142 RELATIONSHIP BETWEEN APOPTOSIS IN BOAR SEMEN AND DNA FRAGMENTATION IN PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 188
Author(s):  
M. Bryla ◽  
M. Trzcinska ◽  
Z. Smorag
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Dna Integrity ◽  
Research Committee ◽  
Boar Semen ◽  
Live Sperm

Although apoptosis in somatic cells and in spermatocytes and spermatids in vivo is well established, the presence and significance of apoptosis in ejaculated animal sperm and its correlation with developmental competence of preimplantation embryos is still unresolved. The aim of this experiment was to study the relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos. Two ejaculates from the same boar were used in the experiment. Both fresh ejaculates were analyzed using Vybrant Apoptosis Assay Kit #4 (Molecular Probes, Inc., Eugene, OR, USA). Then one of them was diluted in Biosolwens plus extender, stored for 5 days at 15°C and analyzed using YO-PRO-1/PI assay, which detect changes in the membrane of boar spermatozoa, based on the slight increase of membrane permeability. Both fresh and stored semen were used for insemination, of superovulated gilts (8 per group). After 5.5 days of insemination embryos were flushed out of the uterus and DNA integrity of obtained embryos were analyzed. DNA fragmentation and caspase-3 activity were detected in embryos using kits, Roche Diagnostics (Mannheim, Germany) and PhiPhiLux G2D2 (Calbiochem, San Diego, CA, USA), respectively. For both the fresh and stored semen, 3 groups of sperm were observed under a fluorescence microscope. In the fresh semen, 3 and 2% of apoptotic sperm, 13 and 9% of necrotic sperm, 84 and 89% of live sperm in first and second ejaculated, respectively, were observed. In stored semen, 14% of apoptotic sperm, 27% of necrotic, and 59% of live sperm were noted. In total, 141 expanded blastocysts for DNA fragmentation were analyzed. The results are summarized in Table 1. In conclusion, apoptosis in fresh boar semen was lower than in stored semen and was correlated with the TUNEL nucleus index in blastocysts. The expression of caspase-3 was positively correlated with cells positive for TUNEL. Table 1. Relationship between apoptosis in boar semen and DNA fragmentation in porcine embryos This study was supported by Polish Research Committee, grant no. 2 P06D 024 30.

The use of butylated hydroxytoluene in cryopreservation of boar semen

2016 ◽  
Vol 12 (2) ◽  
pp. 21-28
Author(s):  
Monika Trzcińska ◽  
Magdalena Baryła
Keyword(s):  
Dna Fragmentation ◽  
Sperm Quality ◽  
Annexin V ◽  
Egg Yolk ◽  
Butylated Hydroxytoluene ◽  
Preimplantation Embryos ◽  
Progressive Motility ◽  
Boar Semen ◽  
Boar Spermatozoa

The objective of the study was to determine the effect of butylated hydroxytoluene (BHT) on the quality and fertilizing capacity of frozen-thawed (FT) boar semen. Semen from five boars (36 ejaculates) was resuspended in lactose-egg yolk-glycerol extender supplemented with 0 (control), 1.0 (R1), 1.5 (R2) or 2.0 mM BHT (R3). Sperm quality was assessed based on motility (CASA; TM: total motility; PM: progressive motility), phosphatidylserine (PS) translocation across the plasma membrane (Annexin-V-FLuos Staining Kit) and DNA fragmentation (TUNEL Assay). The FT semen was also used for intrauterine artificial insemination (AI) of synchronized gilts. The fertilizing capacity of the FT semen was assessed on the basis of the gilt insemination rate and the number of morphologically normal embryos. The quality of the preimplantation embryos was determined by observing a TUNEL-positive reaction. The highest percentage of progressive motile and viable spermatozoa was noted in extender R3 (74.8 ±4.4% and 63.7 ±5.8%), as compared with the control (38.3 ±2.8% and 36.1 ±2.6%). The addition of BHT to the extender did not increase early apoptotic changes in the frozen-thawed spermatozoa with respect to the control. Irrespective of the variant of the extender, cryopreservation and thawing did not induce fragmentation in the boar spermatozoa. The highest number of morphologically normal embryos from inseminated gilts was observed in the case of semen cryopreserved in extender supplemented with 1.5 mM BHT. No significant differences were observed in DNA fragmentation in the expanded blastocysts from gilts inseminated with FT semen cryopreserved in the extenders analysed.

