61 MITOCHONDRIA DISTRIBUTION IN FERTILIZED, PARTHENOGENETIC, AND CLONED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 188
Author(s):  
G. G. Kaiser ◽  
P. J. Ross ◽  
K. Wang ◽  
J. B. Cibelli
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Petri Dish ◽  
Molecular Probes ◽  
Objective Lens ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Morula Stage ◽  
Cloned Bovine

In this study we evaluated mitochondrial distribution of individual bovine embryos after IVF, parthenogenetic activation (PG), and somatic cell nuclear transfer (SCNT). COCs obtained from slaughterhouse ovaries were matured in vitro for at least 18 h in TCM-199 supplemented with hormones, and then divided into 3 groups. SCNT and PG oocytes were stripped by vortexing in HEPES-HECM (hamster embryo culture medium) medium (HH) containing hyaluronidase and metaphase II (MII) oocytes selected by visualization of a polar body. The PG group oocytes were exposed 24h post-maturation to 5 μM ionomycin in HH for 4 min, then rinsed 3 times in HH and allocated to a 4-h culture in 2 mM DMAP in KSOM for activation. The SCNT group oocytes were included in a nuclear transfer procedure performed as previously described (Ross et al. 2006 Biotechniques 41, 741-750). Activation was performed as described for the PG group. The IVF group COCs were co-incubated for 20 h with 106 spermatozoa/mL in IVF-TALP supplemented with heparin. To label mitochondria, 1 mM MitoTracker CMXRos Red (Molecular Probes, Eugene, OR, USA) was added to HH at a final concentration of 0.3 μM. Samples were cultured for 15 min, washed in HH, placed in a glass-bottomed 35-mm Petri dish, and then observed and live photographed by using a spinning disk confocal microscope (Nikon Eclipse TE2000-E + CARV Confocal) equipped with a Cascade 512 B camera (Roper Scientific, Tucson, AZ, USA) using a Nikon 40×, 1.3 NA oil objective lens. Z series images were taken acquiring 15 focal planes at 10-μm intervals. Analysis was performed using Metamorph software. Samples were taken at pronuclear, 4 cell, and morula stages. Each sample was classified for its mitochondrial localization in pericytoplasm, cytoplasm, and perinuclear. Data was analyzed by proc glimmix (SAS, Cary, NC, USA). Significance was set at P < 0.05. A similar pericytoplasmic distribution of mitochondria for all treatments up to the 4-cell stage was observed. At the pronucelar stage, mitochondria distribution was mostly pericytoplasmic, changing to cytoplasmic at the 4-cell stage. At the morula stage there was a significantly higher number of embryos with perinuclear distribution in IVF than in PG and SCNT embryos (Table 1). Our findings demonstrate that mitochondrial reorganization differs in fertilized more-developed embryos compared with their activated counterparts. This may have implications for further embryo development, mainly after SCNT. Table 1.Mitochondria distribution in fertilized, parthenogenetic, and cloned bovine embryos

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

Nuclear transplantation using bovine primordial germ cells from male fetuses

1995 ◽  
Vol 7 (5) ◽  
pp. 1217 ◽  
Author(s):  
F Delhaise ◽  
FJ Ectors ◽  
Roover R de ◽  
F Ectors ◽  
F Dessy
Keyword(s):  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

scholarly journals Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

1995 ◽  
Vol 44 (7) ◽  
pp. 925-933 ◽  
Author(s):  
F.J. Ectors ◽  
A. Delval ◽  
L.C. Smith ◽  
K. Touati ◽  
B. Remy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Bovine Embryos ◽  
Cloned Bovine

38 EFFECTS OF TRICHOSTATIN A ON DNA METHYLATION IN CLONED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 99 ◽  
Author(s):  
D. Iwamoto ◽  
S. Kishigami ◽  
S. Taniguchi ◽  
Y. Abe ◽  
T. Matsui ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Trichostatin A ◽  
Calcium Ionophore ◽  
Serum Starvation ◽  
Image Analyzer ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Cloned Bovine

