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Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full‐term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT. 相似文献
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Xuefang Wang Xiangxing Zhu Xingwei Liang Huiyan Xu Yuying Liao Kehuan Lu Shengsheng Lu 《Reproduction in domestic animals》2019,54(9):1195-1205
As a natural plant‐derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 μM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti‐apoptotic B‐cell lymphoma 2 (BCL‐2) gene and significant downregulation of the pro‐apoptotic BCL2‐associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 μM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 μM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT. 相似文献
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Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development 下载免费PDF全文
W‐J Lee J‐H Lee R‐H Jeon S‐J Jang S‐C Lee J‐S Park S‐L Lee W‐A King G‐J Rho 《Reproduction in domestic animals》2017,52(3):437-445
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
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Ting-Yu ZHANG Jian-Jun DAI Cai-Feng WU Xiao-Long GU Liang LIU Zhi-Qiang WU Yi- Ni XIE Bin WU Hui-Lan CHEN Yao LI Xue-Jin CHEN De-Fu ZHANG 《Animal Science Journal》2012,83(2):103-110
To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs. 相似文献
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不同蔗糖浓度、供体细胞保存温度和融合温度对小鼠体细胞克隆胚胎发育的影响 总被引:1,自引:0,他引:1
以昆明小鼠为实验动物,经过超数排卵后得到卵丘卵母细胞复合体(COCs),以卵丘细胞为核供体细胞,卵母细胞为核受体细胞进行核移植,分析比较了不同蔗糖浓度对小鼠卵母细胞去核时间、去核率及重构胚体外发育的影响,并探讨了供体细胞的保存温度和融合温度对重构胚体外发育的影响.结果表明,各蔗糖浓度组的去核率均达到100%,但3%、4... 相似文献
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Min Jung Kim Geon A Kim Young Kwang Jo Yoo Bin Choi Byeong Chun Lee 《Animal Science Journal》2016,87(1):21-26
The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies. 相似文献
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This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or β-mercaptoethanol (β-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 µM CYS or 100 µM β-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. β-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos. 相似文献
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In-Sun Hwang Hyo-Kyung Bae Hee-Tae Cheong 《Journal of veterinary science (Suw?n-si, Korea)》2013,14(3):235-240
The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels. 相似文献
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Yongjin Lee Joohyeong Lee Sang-Hwan Hyun Geun-Shik Lee Eunsong Lee 《Journal of veterinary science (Suw?n-si, Korea)》2022,23(2)
BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation. 相似文献
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为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。 相似文献
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To obtain data concerning the survival of embryos and calves derived from somatic cell nuclear transfer (SCNT) in Japan, a nationwide survey was carried out in April, 2009. As a result, data concerning 3264 embryo transfers (ETs) with SCNT embryos which produced 301 calves were accumulated and their survival was analyzed. The present survey revealed that survival rates of transferred bovine embryos and produced calves derived from SCNT had not improved over a decade (1998–2007). A remarkable feature of the pregnancies with SCNT embryos was a high incidence of spontaneous abortions. When the decade was divided by the occurrence of bovine spongiform encephalopathy (BSE) in 2001, significant decreases in the ‘after BSE’ period (2002–2007) were observed in the percentages of calves born (P < 0.01), calves living at birth (P < 0.05), calves living for 24 h (P < 0.05) and 6 months (P < 0.01). Abortions that occurred during 61–99 days after ETs were significantly increased (P < 0.01) in the ‘after BSE’ period. Certain kinds of regeneration that occurred in oocytes during the 15–20 h of storage of bovine ovaries at 10–15°C as a part of BSE inspection might have had some negative effects on SCNT embryos when these oocytes were used as recipients of SCNT. 相似文献
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Katsuhiro OHKOSHI Seiya TAKAHASHI Shin-ichiro KOYAMA Satoshi AKAGI Noritaka ADACHI Tadashi FURUSAWA Jun-ichiro FUJIMOTO Yoshiaki IZAIKE Tomoyuki TOKUNAGA 《Animal Science Journal》2003,74(4):269-276
In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO2, 5% O2, 90% N2 for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%). 相似文献
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Lihua Cao Xiaoli Dai Shihai Huang Kaiyuan Shen Deshun Shi Xiangping Li 《Reproduction in domestic animals》2021,56(7):992-1003
Suppressor of variegation 3–9 homolog (Suv39h)1 and 2, Histone H3 lysine 9 trimethylation (H3K9me3)-specific methyltransferases, are mainly involved in regulating the dynamic changes of H3K9me3. Regulating Suv39h expression influences the early development of mice somatic cell nuclear transfer (SCNT) embryos, there are few reports concerning their features in domestic animals. The aim of the present study was to characterize the Suv39h function in early development of Debao porcine SCNT embryos. The global level of H3K9me3 and the expression profiles of Suv39h1/2 in porcine early embryos were analysed by immunohistochemistry and qRT-PCR methods, respectively. Their roles in cell proliferation and histone modification of Debao porcine foetal fibroblast cells (PFFs), and developmental competence of porcine SCNT embryos were investigated by shRNA technology. The methylation levels of H3K9me3 and the expression patterns of Suv39h1 and Suv39h2 were similar (p < .05), and both of them displayed higher levels in Debao porcine SCNT embryos compared with that in PA embryos. The global levels of H3K9me3 and the expressions of G9a, HDAC1 and DNMT1 were decreased by combined inhibition of Suv39h1 and Suv39h2 (p < .05), while the expression of HAT1 was increased (p < .05). Downregulation of Suv39h1/2 also promoted cell proliferation and resulted in a significant increase in the expression of CyclinA2, CyclinB and PCNA in PFFs (p < .05). Furthermore, the use of donor somatic nuclei which depleted H3K9me3 by inhibiting Suv39h1/2 expression markedly increased the cleavage rate, the blastocyst rate and the total cell number of blastocysts of Debao porcine SCNT embryos (p < .05). Altogether, the above results indicate that H3K9me3 levels and Suv39h1/2 expressions display similar patterns in porcine early embryo, and low levels of them are critical to cell proliferation of PFFs and early development of SCNT embryos. 相似文献
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The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 µsec (19 ± 2% vs. 77 ± 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 µsec, although this was still lower than the rate of fusion in the CCCs (33 ± 1% vs. 80 ± 2%). The rates of cleavage (57 ± 5%) and blastocyst formation (1 ± 1%) in the DCC-derived embryos did not differ from those (55 ± 6% and 3 ± 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 ± 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 ± 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts. 相似文献