Microinjection of mouse phospholipase Cζ complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development

2008 ◽  
Vol 20 (8) ◽  
pp. 875 ◽  
Author(s):  
Sylvia J. Bedford-Guaus ◽  
Sook-Young Yoon ◽  
Rafael A. Fissore ◽  
Young-Ho Choi ◽  
Katrin Hinrichs
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Assisted Reproduction Technologies ◽  
Significant Difference ◽  
Developmental Rates ◽  
Advanced Stages ◽  
Consistent Method

Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Cζ (PLCζ) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca2+]i oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCζ complementary (c) RNA (1, 0.5 or 0.25 μg μL–1) into mare oocytes. The frequency of [Ca2+]i oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 μg μL–1 PLCζ cRNA (41.1 ± 5.3, 47 ± 4.0 and 55.4 ± 9.0, respectively). However, [Ca2+]i oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 μg μL–1 PLCζ cRNA (570.7 ± 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCζ (P > 0.05; range 97–100%), but the proportion of oocytes reaching advanced stages of embryonic development (>64 nuclei) was significantly lower for oocytes injected with 0.25 μg μL–1 PLCζ cRNA (3%) than for those injected with 1 μg μL–1 PLCζ cRNA (15%). Based on these results, microinjection of PLCζ may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca2+]i signalling in this species.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

Zygote ◽  
2017 ◽  
Vol 25 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Xiao-Chen Li ◽  
Qing Guo ◽  
Hai-Ying Zhu ◽  
Long Jin ◽  
Yu-Chen Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Formation Rate ◽  
Developmental Competence ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation

SummaryWe examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

234 PARTHENOGENETIC ACTIVATION OF DOMESTIC CAT OOCYTES USING STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 196 ◽  
Author(s):  
C. Wang ◽  
K. Lee ◽  
S. Koh ◽  
Z. Machaty
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Blastocyst Formation ◽  
Formidable Challenge ◽  
Significant Difference

Cloning domestic cats is useful in comparative medicine programs as it may provide insight into unique disease mechanisms and facilitate investigation of new therapeutic options. It is also believed to be beneficial for the conservation of precious animal models. However, as in many species, low birth rates after nuclear transfer remain a formidable challenge. One potential reason for the low efficiency is poor embryo development following activation of the reconstructed oocytes. The number of methods available to induce a transient increase in the oocytes' cytosolic free calcium level to stimulate development is rather limited. Although strontium has been reported to successfully activate the developmental program of mature mouse and rat oocytes, it was without effect in all other species studied. Here we investigated the effect of strontium on mature cat oocytes. Oocytes collected from the cat ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mm cysteine, 0.1 mm cysteamine, 1 IU mL–1 eCG, 2 IU mL–1 hCG, 25 ng mL–1 epidermal growth factor (EGF) for 24 h. For intracellular calcium measurements, mature oocytes were incubated in the presence of 2 µ m fura-2Am, a calcium indicator dye, and 0.02% pluronic F-127 for 40 min. Individual oocytes were transferred into calcium-free HEPES, and SrCl2 was added to the medium at a final concentration of 20 mm. Changes in the intracellular free calcium levels were then monitored using an InCyt Im2™ fluorescence imaging system (Intracellular Imaging, Cincinnati, OH, USA). Preimplantation embryonic development was also evaluated by incubating the oocytes with 20 mm SrCl2 in calcium-free HEPES medium supplemented with 7.5 µg mL–1 cytochalasin B for 6 h. Control oocytes were activated by two 20-µs-long, 100 kV cm–1 direct current pulses and incubated in the presence of 7.5 µg mL–1 cytochalasin B for 6 h. After activation, the oocytes were cultured in FOCM for 6 days. At the end of the culture period, embryonic development was recorded; the nuclear number of the embryos was also determined after staining with Hoechst 33342. Data were subjected to one-way ANOVA, and differences between treatments were analyzed using the Tukey test. We found that strontium triggered a transient rise in the intracellular free calcium concentration in all oocytes tested (N = 20). Strontium treatment also induced cleavage in 49.7% (92/185) of the oocytes, while 4.9% (9/185) of the activated oocytes developed to the blastocyst stage. In the electroporated group, cleavage frequency was 57.1% (104/182) and blastocyst formation was 8.8% (16/182). Data analysis showed that there was no significant difference between the two groups in terms of cleavage frequency and blastocyst formation. This is the first study to demonstrate that strontium can induce cytoplasmic calcium increase in cat oocytes and trigger development up to the blastocyst stage. The results also indicate that SrCl2 may be useful for oocyte activation during cat nuclear transfer. Additional studies are needed to determine whether SrCl2 can trigger development more effectively than current activation techniques.

Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1,2-propanediol

2013 ◽  
Vol 25 (4) ◽  
pp. 609 ◽  
Author(s):  
Dun-Gao Li ◽  
Yan Zhu ◽  
Feng-Ying Xing ◽  
Shan-Gang Li ◽  
Xue-Jin Chen ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Survival Rates ◽  
Slow Freezing ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Artificial Activation ◽  
Pronuclear Formation

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation–activation, embryonic development of oocytes–zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5 h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze–thawing did not affect the later development of zygotes.

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.