Recently, the efficiency of full-term development of somatic cloned mouse embryos was significantly increased by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have shown that TSA treatment improved the rate of development of the cloned bovine embryos to the blastocyst stage (Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 142 abst). Higher levels of DNA methylation have been shown in early cloned bovine embryos than in in vitro-fertilized (IVF) embryos (Dean et al. 2001 Proc. Nat. Acad. Sci. USA 98, 13734–13738; Santos et al. Curr. Biol. 13, 1116–1121). In this study, we examined the effects of TSA on DNA methylation levels in cloned bovine embryos by immunostaining with an antibody to 5-methyl cytosine (5-MeC). Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days before they were used as donor cells. The cells were electrofused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. Atotal of 131 cloned embryos were produced. The NT embryos were exposed to 0 (control) and 50 nmTSA from the start of activation to 48 h post-activation (hpa). They were then cultured in an mSOF medium. At 60 hpa, only embryos developed to the 8-cell stage were used for assessment of DNA methylation levels. Sixteen TSA-treated, 22 non-treated, and 19 IVF embryos were immunostained with 5-MeC antibody. For quantitative analysis of the DNA methylation levels, 5-MeC signals in the fluorescent images were determined using an image analyzer system (Aqua Cosmos; Hamamatsu Photonics, Shizuoka, Japan). The data were analyzed with Tukey-Kramer post hoc test for multiple comparisons following ANOVA. Relative levels of DNA methylation of TSA-treated cloned and IVF embryos did not differ (P > 0.05), but were lower than those of non-treated cloned embryos (P < 0.05). The results indicate that TSA treatment of cloned bovine embryos leads to a reduction of DNA methylation levels of their genome. The data suggest that the TSA treatment decreased the DNA methylation levels of cloned bovine embryos to the levels of IVF embryos, resulting in improved blastocyst development of the cloned embryos.

28 EFFECT OF TRICHOSTATIN A ON IN VITRO EMBRYO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED FROM CAT DONOR NUCLEI AND BOVINE CYTOPLASM

2013 ◽  
Vol 25 (1) ◽  
pp. 161 ◽  
Author(s):  
M. Wittayarat ◽  
Z. Namula ◽  
V. V. Luu ◽  
L. T. K. Do ◽  
Y. Sato ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Cell Stage ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Invaluable Tool ◽  
Morula Stage ◽  
Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus-cytoplasm interactions and may provide an alternative for cloning endangered animals whose oocytes are difficult to obtain. The developmental ability of iSCNT embryos decreases with increases in taxonomic distance between the donor and recipient species. The development of cat-bovine iSCNT embryos is reportedly blocked at the 8-cell stage (Thongphakdee et al. 2008 J. Reprod. Dev. 54, 142–147). Abnormal epigenetic reprogramming, such as DNA methylation or histone modifications, may cause low iSCNT efficiencies. The present study was conducted to evaluate the effect of the histone deacetylase inhibitor trichostatin A (TSA), previously used to enhance nuclear reprogramming following SCNT, on the developmental ability of cat iSCNT embryos using bovine oocytes matured in vitro. The matured bovine oocyte was enucleated by the glass needle and the domestic cat fetal fibroblast used as the donor nuclei was then placed into the perivitelline space adjacent to the plasma membrane of the oocyte. Couplets with bovine ooplasm were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 µs, respectively, using an electro cell fusion generator followed by cycloheximide treatment. Reconstructed cat-bovine embryos were treated with 0, 25, 50, and 100 nM concentrations of TSA for 24 h following fusion. The percentages of embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sin transformation before ANOVA. The TSA treatment at 50 nM contributed significantly higher rates of cleavage and blastocyst formation (n = 139; 84.3 and 4.6%, respectively) compared with untreated embryos (n = 187; 63.8 and 0%, respectively) and embryos treated with 100 nM TSA (n = 172; 71.4 and 0%, respectively; P < 0.05). Development to the morula stage of iSCNT embryos was observed in the TSA treatment groups, whereas no embryos developed beyond the 16-cell stage in the untreated group. In conclusion, our results indicate that TSA treatment for 24 h following fusion improves the development of iSCNT embryos. Specifically, 50 nM TSA treatment provides a beneficial effect on cleavage and development to the blastocyst stage of cat iSCNT embryos using bovine oocytes matured in vitro as recipients and domestic cat fibroblasts as donor nuclei.

scholarly journals 196 IN VITRO DEVELOPMENT OF RECONSTRUCTED WATER BUFFALO (BUBALUS BUBALIS) OOCYTES AFTER FETAL SKIN FIBROBLAST CELL NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 248
Author(s):  
C.R. Meena ◽  
S.K. Das
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Polar Body ◽  
Fibroblast Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Fetal Skin ◽  
Cell Stage ◽  
First Polar Body

The present study was undertaken to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to determine the developmental competence of embryos following transfer of these nuclei to in vitro-matured enucleated buffalo oocytes. Skin cells were isolated from 1–2-month-old fetuses, obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in Dulbecco's PBS) for 15–20 min. The cells were washed four times with Dulbecco's PBS and then once with RPMI-1640 medium + 10% FBS by centrifugation at 600g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5°C for 2–3 days. COCs collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 1 μg mL−1 E-β + 5 μg mL−1 FSH-P + 10 μg mL−1 LH + 10% FBS) for 22–24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body and 10–15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electrofused and incubated in the activation medium (TCM-199 + 8 μg mL−1 cytochalasin-B + 10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199 + 10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5°C for 48 h. Next, the cleaved embryos were co-cultured with buffalo oviductal cells in embryo development medium. Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electrofused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to the 2-cell stage, 3 (3.8%) reached the 4-cell stage, and 1 (1.3%) reached the 8-cell stage. In the synchronized group, out of 100 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (40%) were electrofused, activated, and cultured, out of which 4 (10%) developed to the 2-cell stage, 3 (7.5%) to the 4-cell stage, 2 (5.0%) to early morula stage, and 1 (2.5%) to blastocyst stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro-matured buffalo oocytes.

