278 EFFECTS OF LEPTIN ON IN VITRO DEVELOPMENT AND GENE EXPRESSION IN PORCINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 246 ◽  
Author(s):  
Y.-J. Jeong ◽  
J.-G. Kim ◽  
B. Mohana Kumar ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Leptin Receptor ◽  
Cell Number ◽  
The Other ◽  
Apoptotic Cells ◽  
Mrna Levels ◽  
Female Reproduction ◽  
Expression Of Genes

Recent evidence suggests that leptin is an important signal in female reproduction, including control of ovarian function (Brann et al. 2002 Steroids 67, 95-104). Leptin receptor transcripts are presented in various cell types of humans, rats, and pigs. However, the results have been contradictory on the role of leptin in embryo development. The objective of this study was to determine the effects of different concentrations of leptin during porcine oocyte maturation on subsequent embryo development and expression of genes related to leptin signal transduction or apoptosis. Porcine oocytes were matured for 44 h in TCM-199 with the addition of leptin at 0 (control), 10, 50, 100, and 200 ng/mL (leptin 10, 50, 100, and 200 groups, respectively), and subsequently fertilized with frozen thawed semen (1 � 105 sperm/mL) in mTBM. Presumptive zygotes were then cultured in NCSU-23 medium. Cleavage and blastocyst rates (in seven replicates) were assessed on Days 2 and 7 (the day of IVF was defined as Day 0), respectively. The total cell number in blastocysts was counted and the number of apoptotic cells was determined by TUNEL. Expression of genes encoding leptin receptor (LEPR), cytoplasmic transcription factor (STAT3), pro-apoptotic (Bax), and anti-apoptotic (Bcl2) regulators in blastocysts were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The percentage of MII oocytes (in four replicates) was significantly (P < 0.05) higher in 50 and 100 groups than in the others [80 (216/270) and 84% (236/280) vs. 70 (158/224), 74 (172/230), and 75% (186/248) in the 0, 10, and 200 groups, respectively]. Oocytes matured in the 50 and 100 groups showed a significantly (P < 0.05) greater proportion of cleaved embryos than those in the other groups [82 (368/448) and 84% (380/451) vs. 71 (339/476), 76 (358/470), and 74% (327/441) in 0, 10, and 200 groups, respectively]. Furthermore, the rate of blastocyst formation at Day 7 was significantly (P < 0.05) higher in the leptin 100 group than in the other groups [29% (131/451) vs. 18 (86/476), 23 (109/470), 20 (90/448), and 22% (98/441) in control and leptin 10, 50, and 200 groups, respectively]. Increased cell number and reduced proportion of apoptotic cells were observed in blastocysts originating from the oocytes matured in the presence of 100 ng/mL leptin. Depending on the concentrations used in the maturation medium, leptin increased LEPR, STAT3, and Bcl2 mRNA levels and reduced the Bax mRNA level in blastocysts. The results indicate that addition of leptin at 100 ng/mL during oocyte maturation improved embryonic development with up-regulation of LEPR, STAT3, and Bcl2 genes and suppression of the Bax gene, and thus optimizes the in vitro culture systems for porcine embryos. This work was supported by Grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
J. De la Fuente ◽  
A. Gutiérrez-Adán ◽  
P. Beltrán Breña ◽  
S. S. Pérez-Garnelo ◽  
A. T. Palasz
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Chi Square ◽  
Oh Groups ◽  
In Vitro Development ◽  
Chi Square Analysis ◽  
Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

scholarly journals Exposure of Triclosan in Porcine Oocyte Leads to Superoxide Production and Mitochondrial-Mediated Apoptosis during In Vitro Maturation

2020 ◽  
Vol 21 (9) ◽  
pp. 3050 ◽  
Author(s):  
Hyo-Jin Park ◽  
Bong-Seok Song ◽  
Jin-Woo Kim ◽  
Seul-Gi Yang ◽  
Sun-Uk Kim ◽  
...  
Keyword(s):  
Antioxidant Enzymes ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Superoxide Production ◽  
Mrna Levels ◽  
Female Reproduction ◽  
Mitochondrial Superoxide ◽  
Porcine Oocyte

