In vitro embryo culture in the production of identical merino lambs by nuclear transplantation

1990 ◽  
Vol 2 (6) ◽  
pp. 619 ◽  
Author(s):  
KJ McLaughlin ◽  
L Davies ◽  
RF Seamark
Keyword(s):  
Nuclear Transplantation ◽  
Cell Stage ◽  
Fluid Medium ◽  
Stage Of Development ◽  
Simple Medium ◽  
Pregnancy Status ◽  
Oviduct Fluid ◽  
Similar Stage

This study examined the viability of embryos developed in vitro from 8- to 16-cell stage blastomeres fused with enucleated oocytes. Of 209 blastomeres recovered and subjected to manipulation and electrofusion procedures, 190 (91%) fused successfully, with 86 (45%) of those undergoing cleavage up to the 4- to 16-cell stage when cultured for 66 h in a synthetic oviduct fluid medium. The viability of the embryos was examined by transferring them to recipient ewes and determining the ewes' pregnancy status by ultrasound on Day 45. Of 86 embryos transferred, 14 developed to fetuses in 8 of the 36 recipients, including four sets of identical twins and one set of quads. In contrast, with uncultured and unmanipulated embryos, 15 fetuses developed from 19 embryos transferred at a similar stage of development. The viability of embryos derived from manipulated zygotes cultured in vitro was comparable to that previously reported for studies employing in vivo culture, indicating the potential of in vitro culture systems based on a simple medium for nuclear-transplantation embryos.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

Alteration of extracellular cation concentrations and ratios in culture medium does not affect first cleavage division of hamster zygotes in vitro nor overcome the 'two-cell block'

1989 ◽  
Vol 1 (3) ◽  
pp. 231 ◽  
Author(s):  
BD Bavister ◽  
M Golden
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Cell Block ◽  
Cleavage Division ◽  
Cell Stage ◽  
Wide Range ◽  
Standard Culture ◽  
Simple Medium

In vivo fertilized hamster one-cell eggs (embryos) were cultured in a simple medium that was modified to provide a wide range of concentrations and ratios of the four major cation components (sodium, potassium, calcium and magnesium) while maintaining total osmotic pressure at 290 +/- 5 mosm. Embryos were cultured in these media to find the optimum cation concentrations for supporting the first cleavage division in vitro and to determine if physiologically abnormal cation concentrations and/or ratios in standard culture media could account for the 'two-cell block' to development in vitro in this species. Despite using a broad range of ratios for sodium:potassium (from 45:1 to 5:1) and for calcium:magnesium (from 17:1 to 1:1), there were no significant differences in the proportions of fertilized eggs that underwent the first cleavage division (approx. 60-80% across all treatments), and none of the two-cell embryos underwent further cleavage during extended culture. These data demonstrate that the first cleavage division of hamster embryos in vitro is insensitive to extracellular concentrations and ratios of the major cations, and that the non-physiological concentrations and/or ratios of these cations in the culture medium are not the primary reason for the failure of hamster zygotes to develop past the two-cell stage in vitro.

In vitro assessment of the viability of sheep zygotes after pronuclear microinjection

1990 ◽  
Vol 2 (6) ◽  
pp. 633 ◽  
Author(s):  
SK Walker ◽  
TM Heard ◽  
PJ Verma ◽  
GE Rogers ◽  
CS Bawden ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
In Vitro Culture ◽  
Fluid Medium ◽  
Pronuclear Microinjection ◽  
In Vivo Culture ◽  
Developmental Capacity ◽  
In Vitro Assessment ◽  
Oviduct Fluid

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos

2015 ◽  
Vol 27 (2) ◽  
pp. 385 ◽  
Author(s):  
Yanfang Li ◽  
Ki-Eun Park ◽  
Ryan A. Cabot
Keyword(s):  
Nuclear Import ◽  
In Vitro Assays ◽  
Cell Stage ◽  
Nuclear Trafficking ◽  
Nuclear Localisation ◽  
Cellular Events ◽  
Stage Of Development ◽  
Importin Α

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/β (importin α/β) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/β heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