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

Roles of growth factors in the development of bovine embryos fertilized in vitro and cultured singly in a defined medium

1996 ◽  
Vol 8 (8) ◽  
pp. 1199 ◽  
Author(s):  
JM Lim ◽  
W Hansel
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Defined Medium ◽  
Basic Medium ◽  
Bovine Embryos ◽  
Tgf Beta ◽  
Cell Stage ◽  
Morula Stage ◽  
Beta 2

Bovine embryos at the 8- or 16-cell stage were cultured singly, or in groups (10-12 embryos), in the presence or absence of bovine oviduct epithelial cells (BOEC) in a defined medium which was used as a basic culture medium. A higher (P < 0.05) proportion of 8-cell embryos (48.3-50.8%) cultured singly developed beyond the 8-cell stage after the addition of platelet-derived growth factor (PDGF)-AB (1 ng mL-1) only, or with PDGF-AB + basic fibroblast growth factor (bFGF; 1 ng ml-1) + transforming growth factor (TGF)-beta 1 beta 2 (1 ng mL-1) than in basic medium alone (30.3%). In contrast, a significantly (P < 0.02) higher percentage (62.6-65.8%) of 16-cell embryos developed to the morula stage after the addition of TGF-beta 1 beta 2 only, or the addition of TGF-beta 1 beta 2 + bFGF + PDGF-AB than in basic medium alone (30.2%). These proportions were not significantly (P > 0.05) different from the proportions obtained when embryos were cultured in groups, but were significantly (P < 0.005) lower than the proportions obtained when embryos were cultured in groups on BOEC monolayers. Arachidonic acid (50 ng mL-1), beta-mercaptoethanol (10 microM) and glutathione (10-1000 microM) stimulated the development of 8-cell embryos in the presence of PDGF and TGF-beta 1 beta 2; blastocyst formation was observed for the first time in 8-cell embryos cultured singly in the presence of these embryotrophic substances (2.2-6.2%).

scholarly journals Differential response of IVP, parthenogenetic and nuclear transfer derived bovine embryos upon environmental heat stress-implications for expression of autosomal and X-linked genes

2013 ◽  
Vol 42 (1) ◽  
pp. 1-10
Author(s):  
MA Hashem ◽  
MM Hossain ◽  
A Mohammadi-Sangcheshmeh ◽  
U Cinar ◽  
F Rings ◽  
...  
Keyword(s):  
Heat Stress ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Autosomal Gene ◽  
Bovine Embryos ◽  
Cresyl Blue ◽  
Sexually Dimorphic Expression ◽  
Morula Stage ◽  
Metabolic Activities

The present study was conducted to examine the metabolic activities and expression differences of X-linked and autosomal genes upon exposure to heat stress using different types of bovine embryos. Embryos from in vitro fertilized (IVF), parthenogenetic (PA) and nuclear transfer (NT) were given heat treatment at morula stages at 40°C for 6 hrs. Treated embryos were studied for metabolic activities by quantification of G6PD-activity through Brilliant Cresyl Blue (BCB) staining and were used for X-linked and autosomal gene expression studies. Quantification of G6PD, PGK1, XIST as sex linked and SOX, BAX, OCT4, HSP70.1 as autosomal genes and GAPDH (endogenous control) mRNAs in each cDNA sample from heat treated and untreated embryos were assessed by quantitative PCR. Results of the present study indicate sexually dimorphic expression of selected X-linked and autosomal genes upon exposure to heat stress at morula stage in IVF, PA and NT embryos. Moreover, our study showed that female IVP derived embryos show highly decreased levels of XIST after heat shock. That could be one of the reasons for reduced in vitro developmental competence of female bovine embryos since XIST is the responsible gene for X-chromosome inactivation. Any disturbance would result in dysregulation of all X-Chromosomal encoded genes. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15759 Bang. J. Anim. Sci. 2013. 42 (1): 1-10

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 211-218 ◽  
Author(s):  
Jeong Tae Do ◽  
Kwon Ho Hong ◽  
Bo Yon Lee ◽  
Seung Bo Kim ◽  
Nam-Hyung Kim ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Donor Cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.

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