While triclosan (TCS) exerts detrimental effects on female reproduction, the effect of TCS-derived toxins on porcine oocytes during in vitro maturation (IVM) is unclear. This study investigated the effects of TCS on mitochondrion-derived reactive oxygen species (ROS) production and apoptosis pathways during porcine oocyte maturation. Porcine oocytes were treated with TCS (1, 10, and 100 μM) and triphenylphosphonium chloride (Mito-TEMPO; 0.1 μM), and matured cumulus oocyte complexes (COCs) were stained with orcein, dichlorofluorescein diacetate (DCF-DA), and Mito-SOX. Proteins and mRNA levels of factors related to cumulus expansion and mitochondrion-mediated apoptosis and antioxidant enzymes were analyzed by western blotting and reverse-transcription polymerase chain reaction (RT-PCR), respectively. Meiotic maturation and cumulus cell expansion significantly decreased for COCs after TCS treatment along with an increase in mitochondrial superoxide levels at 44 h of IVM. Further, mitochondrion-related antioxidant enzymes and apoptosis markers were significantly elevated in porcine COCs following TCS-mediated oxidative damage. The protective effect of Mito-TEMPO as a specific superoxide scavenger from TCS toxin improved the maturation capacity of porcine COCs. Mito-TEMPO downregulated the mitochondrial apoptosis of TCS-exposed porcine COCs by reducing superoxide level. In conclusion, our data demonstrate that TCS mediates toxicity during porcine oocyte maturation through superoxide production and mitochondrion-mediated apoptosis.

scholarly journals Effect of various co-culture systems on embryo development in ovine

2013 ◽  
Vol 58 (No. 10) ◽  
pp. 443-452 ◽  
Author(s):  
B. Heidari ◽  
A. Shirazi ◽  
M.-M. Naderi ◽  
M.-M. Akhondi ◽  
H. Hassanpour ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Cell Number ◽  
Culture Conditions ◽  
The Other ◽  
Culture Period ◽  
Hatching Rate ◽  
Embryonic Fibroblast ◽  
One Way Anova

Considering the advent of mesenchymal stem cells (MSCs) as a new source of somatic cells in embryo co-culture system, the current study was aimed to compare in vitro embryo development using embryonic MSCs monolayer with embryonic fibroblast cells (EFCs), oviductal epithelial cells (OECs), and cell-free culture system. The IVM/IVF presumptive sheep zygotes were randomly cultured in different culture conditions as follows: (1) SOFaaBSA medium for the whole culture period (SOF, n = 371), (2) SOFaaBSA medium for the first 3 days followed by co-culturing with MSCs for the next 5 days (SOF-MSCs, n = 120), (3) co-culturing with MSCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (MSCs-SOF, n = 133), (4) co-culturing with MSCs for the whole culture period (MSCs, n = 212), (5) SOFaaBSA medium for the first 3 days followed by co-culturing with EFCs for the next 5 days (SOF-EFCs, n = 132), (6) co-culturing with EFCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (EFCs-SOF, n = 165), (7) co-culturing with EFCs for the whole culture period (EFCs, n = 236), and (8) co-culturing with OECs for the whole culture period (OECs, n = 255). One-Way ANOVA by multiple pairwise comparisons using Tukey&rsquo;s test was performed. Co-culturing in MSCs group had no superiority over EFCs and OECs groups. Though, when co-culturing with MSCs and EFCs was limited to the first 3 days of culture, the embryo development indices were improved compared to the other co-cultured groups. Considering both the hatching rate and total cell number, the application of MSCs for the first 3 days of culture (MSCs-SOF) was superior to the other co-culture and SOF groups. &nbsp;

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

Extracts of forage plants affect the developmental competence of ovine oocytes in vitro

2019 ◽  
Vol 59 (10) ◽  
pp. 1814 ◽  
Author(s):  
Anna Aryani Amir ◽  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
Zoey Durmic ◽  
Dominique Blache ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
The Other ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Reproductive Process ◽  
Forage Plants ◽  
Methanolic Extracts ◽  
Blastocyst Rate

Forage plants may contain secondary compounds that disrupt reproduction in ruminants so, as ‘duty of care’, proposed new forage species need to be tested for harmful effects on reproduction before industrial release. We evaluated the effects of Bituminaria bituminosa, Medicago sativa, Chicorium intybus, Trifolium subterraneum, Trifolium pratense, Biserrula pelecinus and Eremophila glabra, on the in vitro developmental competence of ovine oocytes. Crude methanolic extracts of each plant were added to the medium (final concentrations: 0, 50 or 100 μg dry extract per mL) used for in vitro maturation of cumulus-oocyte complexes derived from abattoir-sourced adult ewe ovaries. After in vitro fertilisation, we quantified cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency, and total blastocyst cell number (TCN). Extract from B. pelecinus, at 50 μg/mL concentration, increased cleavage rate at (P &lt; 0.05), and at 100 μg/mL, increased blastocyst rate and efficiency (P &lt; 0.05). The other plant extracts did not affect these measures. TCN was affected by stage of development and treatment, but not by the interaction between stage and treatment. Within treatments, TCN was increased by C. intybus (at both 50 and 100 μg/mL) but decreased by M. sativa (at both 50 and 100 μg/mL; P &lt; 0.05). We conclude that methanolic extracts of forage plants, present during in vitro oocyte maturation, did not disrupt subsequent fertilisation and embryo development until the blastocyst stage. On the contrary, B. pelecinus appears to improve fertilisation and embryo development. Overall, these observations suggest that these plants will not disrupt in vivo oocyte maturation but further testing is still required, especially for the other stages of the reproductive process.

