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1.
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   

2.
Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.  相似文献   

3.
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro‐produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1‐msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single‐guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.  相似文献   

4.
For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.  相似文献   

5.
The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus–oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.  相似文献   

6.
This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm–oocyte fusion, oocytes and/or sperm were pre‐incubated with laminin or anti‐β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8‐cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm–oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin‐β1 of sperm was involved in sperm–oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm–oocyte fusion, but improves embryonic development. However, only integrin‐β1 is involved in sperm–oocyte binding.  相似文献   

7.
This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL‐I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm ), BL‐I (50 μm ) and association of drugs (ROS 6.25 μm and BL‐I 25 μm ). Oocytes were cultured for 18 h in an agent‐free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL‐I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL‐I and ROS+BL‐I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post‐thawing embryos. Embryos from ROS+BL‐I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re‐expansion (p < 0.05). In conclusion, block of meiosis using BL‐I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL‐I resulted in a better resistance to the embryo cryopreservation process.  相似文献   

8.
The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.  相似文献   

9.
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10.
Although the technique of interspecies somatic cell nuclear transfer can be used to increase the population size of endangered mammals, the mitochondrial heteroplasmy in cloned embryos and animals makes this idea doubtful. In present study, goat–sheep cloned embryos were constructed by fusing goat foetal fibroblasts (GFFs) into sheep oocytes and then cultured in vitro to investigate the capability of sheep oocyte dedifferentiating GFF nucleus. Moreover, at each stage of 1‐ (immediately after fused), 2‐, 4‐, 8‐, 16‐cell, morula and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined using real‐time PCR. The results showed that: 7.4% of the fused cloned embryos can develop to the blastocyst stage; in the process of one cell to the morula stage, the copy number of two kinds of mtDNA was stable relatively; however, in the process of morula to the blastocyst stage, the decreasing in the copy number of GFF‐derived mtDNA, while the increasing in sheep oocyte‐derived, resulted in their ratio of decreasing sharply from 2.0 ± 1.0% to 0.012 ± 0.004%. This study demonstrates that: (i) the goat–sheep cloned embryos have the ability to develop to blastocyst in vitro; (ii) from the morula stage to the blastocyst stage of goat–sheep cloned embryos, goat derived mitochondria can be gradually replaced with those from sheep oocyte.  相似文献   

11.
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12.
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   

13.
This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm ) along with a control (0 mm ) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC‐I) (12–72 hpi) and IVC‐II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC‐I and IVC‐II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.  相似文献   

14.
This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.  相似文献   

15.
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16.
Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus–oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.  相似文献   

17.
The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro‐matured Caprine oocytes. A total of 470 in vitro‐matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro‐matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm‐injected in vitro‐matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non‐activated oocytes.  相似文献   

18.
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19.
The objective of this study was to examine the effects of canthaxanthin (Cx) treatment during in vitro maturation (IVM) of porcine oocytes on embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), on intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in mature oocytes, and on gene expression in both PA‐ and SCNT‐derived blastocysts. To determine the optimal effective concentration of Cx, porcine oocytes were cultured in IVM medium supplemented with various concentrations (0, 20, 40 and 80 μM) of Cx for 22 hr. Compared to other groups, supplementation with 40 μM Cx significantly improved blastocyst formation rates after PA (p < .05), but no significant differences were observed among groups in total blastocyst cell numbers. Subsequently, oocytes were cultured in IVM medium supplemented with or without 40 μM Cx. Oocytes treated with 40 μM Cx showed significantly increased cleavage and blastocyst formation rates after SCNT compared to the control group (p < .05). Moreover, significantly increased intracellular GSH and reduced ROS levels were observed in the Cx‐treated group (p < .05). In addition, both PA‐ and SCNT‐derived blastocysts from the 40 μM Cx‐treated group showed significantly increased mRNA expression of Bcl2 and Oct4 and decreased Caspase3 expression level (p < .05), when compared with the control group. PA‐derived blastocysts from the 40 μM Cx‐treated group also exhibited significantly decreased expression of Bax (p < .05). Our results demonstrated that treatment with 40 μM Cx during IVM improves the developmental competence of PA and SCNT embryos. Improvement of embryo development by Cx is most likely due to increased intracellular GSH synthesis, which reduces ROS levels in oocytes, and it may also positively regulate apoptosis‐ and development‐related genes.  相似文献   

20.
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   

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