236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 197
Author(s):  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Oocyte Activation ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Electrical Pulses ◽  
Blastocyst Rate ◽  
Activation Rates

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer. Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses

1999 ◽  
Vol 11 (8) ◽  
pp. 457 ◽  
Author(s):  
Christopher G. Grupen ◽  
Paul J. Verma ◽  
Zhong Tao Du ◽  
Stephen M. McIlfatrick ◽  
Rodney J. Ashman ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Single Treatment ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
Electrical Pulses ◽  
Number Of Cells

The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm–1 DC pulses of 60 s duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.

scholarly journals 273 OPTIMIZATION OF PROTOCOLS FOR ACTIVATION OF GOAT OOCYTES WITH IONOMYCIN IN COMBINATION WITH 6-DIMETHYLAMINOPURINE

2005 ◽  
Vol 17 (2) ◽  
pp. 286
Author(s):  
J.-H. Tan ◽  
G.-C. Lan ◽  
Z.-L. Chang ◽  
D. Han ◽  
Z.-B. Han ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Cumulus Cell ◽  
Laser Scanning Confocal Microscopy ◽  
Oocyte Activation ◽  
Scanning Confocal Microscopy ◽  
Multiple Comparison Test ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Activation Rates

The protocol of ionomycin in combination with 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstructed embryos of different species. Since numerous abnormalities and impaired development have been observed when oocytes are activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation kinetics and parthenogenetic development of goat oocytes were examined in this study. When goat oocytes matured in vitro in TCM-199 were treated with 5 M ionomycin in PBS for different periods before exposure to 6-DMAP in CR1aa, the activation rate obtained with ionomycin treatment for 1 min (95.2%) was significantly (P < 0.05, Duncan multiple comparison test) higher than with ionomycin treatments for 3, 5, 7, or 9 min. When oocytes were treated with different concentrations of ionomycin for 1 min before exposure to 6-DMAP, activation rates obtained with 0.625, 1.25, 2.5, 5, 10, and 20 M ionomycin (87–95%) did not differ significantly but were significantly higher than that achieved with 0.3125 M ionomycin. Progressive reduction of time for 6-DMAP exposure showed that the duration of 6-DMAP treatment can be reduced to 1 h from the 2nd up to the 4th hour after ionomycin treatment, to produce activation rates greater than 85%. When oocytes were treated with different concentrations of 6-DMAP for the 3rd hour (atotal of 1 h, 3 h after the exposure to ionomycin), activation rates with 4 and 2 mM 6-DMAP (>90%) were significantly higher than those with 1and 0.5 mM. Therefore, the best protocol for goat oocyte activation would be a 1-min exposure to 2.5 M ionomycin followed by 2 mM 6-DMAP treatment for the 3rd hour. When oocytes matured in vitro for different times were stimulated with the best protocol, activation rates of the 27-, 30-, and 33-h oocytes (85, 85, and 91%) were significantly higher than those of the 24-, 26-, and 39-h oocytes. When activated oocytes were co-cultured in CR1aa with cumulus cell monolayers, the highest rates of cleavage (92%) and morulae/blastocysts (23%) were obtained with oocytes activated by the best protocol, and any increase in the intensity of ionomycin treatment and in the duration of 6-DMAP exposure impaired the development of the parthenotes. During anaphase II, chromosomes (the dyads) did not separate into two units in oocytes that were activated by long exposure to 6-DMAP, but they did in oocytes that were activated by short or no exposure to 6-DMAP; as a result, only one pronucleus developed in most of the former but two pronuclei were formed in most of the latter cases. Laser scanning confocal microscopy showed that microtubules also behaved differently in these two groups of activated oocytes. It is therefore concluded that to obtain better activation and development, goat oocytes matured in vitro for 27–33 h should be used, and these should be activated by a 1-min exposure to 2.5 M ionomycin followed by 2 mM 6-DMAP treatment for the 3rd hour. This study was supported by the “973” Project of China Sci. Technol. Ministry (No. G200016108).

Optimisation of porcine oocyte activation following nuclear transfer

Zygote ◽  
2000 ◽  
Vol 8 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Tao Tao ◽  
Zoltán Macháty ◽  
Lalantha R. Abeydeera ◽  
Billy N. Day ◽  
Randall S. Prather
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Free Medium ◽  
Activation Rate

Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent development of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: combined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incubation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development to the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca2+-free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca2+ measurements revealed that the Ca2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 μg/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods.

