143 TRANSCRIPTIONAL PROFILING BY HIGH-THROUGHPUT SEQUENCING OF PORCINE PRE- AND PERI-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 184 ◽  
Author(s):  
S. C. Isom ◽  
J. R. Stevens ◽  
R. Li ◽  
L. D. Spate ◽  
W. G. Spollen ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Expression Profiles ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Regulation Of Transcription

Significant embryo mortality occurs at or around the time of implantation or attachment in virtually all mammalian species studied to date, even in naturally conceived embryos. Embryos resulting from assisted reproductive technologies (ART) are even more susceptible to peri-implantation failure. Herein we describe our effort to characterise the transcriptomes of embryonic disc (ED) and trophoblast (TE) cells from porcine embryos derived from AI, IVF, parthenogenetic oocyte activation (PA) and somatic cell nuclear transfer (NT) on Days 10, 12 and 14 of gestation. The IVF, PA and somatic cell NT embryos were generated using in vitro–matured oocytes, cultured overnight in vitro and then transferred at the 1- to 2-cell stage into appropriately synchronized recipient gilts. On the appropriate collection day, embryos were flushed from the uterus and ED was separated from TE by mechanical dissection. Double-stranded cDNA from the collected samples was sequenced using the GAII platform from Illumina (San Diego, CA, USA). The resulting sequencing reads were aligned to a custom swine transcriptome database (see Isom et al. 2010). A generalized linear model was fit for each of 41 693 genomic regions, for ED and TE samples separately, accounting for embryo type, gestation day and their interaction and using total lane read count as a normalizing offset. Genes with significant embryo type differences (controlling the false discovery rate at 0.10) were subsequently tested for differences between IVF and each of AI, PA and NT. Those genes with significant post hoc differences (either up- or down-regulated compared with IVF) were characterised in terms of gene ontologies and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using a gene set enrichment test. Bone morphogenetic protein signalling was down-regulated (KEGG; P = 0.0099; adjusted to control for FDR at 0.05) in the ED of IVF embryos when compared with AI embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signalling (adj P = 0.031 for both pathways) were aberrantly regulated when compared with AI embryos. Of particular interest is the observation that expression of genes involved in chromatin modification (GO:BiologicalProcess; q-value = 0.00005) and epigenetic regulation of transcription (q = 0.00007) was very significantly disrupted in inner cell mass cells from NT embryos compared with IVF embryos. Surprisingly, no such disruption of the epigenetic machinery was observed in the TE cells from NT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various ART embryo types during peri-implantation development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from ART. Funding was received from NIH R01 RR013438 and Food for the 21st Century (RSP) and the Utah Agricultural Experiment Station (UTA00151 and UTA00560 for S. C. Isom and J. R. Stevens, respectively).

scholarly journals Transcriptional profiling by RNA-Seq of peri-attachment porcine embryos generated by a variety of assisted reproductive technologies

2013 ◽  
Vol 45 (14) ◽  
pp. 577-589 ◽  
Author(s):  
S. Clay Isom ◽  
John R. Stevens ◽  
Rongfeng Li ◽  
William G. Spollen ◽  
Lindsay Cox ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Transforming Growth Factor ◽  
Assisted Reproductive Technologies ◽  
Expression Profiles ◽  
Reproductive Technologies ◽  
Fertilization In Vitro ◽  
Oocyte Activation ◽  
Attachment Development

Substantial mortality of in vitro manipulated porcine embryos is observed during peri-attachment development. Herein we describe our efforts to characterize the transcriptomes of embryonic disc (ED) and trophectoderm (TE) cells from porcine embryos derived from in vivo fertilization, in vitro fertilization (IVF), parthenogenetic oocyte activation (PA), and somatic cell nuclear transfer (SCNT) on days 10, 12, and 14 of gestation. The IVF, PA, and SCNT embryos were generated with in vitro matured oocytes and were cultured overnight in vitro before being transferred to recipient females. Sequencing of cDNA from the resulting embryonic samples was accomplished with the Genome Analyzer IIx platform from Illumina. Reads were aligned to a custom-built swine transcriptome. A generalized linear model was fit for ED and TE samples separately, accounting for embryo type, gestation day, and their interaction. Those genes with significant differences between embryo types were characterized in terms of gene ontologies and KEGG pathways. Transforming growth factor-β signaling was downregulated in the EDs of IVF embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signaling were aberrantly regulated. Expression of genes involved in chromatin modification, gene silencing by RNA, and apoptosis was significantly disrupted in ED cells from SCNT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various embryo types during peri-attachment development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from assisted reproductive technologies.