330 CORRELATION BETWEEN TWO APOPTOTIC MARKERS IN FRESH BOAR SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 280 ◽  
Author(s):  
M. Trzcinska ◽  
M. Bryla ◽  
Z. Smorag
Keyword(s):  
Room Temperature ◽  
Annexin V ◽  
Molecular Probes ◽  
Green Fluorescence ◽  
Research Committee ◽  
Outer Leaflet ◽  
Chi Squared ◽  
Apoptosis Assay ◽  
Live Sperm ◽  
Boar Spermatozoa

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. It is a well-characterized mechanism that allows eukaryotes to eliminate unneeded, senescent, or aberrant cells, but the significance of apoptosis in ejaculated animal sperm is still unresolved. In this experiment, we designed 2 methods to detect early changes in the membranes of boar spermatozoa based on the slight increase in membrane permeability (Vybrant Apoptosis Assay Kit #4; Molecular Probes, Eugene, OR, USA) and on translocation of the phospholipid phosphatidyserine (PS) from the inner to the outer leaflet of the plasma membrane (Annexin-V-FLUOS Staining Kit; Roche, Mannheim, Germany). Detection of early changes in the sperm plasma is very important when designing storage protocols. Three ejaculates from 3 boars were used in the experiment. After collection and separation of the gel, the semen was analyzed using the following assays: (1) Annexin-V-FITC/PI assay: Sperm (2 � 106) were washed and diluted in 100 �L of HEPES buffer; 6 �L of Annexin-V-FITC and 4 �L of PI were added to the sample. The tubes were incubated for 15 min at room temperature in the dark. (2) YO-PRO-1/PI assay: YO-PRO-1 stock solution (1 �L) and 1 �L of PI stock solution were added to the samples. The tubes were gently mixed and incubated for 20 min at room temperature in the dark. After the incubation period, the sperm cell suspensions were analyzed under a fluorescence microscope at 40� magnification. At least 200 spermatozoa per sample were evaluated. Using the YO-PRO-1/PI assay, we observed 3 groups of sperm: apoptotic sperm showed green fluorescence (2–8%), necrotic sperm showed red and green fluorescence (9–37%), and live sperm showed no fluorescence (58–89%). Using the Annexin V-PI assay, 4 sperm subpopulations were easily detectable: apoptotic sperm showed green fluorescence (0.5–7%), early necrotic sperm showed red and green fluorescence (10–35%), necrotic sperm showed only red fluorescence (2–6%), and live sperm showed no fluorescence (57–87%). The results were compared by a chi-squared test. Significant differences (P < 0.01) in the percentage of all sperm subpopulations (apoptotic, necrotic, and live sperm) among boars and among ejaculates from the same boar were observed. We also observed a strong correlation between these 2 methods. Using the Annexin-V-FITC/PI assay, we detected more sperm subpopulations, and this method seems to be more sensitive than the YO-PRO-1/PI assay. However, these 2 methods detect changes in membrane spermatozoa but in different aspects of apoptosis, and this may also cause differences in the frequencies of apoptotic cells found by the different assays. This study was supported by the Polish Research Committee, grant no. 2 P06D 023 30.

scholarly journals Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 230
Author(s):  
Hyo-Gu Kang ◽  
Sanghoon Lee ◽  
Pil-Soo Jeong ◽  
Min Ju Kim ◽  
Soo-Hyun Park ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caspase 3 ◽  
Developmental Competence ◽  
Oxygen Species ◽  
Cell Numbers ◽  
Porcine Embryo ◽  
Apoptosis Pathways ◽  
Vitro Development

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

Anti-cancer Effect of Cyanidin-3-glucoside from Mulberry via Caspase-3 Cleavage and DNA Fragmentation in vitro and in vivo

2017 ◽  
Vol 17 (11) ◽  
Author(s):  
Eugene Cho ◽  
Eun Y. Chung ◽  
Hye-Yeon Jang ◽  
On-Yu Hong ◽  
Hee S. Chae ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
Anti Cancer

73 SUPPLEMENTATION OF FROZEN BOAR SEMEN WITH β-MERCAPTOETHANOL INCREASES THE INCIDENCE OF INTACT CELLS AFTER THAWING

2008 ◽  
Vol 20 (1) ◽  
pp. 117
Author(s):  
H. Funahashi ◽  
S. Yamaguchi ◽  
W. Fujii ◽  
T. Murakami
Keyword(s):  
Liquid Nitrogen ◽  
Dna Fragmentation ◽  
Egg Yolk ◽  
Molecular Probes ◽  
Sperm Cells ◽  
Freezing And Thawing ◽  
Intact Cells ◽  
Boar Semen ◽  
Post Hoc ◽  
Boar Spermatozoa