312 CHEMICAL ACTIVATION OF IN VITRO-MATURED PORCINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 263
Author(s):  
A. Bali Papp ◽  
E. Varga
Keyword(s):  
Cytochalasin B ◽  
Chemical Activation ◽  
Basic Medium ◽  
Ionophore A23187 ◽  
Strontium Chloride ◽  
Oocyte Activation ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Significant Difference

Parthenogenetic oocyte activation is important for nuclear transfer and for the understanding of cell cycle regulation of oocytes. Several chemical agents, including ethanol, cycloheximide, strontium, cytochalasin B, 6 dimethylaminopurine, CaCl2 and ionophore A23187 can induce mammalian oocyte activation in vitro. The objectives of the present study were: (1) to assess the ability of strontium chloride (S), cytochalasin B (CB), cycloheximide (CX), and 6-dimethylaminopurine (D) to induce activation and parthenogenetic development in porcine oocytes; and (2) to verify whether the combinations of treatments (SB group = strontium combined with cytochalasin; SX group = strontium combined with cycloheximide, and SD group = strontium combined with 6-dimethylaminopurine) improves activation and parthenogenetic development rates. Oocytes from slaughterhouse ovaries were matured in vitro for 42 h at 39�C, in 5% CO2 in air. The basic medium used for oocyte maturation was TCM-199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na-pyruvate, 100 �M cysteamine, 0.1 mg/mL streptomycin sulfate, 10 IU/mL pregnant mare serum gonadotropin (PMSG), and 10 IU/mL hCG (Werfft-Chemie GmbH, Vienna, Austria). Denuded MII oocytes were cultured in activation solution for 5 h. Thereafter the oocytes were cultured in NCSU37 for 6 days. At 48 h and 6 days after activation, oocytes, zygotes were fixed in acetic acid:alcohol (1/3 w/v), then stained with 0.1% (w/v) orcein in 45% (v/v) acetic acid, and evaluated under a phase contrast microscope. Each experiment was repeated four times. All data were analyzed by ANOVA, followed by Duncan's multiple range test (P < 0.05). A total of 2243 oocytes were activated in the different groups. In all groups, more than 45% of the oocytes were activated. No significant difference was observed in activation rate among SD (346/170, 49.13%), SX (302/164, 54.3%), and SB (318/182, 57.23%) groups. The activation rate for CB was significantly higher (P < 0.05) than for D or S (323/192, 59.44 � 6.84%; 366/176, 48.09 � 3.43%; and 319/183, 53.29 � 5.39%, respectively). The blastocyst rate for SX was significantly higher (P < 0.05) than that for D, SD, or SB (8.64 � 8.07%; and 0 � 0%; 0 � 0%; and 1.27 � 2.41%, respectively). In conclusion, this study suggests that chemical activation procedure is the most effective in strontium chloride combined with cycloheximide. The lowest oocyte fragmentation rates were in SX (28.40 � 1.26%) and CX (21.05 � 1.12%). This work was supported by the the Hungarian Scientific T 43131 Research Foundation and the Hungarian Science on Technology Foundation E 14/04.

In vitro development of goat parthenogenetic and somatic cell nuclear transfer embryos derived from different activation protocols

Zygote ◽  
2009 ◽  
Vol 18 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Jitong Guo ◽  
Fengjun Liu ◽  
Zekun Guo ◽  
Yu Li ◽  
Zhixing An ◽  
...  
Keyword(s):  
Low Temperature ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Subsequent Development ◽  
Oocyte Activation ◽  
Experimental Conditions ◽  
Electrical Pulses