scholarly journals Pre-Implantation Mouse Embryos Cultured In Vitro under Different Oxygen Concentrations Show Altered Ultrastructures

2020 ◽  
Vol 17 (10) ◽  
pp. 3384
Author(s):  
Manuel Belli ◽  
Paolo Rinaudo ◽  
Maria Grazia Palmerini ◽  
Elena Ruggeri ◽  
Sevastiani Antonouli ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Mouse Embryos ◽  
Human Embryos ◽  
Fertilization In Vitro ◽  
Vitro Fertilization

Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2.

53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
Y. U. Kim ◽  
D. P. Bhandari ◽  
M. S. Hossein ◽  
S. M. Park ◽  
E. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Differential Staining ◽  
In Vitro Production ◽  
Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

scholarly journals 71 DEVELOPMENTAL DELAY OF PRE-IMPLANTATION OVINE IN VITRO CULTURED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 185
Author(s):  
P. Tveden-Nyborg ◽  
T. Peura ◽  
K. Hartwich ◽  
P. Maddox-Hyttel
Keyword(s):  
Somatic Cell ◽  
Developmental Delay ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Germ Layers ◽  
Visceral Mesoderm

Despite advances in the production of somatic cell nuclear transfer (SCNT) embryos, significant embryo losses are persistent, particularly around implantation. Malformations of the placenta and in a variety of organs are seen, and have been linked to deviant epigenetic reprogramming. The aim of the present study was to examine the formation of germ layers, which are prerequisites for formation of the embryo proper and placenta, in invivo-derived (in vivo), partly in vitro-cultured (IVC), and SCNT ovine embryos. Embryos were derived as follows: In vivo embryos (n = 27) were flushed from the uterus on Days 7, 9, 11, and 13. For IVC embryos (n = 22) in vivo zygotes were flushed, followed by culture in the presence of 20% human serum, transfer to the uterus on Day 6, and flushing as in vivo embryos. SCNT embryos (n = 41) were produced by fusion of serum starved granulosa cells with enucleated oocytes, followed by activation, culture in SOF, transfer to the uterus on Day 6, and flushing as described for in vivo embryos. Recovered embryos were processed for light microscopy (LM) and transmission electron microscopy (TEM), and paraffin sections were immunohistochemically labelled for the germ layers: alpha-1-fetoprotein for potential endoderm, cytokeratin-8 for potential ectoderm, and vimentin for potential mesoderm. A consistent delay of the IVC and particularly the SCNT embryos was noted throughout all time points: On Days 7 and 9, differentiation of the inner cell mass into hypoblast and epiblast was evident in 7 out of 12 in vivo embryos, whereas this phenomenon was less prominent or absent in 9 out of 13 IVC and 13 out of 15 SCNT embryos. Furthermore, 6 of the IVC and 12 of the SCNT embryos lacked an identifiable embryonic disc. On Day 11, half of the in vivo embryos had initiated gastrulation, evidenced by localization of endoderm and mesoderm precursor cells between the hypoblast and the epiblast. This feature was noted in only a single IVC and in none of the SCNT embryos. On Day 13, all in vivo embryos had completed gastrulation including the formation of somatic and visceral mesoderm. This feature was noted in only 1 out of 3 IVC and in none of the SCNT embryos. Likewise, amniotic folds were seen in one third of the in vivo embryos at this stage, but not observed in any IVC or SCNT embryos. The immunohistochemical markers displayed the same cell lineage localization in all three groups of embryos, but a developmental delay in the IVC and in particular the SCNT embryos was evident. In conclusion, ovine IVC and SCNT embryos develop at a slower rate than in vivo embryos at least up until Day 13 of gestation.