scholarly journals The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

2021 ◽  
Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Cumulus Cells ◽  
Cholesterol Transport ◽  
Vitro Fertilization ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

P–060 Dose- dependent mitigation of lead acetate toxicity in males on embryo development in female mice

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Dolati ◽  
M J Zamiri ◽  
A Akhlaghi ◽  
Z Jahromi
Keyword(s):  
Adverse Effects ◽  
Embryonic Development ◽  
Embryo Development ◽  
Lead Acetate ◽  
Endocrine System ◽  
Cell Number ◽  
The Other ◽  
Control Group ◽  
Male Mice ◽  
Female Mice

Abstract Study question Does quercetin (75 or 100 mg/kg BW/day) co-administration with lead acetate to male mice affects embryonic development in female mice? Summary answer The low-dose quercetin (75 mg/kg BW/day) ameliorated the adverse effects of lead acetate on mouse embryogenesis. What is known already Lead causes male infertility by impacting on endocrine system and spermatogenesis, and may exert undesirable effects on the offspring. The currently approved treatment for lead poisoning is the use of chelating agents, which form an insoluble complex with lead and shield it from biological targets; thus, reducing its toxicity. One of the main mechanisms of lead-induced toxicity is oxidative stress, and it has been reported that natural antioxidants can reduce the heavy metals toxicity. The aim of the present study was to examine the protective effects of quercetin on the toxicity induced by lead acetate on the embryogenesis in mice. Study design, size, duration Sexually mature (eight-week-old) NMRI male mice (n = 24) were randomly divided into four groups (n = 6 per group) receiving (i) distilled water (control group); (ii) lead acetate (150 mg/kg BW/day) dissolved in deionized water (LA); (iii) lead acetate (150 mg/kg BW/day) + quercetin (75 mg/kg BW/day) (LQ75); (IV) lead acetate (150 mg/kg BW/day) + quercetin (100 mg/kg BW/day) (LQ100). Treatments were applied daily as oral gavages for one cycle of the seminiferous epithelium (35 days). Participants/materials, setting, methods At the end of treatment administration, the males were joined with super-ovulated females, and the retrieved zygotes were cultured for evaluation of the embryo development (at 2-cell, 4-cell, 8-cell, and blastocyst stages), and blastocyst cell number using differential staining (propidium iodide and bisbenzimide). After incubation of capacitated sperm with oocytes, an ultraviolet light microscope was used following 3 min incubation with 25 µg⁄mL bisbenzamide solution for fertilization assessment. Main results and the role of chance Lead acetate (LA) treatment of male mice decreased the 2-cell stage compared with the control group (P &gt; 0.05). There was no difference between control and LQ75, and between LA and LQ100. The other stages of embryonic development were not significantly affected by the treatment. Overall, early embryonic development in the control and LQ75 mice were better than LQ100 and LA mice. The number of cells in the trophectoderm and inner-cell mass were not affected by treatments. However, the total blastocyst cell number in the control was higher than in the other groups; there was no significant difference between LQ100, LQ75 and LA groups. Fertilization rate was not affected by the treatments (P &lt; 0.05). Quercetin acts as a potent antioxidant at low doses, but at high doses exerts a pro-oxidant action. According to previous reports, higher concentrations of quercetin increased apoptosis and necrosis while decreasing the activities of the antioxidant enzymes. Also, it has been suggested that quercetin might disrupt the endocrine system and interfere with Sertoli cell function and sperm motility. Limitations, reasons for caution A limitation of this study is narrow dose selection; more studies are needed to determine the effective dose of quercetin in ameliorating the lead toxicity. There are also side effects of lead-quercetin chelates such as metal redistribution, essential metal loss, accumulation and persistency in intracellular sites, and peroxidation. Wider implications of the findings: Lead administration adversely impacted on the embryogenesis; on the other hand, paternal quercetin co-administration somewhat ameliorated the adverse effects of lead on mice embryogenesis. Trial registration number Not applicable

scholarly journals 28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 114
Author(s):  
Y. Du ◽  
Z. Yang ◽  
B. Lv ◽  
L. Lin ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Cell Number ◽  
Activation Method ◽  
The Other ◽  
Electrical Activation ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Vitro Development

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

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