Recycling bovine embryos for nuclear transfer

1998 ◽  
Vol 10 (8) ◽  
pp. 627 ◽  
Author(s):  
Teija T. Peura ◽  
Alan O. Trounson
Keyword(s):  
Embryonic Development ◽  
Beneficial Effect ◽  
Nuclear Transfer ◽  
First Generation ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Third Generation ◽  
Bovine Embryos ◽  
Serial Transfer ◽  
Donor Nucleus

The terms recycling and serial transfer refer to the use of cells from nuclear transfer embryos as a source of donor nuclei for a subsequent round of nuclear transfer. This approach has two benefits: improved reprogramming of the donor nucleus and an increase in absolute numbers of identical offspring. The beneficial effect of sequential exposure of donor nuclei to the early cytoplasm has been proven in several species, including amphibia, mice and cattle. Transferable embryos and live offspring have also been obtained from cattle and goat embryos produced by three and five rounds of nuclear transfer respectively. Although the limits to recycling have not been determined, embryonic development has been obtained from up to 10 cycles of nuclear transfer. Our results do not indicate differences in the embryonic developmental competence between clones from three generations in bovine embryo recycling. Although one live offspring from the second generation and pregnancies from third generation clones have been obtained, overall lower pregnancy rates and higher fetal losses than from first generation clones have been observed. Application of a novel vitrification method to nuclear transfer recycling removes many of the practical limitations of the technology.

Molecular signatures of bovine embryo developmental competence

2014 ◽  
Vol 26 (1) ◽  
pp. 22 ◽  
Author(s):  
M. Hoelker ◽  
E. Held ◽  
D. Salilew-Wondim ◽  
K. Schellander ◽  
D. Tesfaye
Keyword(s):  
Embryo Quality ◽  
Current Knowledge ◽  
Developmental Competence ◽  
Molecular Signature ◽  
Bovine Embryo ◽  
Molecular Signatures ◽  
Bovine Embryos ◽  
Morphological Criteria ◽  
Developmental Capacity ◽  
New Strategies

Assessment of the developmental capacity of early bovine embryos is still an obstacle. Therefore, the present paper reviews all current knowledge with respect to morphological criteria and environmental factors that affect embryo quality. The molecular signature of an oocyte or embryo is considered to reflect its quality and to predict its subsequent developmental capacity. Therefore, the primary aim of the present review is to provide an overview of reported correlations between molecular signatures and developmental competence. A secondary aim of this paper is to present some new strategies to enable concomitant evaluation of the molecular signatures of specific embryos and individual developmental capacity.

91 Invivo- and invitro-produced bovine embryos have different microRNA profiles after invitro individual culture

2020 ◽  
Vol 32 (2) ◽  
pp. 171
Author(s):  
A. Bridi ◽  
I. Motta ◽  
G. Andrade ◽  
M. Del Collado ◽  
A. Ávila ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Signaling Pathways ◽  
Cross Talk ◽  
Geometric Mean ◽  
Fixed Time ◽  
Bioactive Molecules ◽  
Gnrh Analogue ◽  
Bovine Embryo ◽  
Bovine Embryos

Invivo- and invitro-produced bovine embryos have different metabolic characteristics, embryonic development, and gene transcription. Additionally, pregnancy rates at 30 days (on average 51% and 34% when using fixed-time AI and invitro production, respectively) are different in beef cattle. Between Days 8 and 17 of the oestrous cycle, concurrent with embryo-maternal recognition, is when 40% of embryonic losses occur. These losses may occur due to altered embryo-maternal cross-talk. MicroRNA (miRNA) can be involved in this communication; however, its potentially regulated pathways in invivo and invitro embryos on Day 9 are unknown. Our hypothesis is that bovine embryos produced invivo and invitro contain different miRNA profiles, even after invivo bovine embryo were invitro cultured. Cows had the follicular wave synchronized and were superovulated to produce invivo or invitro bovine embryos. For the invitro group, on Day −8 of the protocol, the dominant follicles were recovered by ovum pickup, and invitro embryo production was performed to obtain embryos. For the invivo group, on Day −8, the cows were inseminated 12 and 24h after GnRH analogue application and on Day 7 after expected oestrus, uterine flushing was performed to obtain the embryos. Embryos from both groups were individually cultured for 48h. Three pools (of 5 embryos each) per group were used for reverse transcription of miRNAs from total RNA using miScript II RT Kit (Qiagen). Relative levels of 383 bovine miRNAs were determined using the geometric mean of miR-99b, RNU43 snoRNA, and Hm/Ms/Rt U1 snRNA by RT-qPCR. Differences in relative levels of miRNAs were determined by Student's t-test. A total of 210 miRNAs were detected in invivo and invitro embryos, and 13 out of 210 were differently identified between the groups. In invivo embryos, 6 miRNAs were up-regulated, whereas 7 miRNAs were up-regulated in invitro embryos. TARGETSCAN software was used to identify genes predicted as modulated by each miRNA. The top 100 genes predicted were used to identify enriched pathways according to DAVID Bioinformatics Resources. The miRNAs (miR-129, miR-132, miR-155, miR-192, miR-215, and miR-377) up-regulated in invivo embryos modulated pathways that include signaling pathways regulating pluripotency of stem cells (16 genes), TGF-β (11), hippo (10), oestrogen (8), and cell cycle (7). Moreover, miR-23a, miR-338, miR-34a, miR-491, miR-92b, miR-940, and miR-1271, which were increased in invitro embryos, regulate PI3K-Akt (17 genes), signaling pathways regulating pluripotency of stem cells (10), oestrogen (9), toll-like receptor (9), Wnt (9), and HIF-1 (7). The results demonstrate that even after 48h of invitro culture, bovine embryos produced invivo and invitro have different miRNA profiles that modulate pathways associated with embryonic development on Day 9. Furthermore, these results suggest that bioactive molecules, such as miRNAs, can modify embryo-maternal cross-talk, depending on the environment where the embryos are produced. Funding was provided by FAPESP 2017/19681-9, 2014/22887-0, and 2018/13155-6.