196 EFFECTS OF DIFFERENT IN VITRO MATURATION SYSTEMS ON BOVINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
S. M. Bernal ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
A. Lucas-Hahn ◽  
B. Timmermann ◽  
...  
Keyword(s):  
Mrna Expression ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Expression Profiles ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Blastocyst Quality

Bovine oocytes and embryos have been established as a valuable model for studying human early development, specifically after assisted reproductive technologies (ART). Efforts for the improvement of ART in the last years have focused on culture media and conditions. Recently, Albuz et al. (2010) reported that the culture of bovine cumulus–oocyte complexes (COC) with cyclic adenosine 3′, 5′-monophosphate (cAMP) modulators, before and during extended in vitro maturation (IVM), improved blastocyst quality and yields in mice and cattle. In this study, we investigated the influence of an extended IVM phase in combination with cAMP modulators on blastocyst yields and quality, the effects on mRNA expression profiles and epigenetic marks. We compared these results to the standard protocol (Wrenzycki et al., 2001) used in our laboratory with oocytes from different retrieval methods. Oocytes were retrieved from slaughterhouse ovaries either by slicing or follicular aspiration. The COC were either subjected directly to IVM using the standard TCM-based protocol for 24 h (TCM24-slicing and TCM24-aspiration, respectively) or oocytes that were retrieved by aspiration were treated with forskolin and IBMX for a 2-h pre-IVM period, followed by an extended IVM phase of 30 h in TCM, supplemented with cilostamide (cAMP30-aspiration). Statistical analyses were performed using 1-way ANOVA followed by the nonparametrical Kruskal–Wallis test. Maturation rates were 79.3 ± 2.6% in TCM24-aspiration, 74.2 ± 8.8% in cAMP30-aspiration and 70.4 ± 5.1% in TCM24-slicing oocytes. Matured oocytes were fertilized in vitro with semen from a bull previously proven to be suitable for IVF. Blastocyst rates from presumptive zygotes were significantly higher (P = 0.003) in the TCM24-aspiration group (32 ± 7%) compared to TCM24-slicing (23 ± 7%) and cAMP30-aspiration (22 ± 5%). Analysis revealed that cell numbers were rather similar in the 3 experimental groups (125 ± 19, 128 ± 15 and 129 ± 9), while in vivo-produced blastocysts possessed slightly more cells (134 ± 17; P ≥ 0.05). RT-qPCR analysis of mRNA expression for a panel of genes indicative of embryo quality including DNMT3a, SLC2A8, COX2 and PCK2, showed that blastocysts derived from both aspiration protocols were similar to in vivo embryos, but were different from blastocysts resulting from the ovary-slicing protocol. Specifically, the expression profile of COX2, which is involved in pregnancy outcome and in the response to growth factors, indicates an enhanced developmental competence of aspirated oocytes. However, the transcript level of EGR1 (early growth response) was significantly higher (P = 0.009) in in vivo-derived blastocysts in comparison to all in vitro treatments. The investigation of the epigenetic status of the in vitro-derived blastocysts based on bisulfite sequencing of 2 satellite repeat sequences is currently underway. Results so far indicate that the method of obtaining the oocytes (slicing vs aspiration) for in vitro production of bovine embryos is of greater influence on blastocyst quality than IVM conditions.

scholarly journals Embryotropic actions of follistatin: paracrine and autocrine mediators of oocyte competence and embryo developmental progression

2014 ◽  
Vol 26 (1) ◽  
pp. 37 ◽  
Author(s):  
Sandeep K. Rajput ◽  
KyungBon Lee ◽  
Guo Zhenhua ◽  
Liu Di ◽  
Joseph K. Folger ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Molecular Mechanisms ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Family Impact ◽  
Oocyte Growth ◽  
Oocyte Competence ◽  
Developmental Progression