SummaryOocyte activation is an essential step in animal cloning to allow subsequent development of the reconstructed embryos. A special activation protocol is required for different animal species. The present study investigated low temperature, electrical pulses, ethanol, ionomycin and strontium for goat oocyte activation in order to optimize the protocols. We found, as a result, effective activation and parthenogenetic development of goat oocytes that had been derived from ionomycin, strontium and electrical pulse groups. Within each group 79.3–81.6%, 2.2–78.8% and 65.5% of the oocytes cleaved and 16.2–24.8%, 0–15.6% and 11.1% of the cleaved embryos developed into blastocysts when the oocytes were activated by ionomycin combined with 6-dimethylaminopurine, strontium plus cytochalasin B and electrical pulses combined with cytochalasin B, respectively. However, low temperature and ethanol were both unable to activate goat oocytes under our experimental conditions. When ionomycin combined with 6-dimethylaminopurine and strontium plus cytochalasin B was applied to activate somatic cell nuclear transfer embryos derived from cultured cumulus, 51.0% and 72.5% of the embryos cleaved, respectively. After transfer of 4-cell embryos into recipients, one (1/19 and 1/7) of the recipients from each group was found to be pregnant as detected by ultrasound, but both of these recipients lost the embryos between 45 and 60 days of pregnancy.

The cyclin-dependent kinase inhibitor, JNJ-7706621, improves in vitro developmental competence of porcine parthenogenetic activation and somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (7) ◽  
pp. 1002 ◽  
Author(s):  
Qing Guo ◽  
Long Jin ◽  
Hai-Ying Zhu ◽  
Xiao-Xu Xing ◽  
Mei-Fu Xuan ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Kinase Inhibitor ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Cyclin Dependent Kinase ◽  
Parthenogenetic Activation ◽  
Cyclin Dependent Kinase Inhibitor

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10 µM JNJ-7706621 for 4 h compared with embryos exposed to 5 µg mL−1 cytochalasin B for 4 h (P < 0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P < 0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P < 0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P < 0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P < 0.05). In conclusion, these results reveal that exposure to 10 µM JNJ-7706621 for 4 h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.

Strategies for activating nuclear transfer oocytes

1998 ◽  
Vol 10 (8) ◽  
pp. 599 ◽  
Author(s):  
Zoltán Macháty ◽  
Randall S. Prather
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Limiting Factor ◽  
Oocyte Activation ◽  
Maturation In Vitro ◽  
A Cell ◽  
Artificial Oocyte Activation ◽  
Transfer Procedures ◽  
Artificial Activation

The technique of nuclear transfer can have enormous applications in the fields of agriculture and biomedicine. This is especially true if a cell line that has been transformed can be used as a source of nuclei for the nuclear transfer. One major aspect of the nuclear transfer procedures is that of oocyte activation. Without oocyte activation the transferred nucleus would never progress to the first interphase. It is therefore of utmost importance that the oocyte be activated in a fashion that is as normal as fertilization. The inability to obtain development after artificial activation of pig oocytes has been a limiting factor in the application of the nuclear transfer technology. Recently, a number of techniques have been developed that result in blastocyst stage embryos after oocyte maturation in vitro and artificial activation. The theories behind normal oocyte activation are reviewed as well as a number of methods of artificial oocyte activation. It is anticipated that such a review will provide the basis for the development of additional methods that are as efficient, or more efficient, at activating the unfertilized oocyte.

scholarly journals Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei

Reproduction ◽  
2001 ◽  
pp. 49-60 ◽  
Author(s):  
T Wakayama ◽  
R Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Cytochalasin D ◽  
Full Term ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Reconstructed Embryos

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.

scholarly journals Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

2007 ◽  
Vol 59 (2) ◽  
pp. 280-287 ◽  
Author(s):  
F. Perecin ◽  
S.C. Méo ◽  
C.L.V. Leal ◽  
J.M. Garcia
Keyword(s):  
Embryonic Development ◽  
Developmental Competence ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Parthenogenetic Activation ◽  
Cyclin Dependent Kinases ◽  
Activation Rate ◽  
Activation Rates ◽  
Bovine Oocytes

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3%) and initial embryonic development (35.2%) than the single ionomycin treatment (69.4% for activation and 21.9% for development); and also lead to a more uniform activation (nearly 90% single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.

scholarly journals 303PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 271
Author(s):  
C.S. Park ◽  
D.I. Jin ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
Y.J. Yi
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Chemical Activation ◽  
Formation Rate ◽  
Penicillin G ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
First Polar Body

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 3h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 3h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 1.5h, and then were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 1.5h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B plus 10μgmL−1 cycloheximide for 3h. Following activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2±0.9) compared to treatment 1, 3 and 4 (27.3±2.2, 30.4±3.8 and 30.9±3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.

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