During the process of freezing and thawing of boar spermatozoa, a large number of the cells appear to be injured by some stresses such as osmotic forces and oxidation, causing reduced viability and penetrability. β-Mercaptoethanol (bME), a strong reducing agent, may ease oxidative stress and rescue sperm cells from those injuries. The aim of this study was to determine the effect of the presence of bME during freezing and thawing of boar spermatozoa on the viability and acrosome status of the sperm cells. Semen samples were collected from 3 boars; only samples with a high motility (more than 80%) were used for this experiment. Each sample was diluted 1:1 with modified Modena solution and kept overnight at 15�C. After centrifugation at 800g for 10 min, the diluent supernatant was removed; spermatozoa were re-suspended at 2 � 109 cells mL–1 in the first diluent (8.8% trehalose solution containing 20% egg yolk and antibiotic) supplemented with 0, 25, or 50 µm bME, and then cooled to 5�C over 2–3 h. At 5�C, semen samples were further diluted 1:1 with the second diluent (same as the first diluent + 5% glycerin + 1.48% Orvus ES Paste (Equex STM; Minitube, Verona, WI, USA)) supplemented with 0, 25, and 50 µm bME, respectively. After packaging the semen into 0.5-mL straws, it was frozen by keeping the straws 4 cm above the surface of liquid nitrogen for 15 min and then storing them in liquid nitrogen until use. After thawing at 37�C for 30 s, semen samples were re-suspended in 10 mL of BTS solution containing 1.15 mm caffeine and 4 mm Ca chloride, and incubated at 37�C under 5% CO2 in air for 90 min. Viability, DNA fragmentation, and acrosome status of spermatozoa were assessed by flow cytometry after staining with SYBR�14/PI (Molecular Probes, Inc., Eugene, OR, USA), acridine orange, and PNA/PI, respectively. Statistical analyses of data from at least 3 replicated trials were carried out by ANOVA and Fisher's protected least-squares difference (PLSD) post-hoc test. Just after thawing, no differences in viability (45.6–51.1%; P = 0.67), DNA fragmentation (0.7–0.9%; P = 0.76), and acrosome status (intact acrosome: 79.2–83.0%; P = 0.26) of the spermatozoa were observed when sperm cells were frozen and thawed in 0, 25, and 50 µm bME. After culture for 90 min, however, the incidence of spermatozoa with an intact acrosome was significantly higher (P < 0.05) when the semen was frozen and thawed in the presence of 50 µm bME (70.9%), compared with 0 (61.7%) and 25 µm bME (61.0%). Chlortetracycline (CTC) analyses were peformed to confirm that the incidence of intact spermatozoa was higher (P < 0.01) in 50 µm bME (67.6%) than that of non-supplementation controls (51.4%). These results demonstrate that supplementation of semen with 50 µm bME during freezing and thawing processes reduces acrosome damage of boar spermatozoa.

69 QUALITY OF IN VITRO PRODUCED AND CRYOPRESERVED PORCINE EMBRYOS ASSESSED BY CELL NUMBER, TUNEL-POSITIVE NUCLEI, AND CASPASE-3 ACTIVITY

2011 ◽  
Vol 23 (1) ◽  
pp. 140
Author(s):  
M. Bryla ◽  
M. Trzcinska ◽  
B. Gajda
Keyword(s):  
In Vitro Culture ◽  
Caspase 3 ◽  
Preimplantation Embryos ◽  
Lower Percentage ◽  
Cell Detection ◽  
Large White ◽  
Number Of Cells