In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 223-227 ◽  
Author(s):  
Gang Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Negative Effects ◽  
Cell Stage ◽  
Kunming Mouse ◽  
Donor Cells ◽  
Significant Difference ◽  
Developmental Rates

SummaryIn this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2–4, 5–7 or 8–10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p > 0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p > 0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p < 0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.

scholarly journals Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa

Reproduction ◽  
2003 ◽  
pp. 489-499 ◽  
Author(s):  
SJ Bedford ◽  
M Kurokawa ◽  
K Hinrichs ◽  
RA Fissore
Keyword(s):  
Embryonic Development ◽  
Intracellular Calcium ◽  
Intracytoplasmic Sperm Injection ◽  
Animal Species ◽  
Mammalian Species ◽  
Calcium Oscillations ◽  
Large Animal ◽  
Oocyte Activation ◽  
Mouse Oocytes ◽  
Species Specific

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

Dynamic reprogramming and function of RNA N6-methyladenosine modification during porcine early embryonic development

Zygote ◽  
2021 ◽  
pp. 1-10
Author(s):  
Tong Yu ◽  
Xin Qi ◽  
Ling Zhang ◽  
Wei Ning ◽  
Di Gao ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Pluripotent Stem Cells ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Early Embryonic Development ◽  
Parthenogenetic Activation ◽  
Methylation Inhibitor ◽  
Induced Pluripotent ◽  
And Function

Summary N6-Methyladenosine (m6A) regulates oocyte-to-embryo transition and the reprogramming of somatic cells into induced pluripotent stem cells. However, the role of m6A methylation in porcine early embryonic development and its reprogramming characteristics in somatic cell nuclear transfer (SCNT) embryos are yet to be known. Here, we showed that m6A methylation was essential for normal early embryonic development and its aberrant reprogramming in SCNT embryos. We identified a persistent occurrence of m6A methylation in embryos between 1-cell to blastocyst stages and m6A levels abruptly increased during the morula-to-blastocyst transition. Cycloleucine (methylation inhibitor, 20 mM) treatment efficiently reduced m6A levels, significantly decreased the rates of 4-cell embryos and blastocysts, and disrupted normal lineage allocation. Moreover, cycloleucine treatment also led to higher levels in both apoptosis and autophagy in blastocysts. Furthermore, m6A levels in SCNT embryos at the 4-cell and 8-cell stages were significantly lower than that in parthenogenetic activation (PA) embryos, suggesting an abnormal reprogramming of m6A methylation in SCNT embryos. Correspondingly, expression levels of m6A writers (METTL3 and METTL14) and eraser (FTO) were apparently higher in SCNT 8-cell embryos compared with their PA counterparts. Taken together, these results indicated that aberrant nuclear transfer-mediated reprogramming of m6A methylation was involved in regulating porcine early embryonic development.

scholarly journals 315THE REDUCTION OF MATURATIONAL COMPETENCE BY STREPTOMYCIN DURING IN VITRO MATURATION OF GOAT FOLLICULAR OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 277 ◽  
Author(s):  
J.K. Kang ◽  
J.H. Yang ◽  
K. Naruse ◽  
C.S. Park ◽  
K.S. Min ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Maturation Medium ◽  
Significant Difference ◽  
Activation Potential

Antibiotics are commonly added to mammalian oocyte maturation media, but their effects on oocytes maturation have not been examined thoroughly. Goat follicular oocytes were used to investigate whether penicillin, streptomycin or gentamycin affect maturational competence of oocytes and subsequent parthenogenetic activation potential in vitro. Cumulus-oocyte complexes collected from a local abattoir were matured for 24h in five treatments, and matured oocytes were cultured for 48h in five treatments after parthenogenetic activation by treatment with ionomycin, followed by immediate exposure to 6-diethlaminopurine; (1) Control: TCM-199 medium with no antibiotics, (2) TCM-199 with 100IU/mL−1 penicillin (P-4687, Sigma, St. Louis, MO, USA), (3) TCM-199 with 50μgmL−1 streptomycin (S-1277, Sigma), (4) TCM-199 with 50μgmL−1 gentamycin (G-1264, Sigma) and (5) TCM-199 with both 100IUmL−1 penicillin and 50μgmL−1 streptomycin. Maturation rates at 24h post-in vitro maturation and parthenogenetic cleavage development at 48h post-activation were evaluated. Data were analyzed by ANOVA and Student’s t-test. Penicillin and gentamicin treatment groups did not affect maturation rates and percentages of cleavage to 2–4 cell stage at 48h post-chemical oocyte activation. However, when streptomycin was present in the maturation medium, the percentages of matured oocytes at 24h post-in vitro maturation of immature goat oocytes were significantly lower than those from the other groups. However, among the five treatments, there was no significant difference in cleavage rates of matured oocytes at 48h post-activation (Table 1). Therefore, streptomycin did interfere with the maturation of immature goat oocytes, but did not affect the subsequent development of matured goat oocytes. The mechanism by which streptomycin affects the maturation of goat follicular oocytes needs to be investigated further. We conclude that streptomycin in oocyte maturation medium can be detrimental during in vitro maturation of goat follicular oocytes. Table 1 Effect of antibiotics on maturational competence of goat follicular oocytes and subsequent parthenogenetic activation potential in vitro

scholarly journals Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 319-330
Author(s):  
Naoki Hirose ◽  
Sayaka Wakayama ◽  
Rei Inoue ◽  
Junya Ito ◽  
Masatoshi Ooga ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Activation Process ◽  
Oocyte Activation ◽  
Basic Study ◽  
Injection Method ◽  
Significant Difference

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

189 THE EFFECTS OF RESVERATROL ON PORCINE OOCYTES IN VITRO MATURATION AND SUBSEQUENT DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 207 ◽  
Author(s):  
S. S. Kwak ◽  
S. A. Jeong ◽  
Y. B. Jeon ◽  
S. H. Hyun
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Significant Difference

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus–oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0 μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0 μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (P < 0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0 μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0 μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0 μM resveratrol (0.4) was significantly (P < 0.05) decreased compared to other groups (control: 1.0; 0.5 μM: 0.6; and 10.0 μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0 μM resveratrol when compared with that of the control (P < 0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0 μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0 μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0 μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0 μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5 μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0 μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0 μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0 μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

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