374 SEX-DEPENDENT METABOLIC DIFFERENCES OF BOVINE PREIMPLANTATION EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 343
Author(s):  
R. G. Sturmey ◽  
P. Bermejo-Alvarez ◽  
A. Gutierrez-Adan ◽  
D. Rizos ◽  
H. J. Leese ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cellular Damage ◽  
Preimplantation Embryos ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Metabolic Marker

Sex-dependent differences in mammalian embryo phenotype are apparent at the preimplantation stage of development, before the appearance of sex-specific cells. The ratio of male:female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling (AAP), a defined, non-invasive metabolic marker of developmental potential to compare the effect of sex on the metabolism of bovine preimplantation blastocysts and expanded blastocysts conceived in vivo (n = 35) or produced in vitro (n = 172). Blastocysts were incubated individually for 24 h in synthetic oviduct fluid medium plus a close-to-physiological mixture of amino acids. The depletion or appearance of 18 amino acids was measured using high-performance liquid chromatography. Blastocysts were then sexed by PCR and the outcome related to AAP. Amino acid depletion by in vitro-produced blastocysts was higher than in embryos conceived in vivo (P = 0.02). Net appearance of amino acids was higher in the medium from early blastocysts produced in vitro (P = 0.018) although this rise was lost at the expanded stage. There were marked differences in the amino acid profiles of male and female embryos produced in vitro: female embryos exhibited significantly increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited gender-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced blastocysts exhibited differences in 2 out of 18 amino acids. These differences had disappeared by the expanded blastocyst stage. Our experiments reveal striking differences in the metabolism of preimplantation embryos conceived in vivo and in vitro, some of which, particularly in the case of the in vitro-produced embryos, are dependent on embryo sex. Moreover, in vivo-derived embryos tend to have a reduced metabolism consistent with the Quiet Embryo Hypothesis, which proposes that higher quality embryos have less molecular and cellular damage than those of a lower quality and thus have a reduced need to take up nutrients for repair processes. Supported by a Wellcome-VIP/University of York Fellowship to RGS.

142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 175
Author(s):  
E. M. Razza ◽  
I. P. Emanuelli ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Aggregation Rate ◽  
Stage Of Development

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

scholarly journals 252A COMPARATIVE EXPRESSION ANALYSIS OF GENES IN PREIMPLANTATION DEVELOPMENTAL STAGES OF BOVINE EMBRYOS PRODUCED IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
D. Tesfaye ◽  
K. Wimmers ◽  
M. Gilles ◽  
S. Ponsuksili ◽  
K. Schellander
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
The Novel ◽  
Cell Stage ◽  
Novel Transcript ◽  
Stage Of Development ◽  
Significant Difference

A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P&lt;0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P&lt;0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P&lt;0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P&lt;0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.

Glucose utilization by sheep embryos derived in vivo and in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 571 ◽  
Author(s):  
JG Thompson ◽  
AC Simpson ◽  
PA Pugh ◽  
RW Wright ◽  
HR Tervit
Keyword(s):  
Glucose Oxidation ◽  
Glucose Utilization ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stage ◽  
Glycolytic Activity ◽  
Total Utilization ◽  
Oxidation Of Glucose

Embryos were collected from superovulated donors at various intervals from onset of oestrus, ranging from Day 1.5 to Day 6. In addition, blastocysts obtained from the culture of 1-cell embryos collected in vivo or of oocytes matured and fertilized in vitro were used to assess the effects of in vitro manipulation and culture on glucose utilization. Glycolytic activity was determined by the conversion of [5-3H]glucose to 3H2O, and oxidation of glucose was determined by the conversion of [U-14C]glucose to 14CO2. Glucose utilization increases significantly from the 8-cell stage and during compaction and blastulation. Glucose oxidation was at a relatively low level (5-12% of total utilization) compared with glycolysis. No difference was observed between the glycolytic activity of blastocysts derived from in vivo or in vitro sources. However, glucose oxidation was lower (P less than 0.05) in blastocysts derived from the culture of 1-cell embryos or from oocytes matured and fertilized in vitro. Exogenous tricarboxylic acid cycle substrates (i.e. pyruvate and lactate supplied in the medium) affected the level of glucose oxidation.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

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