P<0.05) than those of the single treatment groups. Although there were no significant differences in the activation rates of the combined treatment groups, the developmental rate to blastocysts in the ION + CH treatment group (36.1%) was significantly higher (P<0.05) than the other combined treatment groups (14.6-24.7%). Subsequently, we investigated the in vitro development and distribution of microfilaments in SCNT embryos. The developmental rate to blastcysts of the SCNT embryos in the ION + CH treatment group (11.3%) was significantly higher (P<0.05) than in the ES and ION + ES + CH treatment groups (4.5 and 5.2%, respectively). The rate of normal actin filament distribution in the SCNT embryos activated with ION + CH was significantly higher (P<0.05) than those activated with ES or ION + ES + CH treatment (63.3 vs. 46.8 or 46.4%). In addition, the fragmentation rate of the SCNT embryos activated with ION + CH was significantly lower (P<0.05) than those activated with ION + ES + CH (14.9 vs. 26.1%). The present results suggest that an activation treatment of ionomycin combined with cycloheximide may avoid physical damage to microfilaments and result in improved subsequent development of miniature pig SCNT embryos.  相似文献   

8.
The effect of linoleic acid-albumin (LAA) supplementation to the media for IVM, enucleation, and activation on the developmental potential of bovine embryos produced by nuclear transfer (NT) into frozen-thawed cytoplasts was investigated. Blastomeres derived from morulae was placed in the perivitelline space of frozen-thawed cytoplasts, which were then fused by a DC pulse. The proportion of fused embryos was similar between groups with and without LAA (87 vs. 90%). The proportion of development to blastocysts of NT embryos derived from the media with LAA (14%) was higher than that without LAA (4%), indicating that LAA treatment of bovine oocytes during IVM, enucleation and activation can improve the ability of such cytoplasts after freezing and thawing to develop into blastocysts after NT.  相似文献   

9.
The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.  相似文献   

10.
We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.  相似文献   

11.
The objective of this study was to determine if the transfection of human prourokinase (ProU) gene and passage number of transfected ear fibroblasts affected in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human ProU was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker and human ProU gene into a pcDNA3 plasmid and transfected into bovine ear fibroblasts using a lipid mediated method. Abattoir derived oocytes were enucleated at 18-20 hr post maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the couplets were cultured in modified synthetic oviductal fluid (mSOF) medium for 168 hr. In Experiment 1, significantly lower rate in blastocysts formation (10.3%) was observed in transfected donor cells at early passage than that in nontransfected counterparts (22.1%, P<0.05). In Experiment 2, development to blastocysts and GFP expression in blastocysts were not significantly different between early (3-7) and late (8-12) passage donor cells (10.3 vs. 11.3% and 54.5 vs. 41.7%, respectively). This study indicates that in vitro development of bovine transgenic NT embryos is negatively influenced by transfection of human ProU gene into donor fibroblasts. However, passage number of transfected ear fibroblasts does not affect in vitro development of bovine transgenic NT embryos.  相似文献   

12.
The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.  相似文献   

13.
The overall aim of the present study was to evaluate in vitro development ability of oocytes recovered from 56 Holstein Frisian heifers with low [group 1 (G1): <13 mg /dl], moderate [group 2 (G2): 13–16 mg /dl] and high [group 3 (G3): >16 mg /dl] plasma urea nitrogen (PUN) concentrations, to determine whether PUN concentrations affect the competence of oocytes to progress to blastocysts after in vitro fertilization. In vitro oocyte and embryo development was assessed by blastocyst rates, embryo total cell numbers and apoptosis. Blood samples for the determination of PUN were collected 24 h prior to collection of the ovaries at the slaughter. A total of 112 ovaries were collected at a local abattoir and oocytes (n = 697) were aspirated, in vitro matured and fertilized. On day 8, blastocysts were assigned to the terminal dUTP nick end labelling assay. Cleavage rates were significantly higher (p < 0.001) for groups 1 and 2 than for group 3 (i.e. 72.5% and 72.2% vs 61.7%, respectively). The proportion of fertilized oocytes that developed into blastocysts was higher (p < 0.05) for group 1 than for group 3 (34.0% vs 23.0%, respectively). Day 8 blastocysts showed higher total cell counts (p < 0.05) for group 1 than for group 3 (123.7 vs 76.3), and a higher (p < 0.05) total apoptotic cell rate was found in group 3 (25.9 and 19.0 vs 43.2 for G1, G2 and G3, respectively). In conclusion, the ability of oocytes from heifers with increased levels of PUN to develop to the blastocyst stage was significantly reduced when standard routines for in vitro maturation, fertilization and culture were followed. These detrimental effects can be mediated in part through direct effect of urea and/or by the metabolic products on the process of follicle-enclosed oocyte nuclear and cytoplasmic development.  相似文献   

14.
The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.  相似文献   

15.
The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro‐matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) (‘ET‐vivo’ embryos), or cultured in vitro for 5 or 6 days (‘IVC’ embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET‐vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term.  相似文献   

16.
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   

17.
The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.  相似文献   

18.
Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode's medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.  相似文献   

19.
To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromosome complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or IVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.  相似文献   

20.
The present experiment was designed to identify possible effects of sera and steroid hormones added to a co-culture with bovine oviduct epithelial cells on embryonic development in vitro. Bovine oocytes were matured in vitro for 24 h and then fertilized in vitro using swim-up and heparin-treated, frozen-thawed spermatozoa. At 18 and 20 h after insemination, oocytes were cultured for 3 or 7 d in a co-culture system with bovine oviduct epithelial cells containing either fetal calf serum (FCS) or estrous cow serum (ECS) and one of six hormonal additions (none, 1 or 10 micrograms/ml estradiol [E]; 1 microgram/ml progesterone [P]; 1 microgram/ml E + P; and 10 micrograms/ml E + P). A total of 2,666 oocytes were cultured for 3 d and examined for cleavage. Of those, 2,280 oocytes were cultured up to 7 d for development to the late morula or blastocyst stage. Greatest cleavage rates for 2- to 8-cell and 8-cell stages were observed in FCS (71 and 24%) and ECS (66 and 23%) without steroid addition. For development into blastocysts, no serum effect was observed. Greatest rates for development into blastocysts were observed in FCS (14%) and ECS (16%) without steroid addition. These results indicate that addition of E and P at the doses and combinations tested did not enhance developmental capacity of in vitro fertilized bovine oocytes. Compared with FCS, ECS tended to increase cleavage rates and development into blastocysts.  相似文献   

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