Alpha lipoic acid (ALA) is a biological membranes compound. As the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E, and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM ALA increased significantly (P < 0.05) and exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50 uM of ALA during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the cytoplasmic maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

Ferulic Acid (FA) is a metabolite of phenylalanine and tyrosine, a phenolic compound commonly found in fruits and vegetables. Several studies have shown that FA has various functions such as antioxidant effect, prevention of cell damage from irradiation, protection from cell damage caused by oxygen deficiency, anti-inflammatory action, anti-aging action, liver protective effect and anti-cancer action. In this study, we investigated the maturation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) of porcine oocytes by adding FA to the in vitro maturation (IVM) medium and examined subsequent embryonic developmental competence at 5% oxygen through parthenogenesis. There is no significant difference between the control group (0μM) and treatment groups (5μM, 10μM, 20μM) on maturation rates. Intracellular GSH levels in oocyte treated with 5μM of FA significantly increased (P < 0.05), and 20μM of FA revealed significant decrease (P < 0.05) in intracellular ROS levels compared with the control group. Oocytes treated with FA exhibited significantly higher cleavage rates (79.01% vs 89.19%, 92.20%, 90.89%, respectively) than the control group. Oocytes treated with 10μM showed significantly higher blastocyst formation rates (28.3% vs 40.3%, respectively) after PA than the control group. Total cell numbers in blastocyst of 10μM FA displayed significantly higher (39.4 vs 51.9, respectively) than the control group. In conclusion, these results suggested that treatment with FA during IVM improved the developmental potential of porcine embryos by increasing intracellular GSH synthesis and reducing ROS levels. Also, there was an improvement of cleavage rate, blastocyst formation and total cell numbers in blastocysts. It might be associated with Keap1-Nrf2 pathway as an antioxidant regulate pathway that plays a crucial role in determining the sensitivity of cells to oxidative damages by regulating the basal and inducible expression of enzymes which is related to detoxification and anti-oxidative effects, stress response enzymes and/or proteins and ABC transporters.

Oocytes from small antral follicles (< 3 mm in diameter; SAFO) show lower developmental competence compared to those from medium antral follicles (3-8 mm in diameter; MAFO) in pigs. This study was designed to evaluate the effect of various macromolecules such as fetal bovine serum (FBS), porcine follicular fluid (PFF), bovine serum albumin (BSA) and polyvinyl alcohol (PVA) in in vitro growth (IVG) medium on oocyte growth, maturation, and embryonic development after parthenogenesis (PA). The base medium for IVG was α-MEM supplemented with dibutyryl cyclic AMP, pyruvate, kanamycin, hormone. This medium was further supplemented with 10% FBS, 10% PFF, 0.4% BSA, or 0.1% PVA. The in vitro maturation (IVM) medium was medium-199 supplemented with 10% PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. SAFO were cultured for 2 days for IVG and then cultured for 44 h for IVM. After IVG, the mean diameter of SAFO treated with FBS, PVA, and no IVG-MAFO (114.1, 113.0, and 114.8 μm, respectively) was significantly larger (P<0.01) than that of no IVG-SAF (111.8 μm). Oocyte diameter after IVM was greater (P<0.01) in SAFO treated with FBS, BSA and PVA (112.8, 112.9 and 112.6 μm, respectively) than other groups (110.4, 109.6, and 109.8 μm for no IVG-MAFO, no IVG-SAFO and PFF, respectively). Intraoocyte GSH content was not influenced by the macromolecules in IVG medium (0.92, 0.93, 1.05, and 1.12 pixels/oocyte for FBS, PFF, BSA and PVA, respectively). The proportion of oocytes reached the metaphase II stage was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but not different from that of FBS treatment (61.5%). The cumulus expansion score of oocytes after IVG was significantly influenced (P<0.01) by the macromolecules (2.94, 2.24, 1.84, and 1.38 for PFF, FBS, PVA, and BSA treatments, respectively). Blastocyst formation of PA oocytes that were treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) during IVG was higher (P<0.05) than that of BSA-treated oocytes (20.6%) but was not significantly different from that (54.8%) of no IVG-MAFO oocytes. Our results demonstrated that growth, maturation, and embryonic development of SAFO are greatly influenced by macromolecules in IVG medium and that PFF or FBS can be replaced with a chemically defined synthetic macromolecule PVA.

