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1.
This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague‐Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co‐cultured with autologous cumulus cells. We also experimented with co‐culture in 100 µL drops. Drop co‐culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co‐cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post‐transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co‐culture in Cook medium with cumulus cells compared to in vivo‐derived blastocysts. The advantage of the co‐culture system is in generating more blastocysts available for transfer.  相似文献   

2.
Currently, in vitro‐produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one‐third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU‐IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF‐ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF‐ITS (EGF‐ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF‐ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF‐ITS improved the embryo quality when smaller groups of embryos were cultured.  相似文献   

3.
The objective of this study was to investigate the effects of beta‐mercaptoethanol (β‐ME) on post‐thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open‐pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze–thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 μm β‐ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 β‐ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of β‐ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of β‐ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen–thawed mouse PN embryos after different cryopreservation protocols.  相似文献   

4.
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5.
In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.  相似文献   

6.
The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre‐implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.  相似文献   

7.
Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.  相似文献   

8.
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9.
Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10?12, 10?9, 10?4 m ; Experiment 4: 0, 10?3 m ), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat‐stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10?4 m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non‐HSO without melatonin. Melatonin did not have any effect in embryos from non‐HSO groups compared with the control. In Experiment 4, 10?3 m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non‐HSO when compared to melatonin‐untreated oocytes/embryos. In conclusion, 10?4 m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.  相似文献   

10.
Vitamin C (Vc) is a natural compound supplemented to culture media to guarantee the appropriate reactive oxygen species (ROS) level, as well as protect cells from oxidative damage and apoptosis. The current study was conducted to determine the effects of Vc (0, 2.5, 5, 10, 20 and 40 μg/ml) on the ROS production, developmental ability and quality of in vitro produced porcine parthenotes. The results show that: (i) the ROS levels in the embryos significantly decrease in the Vc‐treated groups compared with the control (p < 0.05), (ii) the rates of blastocyst formation and total cell numbers in each blastocyst are significantly higher in the Vc‐treated groups than in the control (p < 0.05); the optimum concentration of Vc is 20 μg/ml, (iii) the relative expression of Bcl‐xL significantly increases and that of Bax is downregulated after Vc treatment. Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling analysis indicates that the ratio of apoptotic cells in the blastocyst is also significantly lower in Vc‐treated groups (p < 0.05) and (iv) Vc treatment can also increase the expression of the Nanog gene in porcine embryos, with a fivefold increase in 20 μg/ml Vc treatment compared with the control (p < 0.05). Therefore, Vc improves the development of porcine embryos by reducing the ROS levels. Vc addition in PZM‐3 medium can decrease the number of apoptotic cells and increase the cell numbers in blastocysts to produce high‐quality porcine embryos in vitro.  相似文献   

11.
Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO2, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO2 incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.  相似文献   

12.
Leucemia inhibitory factor (LIF) is involved in various reproductive processes, including sperm development, regulation of ovulation, as well as blastocyst formation, hatching and implantation in embryos. Moreover, LIF has also been shown significantly to enhance the blastocyst formation rates of bovine embryos, a finding that remains controversial. Our purpose was to investigate time‐dependent effect of LIF on bovine embryo culture, especially in terms of addition timing. Presumptive zygotes were cultured in five different groups. In this study, 100 ng/ml LIF was added to the culture medium were as follows; control: SOF alone, group A: at day 0 (fertilization day), group B: at day 4 post‐insemination (p.i.), group C: at day 4 to 7 (p.i. before vitrification) and group D: at day 8 (p.i. after thawing). Addition of LIF to the culture medium at day 4 significantly increased the percentage of blastocyst rate when compared day 0, day 4 at 6/7 and control group (41.8% versus 24.3%, 19.7%, 34.6%). In conclusion, the addition of LIF only on day 4 (p.i.) to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts rate, re‐expansion rate and cellular cryotolerance after vitrification.  相似文献   

13.
The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre‐implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus‐oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus‐oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39°C in an atmosphere of 5% (v/v) CO2 in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39°C in a humidified atmosphere of 5% (v/v) CO2, 5% (v/v) O2 and 90% (v/v) N2. In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO2 in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 μg/ml RT and 0.5 μm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre‐implantation development of goat embryos and can be used to enhance in vitro embryo production.  相似文献   

14.
Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.  相似文献   

15.
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   

16.
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17.
The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B4 isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.  相似文献   

18.
The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro‐produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre‐incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre‐incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.  相似文献   

19.
In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low‐oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low‐oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium.  相似文献   

20.
The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short‐ vs long‐term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP‐Group), or after IVF and 2–3 days of IVC, 4–8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo‐Group) or IVM oocytes were co‐incubated with spermatozoa for 3–4 h and transferred into the oviducts of synchronized heifers (GIFT‐Group). Embryos of the In Vivo‐Group and the GIFT‐Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa‐medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP‐Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo‐survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo‐tolerance. However, the duration of in vivo culture crucially influenced the cryo‐tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.  相似文献   

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