Despite several decades since the birth of the first test tube baby and the first calf derived from an in vitro-fertilised embryo, the efficiency of assisted reproductive technologies remains less than ideal. Poor oocyte competence is a major factor limiting the efficiency of in vitro embryo production. Developmental competence obtained during oocyte growth and maturation establishes the foundation for successful fertilisation and preimplantation embryonic development. Regulation of molecular and cellular events during fertilisation and embryo development is mediated, in part, by oocyte-derived factors acquired during oocyte growth and maturation and programmed by factors of follicular somatic cell origin. The available evidence supports an important intrinsic role for oocyte-derived follistatin and JY-1 proteins in mediating embryo developmental progression after fertilisation, and suggests that the paracrine and autocrine actions of oocyte-derived growth differentiation factor 9, bone morphogenetic protein 15 and follicular somatic cell-derived members of the fibroblast growth factor family impact oocyte competence and subsequent embryo developmental progression after fertilisation. An increased understanding of the molecular mechanisms mediating oocyte competence and stage-specific developmental events during early embryogenesis is crucial for further improvements in assisted reproductive technologies.

scholarly journals Factors associated with subchorionic hematoma formation in pregnancies achieved via assisted reproductive technologies

2020 ◽  
Vol 37 (2) ◽  
pp. 305-309
Author(s):  
Brady T. West ◽  
Parviz K. Kavoussi ◽  
Kate C. Odenwald ◽  
Krista London ◽  
Caitlin L. Hunn ◽  
...  
Keyword(s):  
Logistic Regression ◽  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Assisted Hatching ◽  
Factors Associated ◽  
Uterine Surgery ◽  
Multivariable Logistic Regression ◽  
Significant Difference

Abstract Purpose To determine if certain clinical and/or embryologic factors are independently associated with the increased prevalence of subchorionic hematoma (SCH) among pregnancies achieved via in vitro fertilization (IVF) with fresh embryo transfer (ET). Design Retrospective chart review. Methods In this retrospective study, data were abstracted from 210 autologous oocyte IVF clinical pregnancies that resulted from fresh ET at a single fertility center from January 2012 through December 2016. Clinical and embryology laboratory variables were analyzed as possible factors associated with the presence or absence of SCH in IVF pregnancies via bivariate associations and multivariable logistic regression analyses. Independent variables included prior uterine surgery versus no uterine surgery, peak estradiol, and progesterone levels, day 3 (n = 92) versus day 5 (n = 118) ET, and assisted hatching versus no assisted hatching. Among the day 5 ET subgroup of 118 patients, 117 had data for the variables inner cell mass (ICM) grading and trophectoderm (TE) because one day 5 ET was at the morula stage. Results We found a significant bivariate association between TE grading and SCH, where cases with TE grade “A” were significantly less likely to have SCH compared with cases with grades “B” or “C.” This significant difference remained when adjusting for the other factors considered in a multivariable logistic regression model for the probability of SCH. Conclusions The data analyzed here suggest that a less-advanced trophectoderm grade may be a potential factor that is associated with the presence of SCH in pregnancies achieved via IVF.

105 COMPARATIVE ULTRASTRUCTURE OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS (BOS INDICUS) DERIVED FROM IN VITRO FERTILIZATION, SNC, AND PARTHENOGENESIS

2013 ◽  
Vol 25 (1) ◽  
pp. 200
Author(s):  
F. Oliveira ◽  
F. Perecin ◽  
F. Meireles ◽  
J. Sangalli ◽  
Y. Watanabe ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
First Trimester ◽  
High Rate ◽  
Normal Embryo ◽  
Vitro Fertilization ◽  
Parthenogenetic Embryos