In this study, to improve the in vitro development of various cells including cloned embryos, the effects that isoproterenol and melatonin have on in vitro development of porcine parthenogenetic oocytes were investigated. Parthenogenetic activation was induced with electrical stimulation, BSA and 6-DMAP treatment. 10-7 M of melatonin and isoproterenol (10-10, 10-12 and 10-14 M) were supplemented for in vitro maturation (IVM) and in vitro culture (IVC) medium, with different concentrations. When isoproterenol and melatonin were supplemented in IVM medium with different concentrations, there was no significant (P<0.05) difference of maturation rate in the treatment groups as well as in that of only melatonin. As isoproterenol and melatonin were supplemented in IVM medium with different concentrations, blastocyst rates of isoproterenol 10-12 M treatment group (37.1%) were significantly (P<0.05) higher than control group (26.0%). Isoproterenol and melatonin were supplemented in IVC medium with different concentrations, then the cleavage rate of 10-12 M isoproterenol treatment group (82.2%) was significantly (P<0.05) higher than the group that melatonin was only supplemented (70.9%). There was no difference of blastocyst rate between the treatment groups. When isoproterenol and melatonin were supplemented for IVM+IVC medium with different concentrations, the cleavage rate of 10-12 M isoproterenol treatment group (92.5%) was significantly (P<0.05) higher than the control group (82.8%) and the group that melatonin was only treated (81.6%). The blastocyst rate of 10-12 M as 45.6% was significantly (P<0.05) higher than control group (25.2%) and melatonin treatment group (31.2%). The cell number of blastocyst in 10-12 M isoproterenol treatment group 35.5±3.4 was significantly (P<0.05) highest. The results of this study showed that the development rate of IVC when both isoproterenol and melatonin were supplemented was higher than when melatonin was only supplemented. Therefore, it is concluded that isoproterenol is rather effective in the activation of melatonin. 10-7 M melatonin and 10-12 M isoproterenol were considered suitable concentration.

This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of cloned mammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In this study, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activated after 6 h regardless of cytochalasin B (CB) concentration. CB treatment (2 μg/ml) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increased during 6 h incubation. The small fragments were exhibited in the 5 μg/ml treatment of CB, but it was not found in lower concentration groups (> 2.5 μg/ml). To examine effects of SrCl2 on the adult cumulus cells, somatic cell NT oocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposure group (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride (SrCl2) groups (84.1% and 64.3%) than 5, 10 m MSrCl2. The implantation rate was not significantly difference among 5, 7.5 and 10 m MSrCl2 group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavier than IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones. (Key words : parthenogenetic oocytes, cytochalasin B, cloned mice)

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and -mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