The aim of this experiment was to investigate the quality of in vitro cultured and cryopreserved porcine expanded blastocysts from Day 5, 6, and 7 of culture. The quality of the preimplantation embryos was determined by counting the number of cells, observing a TUNEL-positive reaction (TUNEL reagent; In Situ Cell Detection kit, Roche Diagnostics, Germany) and by caspase-3 labelling (PhiPhiLuxG2D2 Kit, Calbiochem, Germany). Embryos were collected from 32 superovulated donor gilts. All were crossbreds of Polish Landrace and Large White, age 6–8 months, weighing 90–100 kg. The experiment was done on 2–4 cell embryos produced in vivo and cultured in vitro for 7 days in NCSU-23 medium until expanding blastocyst stage. The embryos of this stage were obtained on Day 5, 6, and 7 of in vitro culture and divided into two groups: control-(1) 210 nonvitrified (NV) embryos and -(2) vitrified/thawed (VT) 169 embryos. The expanded blastocysts were vitrified the open pulled straw (OPS) method (Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). The results were analyzed by Student’s t-test, and all values were significant at P ≤ 0.05. The NV group of embryos showed significant differences in the number of cells (66.5 ± 24.0 v. 54.8 ± 15.9) and in TUNEL-positive nuclei (8.8 ± 12.5 v. 16.2 ± 14.9) between Day 5 and Day 7 of culture, respectively. Analysis of VT embryos also revealed significant differences in the number of cells (65.2 ± 17.4 v. 55.5 ± 14.3) and in TUNEL-positive nuclei (25.5 ± 16.4 v. 35.8 ± 19.3) between Day 5 and Day 7 of culture, respectively. Lower percentage of NT and VT blastocysts produced on Day 5 of culture revealed caspase-3 activity (51.3 v. 64.8%) compared with embryos on Day 7 (76.8 v. 89.3%), respectively. In conclusion, blastocysts cultured in vitro for 5 days consist of a high number of nuclei, have a low incidence of TUNEL-positive nuclei, and low caspase-3 activity compared with blastocysts cultured for 6 and 7 days in all analysed groups. Our results revealed that expanding blastocysts produced on Day 5 of in vitro culture had higher ability to survive vitrification/thawing procedure. This work was supported by Grant NR 12 0036 06 from the National Centre of Research and Development, Poland.

First report on in vivo fertility trial of frozen thawed boar semen in India#

2016 ◽  
Author(s):  
S. K. Baishya ◽  
R. K. Biswas ◽  
G. Kadirvel ◽  
B. C. Deka ◽  
Suresh Kumar ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Sperm Quality ◽  
Quality Parameters ◽  
Frozen Semen ◽  
Boar Semen ◽  
The Mean ◽  
Live Sperm ◽  
Farrowing Rate ◽  
Size At Birth

The present study was conducted to evaluate the in vivo fertility of frozen thawed boar semen. Twenty ejaculates collected from six mature boars were frozen in a programmable freezer. After freezing the semen was evaluated for different sperm quality parameters. Twenty five sows were inseminated artificially utilizing frozen thawed semen. The percentage of sperm motility, live sperm, live intact acrosome, plasma membrane intact sperm, HOST-reacted sperm, live sperm with high mitochondrial membrane potential, lipid peroxidised sperm and DNA-damaged sperm of frozen semen utilised for AI were 56.25 ± 0.96, 63.75 ± 1.47, 59.88 ± 1.09, 41.08 ± 1.01, 40.31 ± 1.02, 86.23 ± 1.29, 9.28 ± 0.83 and 4.20 ± 0.29 respectively. The farrowing rate was 44.00 per cent and the mean litter size at birth was 5.91 ± 0.69. It could be concluded that the freezing and insemination protocol of boar semen used in the present study resulted in moderate fertility of frozen boar semen and it would help in further improvement and utilization of frozen boar semen for AI in India.

The Bcr-Abl Kinase Inhibitor INNO-406 Induces Autophagy and Different Modes of Cell Death Execution in Bcr-Abl-Positive Leukemias.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2951-2951
Author(s):  
Yuri Kamitsuji ◽  
Junya Kuroda ◽  
Shinya Kimura ◽  
Ken-ichiro Watanabe ◽  
Eishi Ashihara ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Caspase 3 ◽  
Kinase Inhibitor ◽  
Cellular Growth ◽  
Mitochondrial Outer Membrane ◽  
Caspase Activity ◽  
Cell Death Pathway