scholarly journals Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e108139 ◽  
Author(s):  
Maria Jesús Cánepa ◽  
Nicolás Matías Ortega ◽  
Melisa Carolina Monteleone ◽  
Nicolas Mucci ◽  
German Gustavo Kaiser ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Expression Profile ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Vitro Fertilization

The requirement for protein kinase C delta (PRKCD) during preimplantation bovine embryo development

2016 ◽  
Vol 28 (4) ◽  
pp. 482 ◽  
Author(s):  
Qi-En Yang ◽  
Manabu Ozawa ◽  
Kun Zhang ◽  
Sally E. Johnson ◽  
Alan D. Ealy
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Embryonic Development ◽  
Embryo Development ◽  
Early Embryonic Development ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Kinase C

Protein kinase C (PKC) delta (PRKCD) is a member of the novel PKC subfamily that regulates gene expression in bovine trophoblast cells. Additional functions for PRKCD in early embryonic development in cattle have not been fully explored. The objectives of this study were to describe the expression profile of PRKCD mRNA in bovine embryos and to examine its biological roles during bovine embryo development. Both PRKCD mRNA and protein are present throughout early embryo development and increases in mRNA abundance are evident at morula and blastocyst stages. Phosphorylation patterns are consistent with detection of enzymatically active PRKCD in bovine embryos. Exposure to a pharmacological inhibitor (rottlerin) during early embryonic development prevented development beyond the eight- to 16-cell stage. Treatment at or after the 16-cell stage reduced blastocyst development rates, total blastomere numbers and inner cell mass-to-trophoblast cell ratio. Exposure to the inhibitor also decreased basal interferon tau (IFNT) transcript abundance and abolished fibroblast growth factor-2 induction of IFNT expression. Furthermore, trophoblast adhesion and proliferation was compromised in hatched blastocysts. These observations provide novel insights into PRKCD mRNA expression profiles in bovine embryos and provide evidence for PRKCD-dependent regulation of embryonic development, gene expression and post-hatching events.

174 DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER EXPOSURE TO TRYPLE™ EXPRESS (RECOMBINANT TRYPSIN)

2006 ◽  
Vol 18 (2) ◽  
pp. 195 ◽  
Author(s):  
J. H. Pryor ◽  
K. R. Bondioli ◽  
S. K. Wood ◽  
M. D. Givens ◽  
C. R. Looney
Keyword(s):  
Embryonic Development ◽  
Animal Origin ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Average Cell ◽  
Treatment Groups ◽  
Cell Counts ◽  
Chi Square Analysis ◽  
Normal Embryonic Development

TrypLE™Express (1X; GIBCO, Grand Island, NY, USA) is a recombinant fungal trypsin-like protease that may provide an alternative to animal-origin trypsin for inactivation of embryo-associated virus. This experiment was designed as an embryo safety study to determine if different exposure times of TrypLE™Express on 7-day bovine in vitro-fertilized (IVF) embryos would inhibit normal embryonic development in vitro. Good quality oocytes were harvested from ovaries of slaughtered animals and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). Oocytes were fertilized (Day 0) with frozen-thawed, Percoll-separated Brangus spermatozoa in TALP-fertilization medium for 18 h. Presumptive zygotes were cultured in 0.5 mL of G1.3/G2.3 medium (Lane et al. 2003 Theriogenology 60, 407) supplemented with 8 mg/mL of Pentex BSA (Serologicals Corporation, Norcorss, GA, USA) under oil for 3 days in a 5% CO2, 5% O2, 90% N2 humidified modular incubator (Billups-Rothenberg, Inc., Del Mar, CA, USA) at 38.5°C. On Day 4, embryos were washed and moved to new culture wells containing G2.3 medium. Cleavage rates of 74% (750/1014) from three replicates produced 322 viable embryos on Day 7 which were then evenly distributed by grade and stage into three treatment groups (1-, 5-, and 10-minute exposure times to TrypLE™Express). Embryos were washed in accordance with International Embryo Transfer Society standards (Stringfellow, IETS Manual, 1998, 79–84) substituting TrypLE™Express with phenol red for the porcine-origin trypsin. Washed embryos were cultured in G2.3 medium with hatched rates assessed at 24, 48, and 72 h. Hatched embryos were fixed and stained using a modified Hoechst method (Damiani et al. 1996 Mol. Reprod. Dev. 45, 521–534) to count cells. The results are presented in Table 1. There were no significant differences (P < 0.05, chi-square analysis) between treatment groups for hatched rates or average cell counts. The results indicate that exposure to TrypLE™Express did not inhibit normal embryonic development. The efficacy of TrypLE™Express to remove or inactivate certain embryo-associated viruses is being investigated. Establishing an appropriate protocol for treatment of bovine embryos with TrypLE™Express will depend on the safety and efficacy of exposing embryos to this recombinant protease. Table 1. Comparison of hatched IVF bovine embryo rates and average cell counts after exposure to TrypLE™Express This work was funded by Invitrogen Corporation.