Efficient oocyte activation is a key step for the success of nuclear transfer in cloning. Ionomycin sequentially combined with 6-DMAP is now widely used to activate normal oocytes for analytical studies of oocyte activation and to activate reconstructed oocytes after nuclear transfer. The present study investigated sources of oocytes, duration of ionomycin and 6-DMAP, laser and electric stimulation in goat oocyte activation in order to optimize the protocols. Goat ovaries were collected in individual abattoirs during the breeding season and were delivered to the laboratory within 6 h in saline with 100 IU/ml streptomycin and 0.05 mg/ml penicillin. The oocytes were denuded from the cumulus cell by pipetting with 0.2% hyaluronidase in PBS at 20~22 hr post maturation. Oocytes with the polar body were selected and assigned to four groups for parthenogenetic activation. To examine the effect of duration of ionomycin treatment, oocytes after 20~22 hr of maturation were treated with 2.5 uM ionomycin for 1 or 5 min times and then cultured in 2 mM 6-DMAP for 2 or 4 hr. The activated oocytes were cultured in mSOF at in 5%, 5% and 90% multi incubator. Cleavage and blastocyst development was observed at 48 hr and day 8 of culture , respectively. Activation rates of oocytes exposed to ionomycin for 1 min(86.4%) were significantly higher than those treated for 5 min(74.3%) duration. This indicated that 1 min ionomycin treatment was most suitable for activation of goat oocytes. The duration of 6-DMAP treat duration was in 2 mM 6-DMAP for 2 hr after 1 min exposure to 2.5 uM ionomycin. The activation rate of oocytes incubated in 6-DMAP for 2 hour(82.5%) was significantly higher than those in oocytes treated with 4 hr(75.5%).

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

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 expressions in matured oocytes, cumulus cells, and IVF-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). 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 (84.1%), but the group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (p<0.05). The groups of 0.5 and 2.0 μM showed a significant (p<0.05) increase in intracellular GSH levels compared to the control and 10.0 μM groups. Intracellular ROS level of oocytes matured with 2.0 μM resveratrol was significantly (p<0.05) decreased compared to the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rate, and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) compared to the control group. Cumulus-oocytes complex (COCs) treated with 2.0 μM resveratrol were showed lower (p<0.05) expressions of apoptosis-related genes in both matured oocytes (Bax, Bak, and Caspase-3) and cumulus cells (Bax). In IVF-derived blastocysts derived from 2.0 μM resveratrol treated oocytes had also decreased (p<0.05) expression of Bak compared to the control. In conclusion, the 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating apoptosis-related genes expression during oocyte maturation.

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at with 5% in humidified air. The matured oocytes (n = 248) were activated with 5 ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at with 5% in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was . The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was . Parthenogenetic activation, evidenced by formation of pronucleus, occurred in of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

This study was designed to investigate the effect of kinetin on in vitro development of parthenogenetic porcine oocytes exposed to demecolcine prior to activation. In vitro matured metaphase II stage oocytes were incubated in 0 or 2 g/ml demecolcine supplemented defined culture medium for 3 h and the oocytes were activated electrically. The parthenogenetic porcine embryos were then cultured in 0 or 200 M kinetin supplemented defined culture medium for 7 days. Regardless of demecolcine treatment, kinetin supplementation increased blastocyst rates significantly (7.0% versus 12.1% and 4.9% versus 8.5%; Control versus Kinetin and Demecolcine versus Kinetin + Demecolcine, respectively, p<0.05). Demecolcine treatment before activation tended to decrease blastocyst rates regardless of kinetin supplementation although it is not statistically significant. Total cell numbers in the blastocysts also tended to be elevated in embryos when supplemented with kinetin, however only the result between Kinetin and Demecolcine groups is statistically significant (37.6 7.2 versus 28.1 9.5, respectively, p<0.05). In conclusion, the present report shows that kinetin enhances developmental competence of parthenogenetic porcine embryo regardless of demecolcine pre-treatment before parthenogenetic activation when they were developed in defined culture condition.

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), follicle stimulating hormone (FSH), luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at in an atmosphere of 5% and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

본 연구에서 돼지 난포란에서 채취된 난모 세포들을 체외성숙 후 형태적으로 선별하거나 극체 방출란을 선별하여 활성화 처리 후 48시간째에 분할란을 선별할 때 배발달율이 어느정도 향상되는지를 검토하였다. 난모 세포를 48시간 성숙 배양 후 형태적 선별과 극체의 방출 유무를 검사하고, 선별된 난모 세포들을 시간 추가 배양한 후 7% ethanol로 활성화시키고 cytochalasin B에 5시간 노출 후 PZM-5 배 양액으로 7일간 배양하였으며, 배양 중