It is known that embryos produced in vitro may have structural alterations that often compromise the normal embryo development, generating a high rate of pregnancy loss. The study of these changes is of great importance because it may elucidate the cause of embryonic loss during the first trimester of pregnancy. Thus, the objective of this study was to characterize and compare ultrastructurally bovine blastocysts in the 7th day of development produced by IVF, cloning by somatic cell nuclear transfer (SCNT), and parthenogenesis. In vitro-produced embryos were derived from in vitro-matured oocytes. The somatic cell used to make cloning was fibroblasts of adult cows, and the protocol for parthenogenetic activation of the embryos was done with ionomycin-DMAP. The blastocysts derived from the different experimental groups were fixed in 2.5% glutaraldehyde and processed for transmission electron microscopy evaluation. The results showed that blastocysts derived by SNC and parthenogenesis exhibited a significantly reduced size; the inner cell mass and the blastocoel were not well defined compared with IVF embryos, indicating a less-advanced state of development. Furthermore, organelles of blastocysts derived from SCNT and parthenogenesis were fewer in number and had changes in form, when compared with IVF blastocysts. In parthenogenetic embryos there was the presence of phagosomes, suggesting a high degradation activity of cellular. Mitochondria showed the most significant changes. Although they occur in large quantities in all blastocysts, the morphology of them was impaired in SNC and parthenogenetic embryos (vacuolization, abnormal shape). Such modifications could suggest changes in mitochondria functionality, which may decrease cellular metabolic activity. Thus, we find that the D7 blastocysts derived from SCNT and parthenogenesis showed several ultrastructural differences compared with IVF embryos, with particular reference to a reduced number and morphology of embryo organelles.

405. Isolation and preliminary characterisation of putative sheep embryonic stem cells

2008 ◽  
Vol 20 (9) ◽  
pp. 85
Author(s):  
B. M. Murray ◽  
C. M. O.'Brien ◽  
J. L. Johnson ◽  
B. J. Conley ◽  
P. Bello ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Animal Health ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Well Being ◽  
Reproductive Technologies ◽  
Undifferentiated State

The establishment of true, fully characterised embryonic stem (ES) cells from livestock, (eg sheep) has yet to be reported. Such cells could make a significant impact on assisted reproductive technologies, vaccine delivery, and animal health and well being in the livestock industries. To date in sheep, there is a single report of putative ES cells which were maintained in an undifferentiated state for only 2 passages. Here we report the isolation and culture of pluripotent ES-like cells from in vivo derived, vitrified, sheep blastocysts. The inner cell mass of blastocysts were isolated by immunosurgery, cultured in Stem Cell Sciences' (SCS) novel inhibitor-based media, on a feeder layer of mouse embryonic fibroblasts (MEFs), resulting in putative ovine ES cells proliferating to at least passage 5. One cell line, BMCOV002, was established out of four thaw-recovered embryos. The colonies formed compact, near homogenous, small cell, multilayered, and well defined dome shaped masses that were morphologically similar to both mouse and human ES cell colonies. A peripheral halo of filamentous differentiated cells was detected in selected colonies from passage 3 to passage 5. The putative ovine ES-like cells were passaged by mechanical excision between days 6–7, and these colonies stained positive for alkaline phosphatase at both passage 3 and passage 5. Expression levels of genes encoding the pluripotent transcription factors OCT4, SOX2, REX1 and NANOG are shown using RT PCR in cells from passage 3. An important first step in studying the properties of ovine ES-like cells is the ability shown here to isolate and culture cells. Our attention now is focussed on maintaining these cells for some months in an undifferentiated state, and on being able to successfully cryopreserve and regenerate these cell lines.

scholarly journals Generation of monogenetic cattle by different techniques of embryonic cell and somatic cell cloning - their application to biotechnological, agricultural, nutritional, biomedical and transgenic research

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Maria Skrzyszowska ◽  
Marcin Samiec
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Embryonic Cell ◽  
Cell Cloning ◽  
Genetic Rescue ◽  
The Family ◽  
Current Article

AbstractThe development of effective approaches for not only the in vitro maturation (IVM) of heifer/cow oocytes and their extracorporeal fertilization (IVF) but also the non-surgical collection and transfer of bovine embryos has given rise to optimizing comprehensive in vitro embryo production (IVP) technology and improving other assisted reproductive technologies (ARTs), such as cattle cloning by embryo bisection, embryonic cell nuclear transfer (ECNT) and somatic cell nuclear transfer (SCNT). The primary goal of the present paper is to demonstrate the progress and achievements in the strategies utilized for embryonic cell cloning and somatic cell cloning in cattle. Moreover, the current article is focused on recognizing and identifying the suitability and reliability of bovine cloning techniques for nutritional biotechnology, agri-food and biopharmaceutical industry, biomedical and transgenic research and for the genetic rescue of endangered or extinct breeds and species of domesticated or wild-living artiodactyl mammals (even-toed ungulates) originating from the family Bovidae.

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