50 PRODUCTION OF THIRD-GENERATION CLONES OF A PIG BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 133 ◽  
Author(s):  
M. Kurome ◽  
R. Tomii ◽  
S. Ueno ◽  
K. Hiruma ◽  
H. Saito ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
First Generation ◽  
Average Weight ◽  
Donor Strain ◽  
Third Generation ◽  
Large White ◽  
Average Birth Weight ◽  
Average Size

There has been a dominant view that serial cloning, i.e., cloning of a cloned animal, is only possible for a few generations. In this study, we examined the reproduction efficiency and normality of porcine offspring generated by serial somatic cell cloning. Salivary gland progenitor (SGP) cells were collected from a 4-month-old female cloned Landrace large white Duroc (LWD) pig (first generation, G1), which had been cloned from a fibroblast, and used as nuclear donors for second-generation clones (G2). The third generation of clones (G3) was produced by nuclear transfer using SGP cells from the G2 clones. Nuclear transfer was carried out by electric cell fusion using in vitro matured oocytes as recipients. Reconstructed embryos were electroactivated 1 to 1.5 hr after nuclear transfer, cultured for 1 to 2 days, and transplanted to the oviducts of estrus-synchronized surrogate gilts. A total of 391 embryos cloned from G1 animals were transplanted to three surrogates. All of the surrogates became pregnant and gave birth to a total of 13 (3.3%) of G2 clones (including two stillbirths). The average birth weight and size of eleven live piglets were 1203.6 � 113.5 g and 27.1 � 1.2 cm, both within the standard ranges of the original donor strain (LWD). Their growths until 8 months old were comparable to those of normal piglets of the same strain. For the generation of G3 clones, transplantation of 242 G2-derived embryos to two surrogate gilts resulted in one pregnant surrogate and three G3 clones (1.2%; average weight 1196.7 � 267.1 g and average size 35.7 � 2.3 cm), including a stillbirth. These results indicate that porcine serial cloning can efficiently generate up to three generations of apparently healthy clones, when SGP cells are used as nuclear donors. This study was supported by PROBRAIN.

Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 111-116 ◽  
Author(s):  
XiuZhu Sun ◽  
ShuHui Wang ◽  
YunHai Zhang ◽  
HaiPing Wang ◽  
LiLi Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Statistical Difference ◽  
Treatment Time ◽  
Day Treatment ◽  
Contact Inhibition ◽  
Developmental Competence ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Cell Cycle Synchronization

SummaryThe purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 ± 2.70 versus 87.40 ± 5.13; 44.10 ± 8.62 versus 58.38 ± 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.

Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation

2011 ◽  
Vol 23 (4) ◽  
pp. 591 ◽  
Author(s):  
Rémi Labrecque ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Embryo Development ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Cdna Array ◽  
Developmental Competence ◽  
Time Interval ◽  
Bovine Embryo ◽  
Sperm Entry

The processes underlying the very first moments of embryonic development are still not well characterised in mammals. To better define the kinetics of events taking place following fertilisation, it would be best to have perfect synchronisation of sperm entry. With fertilisation occurring during a time interval of 6 to 12 h in the same group of fertilised oocytes, this causes a major variation in the time of activation of embryonic development. Bovine parthenogenesis could potentially result in better synchronisation and, if so, would offer a better model for studying developmental competence. In the present study, bovine oocytes were either parthenogenetically activated or fertilised and cultured in vitro for 7 days. Gene expression analysis for those two groups of embryos at early and expanded stages was performed with BlueChip, a customised 2000-cDNA array developed in our laboratory and enriched in clones from various stages of bovine embryo development. The microarray data analysis revealed that only a few genes were differentially expressed, showing the relative similarity between those two kinds of embryos. Nevertheless, the fact that we obtained a similar diversity of developmental stages with parthenotes suggests that synchronisation is more oocyte-specific than sperm entry-time related. We then analysed our data with Ingenuity pathway analysis. Networks of genes involved in blastocyst implantation but also previous stages of embryo development, like maternal-to-embryonic transition, were identified. This new information allows us to better understand the regulatory mechanisms of embryonic development associated with embryo status.

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