Abstract The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl+ cells. While they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we assessed the regulatory mechanisms for cellular survival and death of Bcr-Abl+ leukemias more precisely, using a novel Bcr-Abl tyrosine kinase inhibitor, INNO-406 (formerly NS-187) which is more selective and 25-55-fold more active than imatinib (Kimura S, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl), and in vivo CML mouse model. INNO-406 induces apoptosis in all lines examined, as were demonstrated by typical apoptotic morphology, loss of mitochondrial outer membrane potential (reduction of DiOC6 uptake), increase of cells in subG1 fraction by propidium iodide (PI) staining, DNA fragmentation and caspase-3 activation. However, when we inhibit caspase activity by zVAD-fmk (zVAD), a pan-caspase inhibitor, two modes of cell death execution were observed. In K562, KT-1 and BV173 cells treated with INNO-406, zVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic, so-called caspase-independent necrosis-like cell death (CIND). While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases’ activity, the inhibition of the cell death by zVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of alter cell death pathway which is caspase-independent apoptosis (CIA) in MYL and Ba/F3/wt bcr-abl. The propensity towards CIND or CIA in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity, that is CIND with a high apoptosome activity potential while CIA with low. Freshly isolated leukemic cell samples from Bcr-Abl+ leukemia patients also had either low or high apoptosome activity potential. Moreover, cells undergoing CIND exhibited hallmarks of autophagy (i.e. the autophagosome formation, punctate formations of LC3 and the accumulation of LC3-II isoform), suggested the participation of autophagy in response to Bcr-Abl blockade. Inhibition of autophagy with chloroquine enhanced INNO-406-induced cell death, which indicates that the autophagic response of the tumor cells is protective. While, in vivo CML model, INNO-406 treatment increased apoptotic cells regardless of the caspase-3 activation, further implicating the involvement of caspase-independent cell death regulatory pathway in vivo in primary Bcr-Abl+ leukemic cells. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics

2013 ◽  
Vol 25 (6) ◽  
pp. 935 ◽  
Author(s):  
C. Tomás ◽  
E. Blanch ◽  
A. Fazeli ◽  
E. Mocé
Keyword(s):  
Dna Fragmentation ◽  
Sperm Quality ◽  
Epithelial Cell Line ◽  
Dna Integrity ◽  
Sperm Longevity ◽  
Boar Sperm ◽  
Fragmentation Dynamics ◽  
Freezing Treatment

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze–thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P < 0.05) immediately after thawing, these differences disappeared (P > 0.05) after long-term incubation (26 h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P > 0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P < 0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary­–isthmic junction in vivo. Additionally, frozen–thawed spermatozoa can be stored at 16°C for at least 6 h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

51 DELAYED NUCLEOLOGENESIS IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 183
Author(s):  
R. S. Deshmukh ◽  
O. Østrup ◽  
E. Lemme ◽  
B. Peterson ◽  
A. Lucas-Hahn ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Condensed Chromatin ◽  
Preimplantation Embryos ◽  
Metaphase Chromosomes ◽  
Cell Stage

Nucleolus is known to be a well-suited morphological marker for embryo technologies. Failure in de novo nucleolar formation during embryonic genome activation (EGA) has been observed in many species. The aim of the present study was to investigate nuclear changes and nucleolar formation during EGA in the porcine preimplantation embryos developed in vivo and produced by somatic cell nuclear transfer (SCNT). Embryos were collected at early and late 1-cell stage, 2-, 4-, and 8-cell stage, early and late blastocyst stage, fixed in 3% glutaraldehyde for 1 h, and processed for transmission electron microscopy. In vivo embryos from 1- and 2-cell stages showed electron dense, spherical nucleolar precursor bodies (NPB) in centrally located nuclei with well-developed nuclear envelope and condensed chromatin. Two 1-cell-stage embryos, however, had represented metaphase chromosomes in the periphery. At the 4-cell stage, in vivo embryos displayed fibrillo-granular nucleoli containing all 3 functional nucleolar compartments: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC). The nuclei were centrally located, round, and had complete nuclear envelopes. The same types of nuclei and nucleoli were observed for all following stages. On the other hand, embryos produced by SCNT at early 1-cell stage showed centrally located, irregular-shaped nuclei with incomplete nuclear envelopes and condensed chromatin with large intact NPB. Exceptionally, 1 out of the 5 embryos presented a peripheral nucleus with partially condensed chromatin lacking nuclear envelope and fibrillo-granular nucleolus probably persisting from donor fibroblast. Only 2 out of 5 late-1-cell SCNT embryos showed nuclear structures. The nuclei had irregular shapes, complete nuclear membranes, and contained large NPB. At the 2- and 4-cell stages, the embryos presented central nuclei with complete nuclear envelopes. Some of the embryos showed more than one nucleus of varying shapes and sizes. The fibrillo-granular nucleoli were first observed toward the 8-cell stage. The embryos from this stage contained irregularly shaped nuclei with well-developed nuclear envelopes. The nucleoli displayed fibrillar and granular compartments in SCNT 8-cell stage embryos, but lacked the typically structured functional nucleoli observed in in vivo embryos. The absence of formation of functional nucleoli at the 4-cell stage and altered nuclear ultrastructure during the EGA in SCNT embryos, thus, may be one of the main reasons for decreased developmental competence of SCNT embryos.

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