• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.181-185
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• 2004

This study was conducted to examine whether activation treatments, source of oocytes and culture conditions affect in vitro developmental ability of caprine oocytes. Mature Korean native goats were pretreated with intravaginal CIDR for 10 days. The goats were then treated with a single intramuscular injection of 1,000 IU PMSG on Day 8 or twice daily injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR insertion for superovulation. All the goats were injected with 10 mg PGF/sub 2a/ on Day 8 and 400 IU hCG on Day 10 of CIDR. Oocytes were surgically collected by oviduct flushing(in vivo maturation) or direct follicle aspiration(in vitro maturation) through mid-ventral incision at 35 h after hCG injection. Fifteen to twenty oocytes were placed in TCM-199 medium containing 25 mM Hepes and hormones under mineral oil at 39℃ in a humudified atmosphere of 5% CO₂ in air for 22 to 24 h. After maturation, the oocytes were activated by electric stimulation or ionomycin + 6-DMAP. The activated oocytes were then cultured in M16, TCM-199 and mSOF media supplemented with proteins at 39℃ for 6 to 7 days. Activation treatments did not affect cleavage of the oocytes. The cleavage rates were 64.1% (41/64) in oocytes activated by electric stimulation and 76.5% (218/285) in oocytes activated by ionomycin + 6-DMAP. The proportion of development to blastocyst was 15.6% (34/218) in oocytes activated by ionomycin + 6-DMAP, but activation by electric stimulation did not support embryos developed beyond morula stage. There were no differences in the cleavage rates of activated oocytes experiencing in vivo (86.8%, 66/76) and in vitro maturation (69.0%, 127/184). However, the development rate to blastocyst stage was significantly (P<0.05) higher for oocytes matured in vivo (50.0%, 33/66) compared to in vitro (0.8%, 1/127). Culture conditions did not affect the cleavage of -activated oocytes. The cleavage rates were 51.6% (49/95) in M16, 64.3% (18/28) in TCM-199 and 81.0% (145/179) in mSOF, respectively. By contrast, the development rate of activated oocytes to stage was greater (P<0.05) for oocytes cultured in mSOF medium (23.4%, 34/145) than in M16 or TCM-199 (0.0%). Our results suggest that source of oocytes and culture conditions are major factors affecting in vitro development of caprine parthenogenetic oocytes.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.23-31
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• 2000

This study was conducted to investigate the effects of quiescent treatment of donor cells and activation treatment time of recipient cytoplasm on nuclear remodeling and in vitro development of somatic cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling adult skin cells were teansferred into enucleated oocytes. Nuclear transfer oocytes were activated at 30 min, 1 and 2 hrs after electrofusion. Some nuclear transfer embryos(23% to 35%) extruded a polar body, which was not affected by quiescent treatment of donor cells and activiation time of recipient cytoplasm. About 68% of nuclear transfer embryos fused with a serum starved cells has a chromatin clump, but which was not different from embryos fused with confluent(51%) and nonquiescent(47%) cells. The proportion of embryos with a single chromatin clump was sightly increased when nuclear transfer embryos were activated within 30 min after fusion(69%) compared to those were activated at 1 and 2 hrs after fusion, but there was not significantly different. Development rates to the blastocyst stage were 8.6% and 15.9% when serum starved and confluent cells were transferred, which were higher than that of control group. Developmental rate to the blastocyst stage was higher in embryos were activated within 30 min after fusion (17.3%) compared to those of embryos were activated at 1 and 2 hrs after fusion (P<0.05). From the present result, it is suggested that quiescent treatment of donor cells and activation time of recipient cytoplasm can affect the in vitro development. Quiescent plasm activation within 30 min after fusion could increase the number of embryos with a normal chromation structure, which results in increased in vitro development.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.231-238
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• 1999

The objective of this study was to examine the effect of developmental stage and embryo age of in vitro produced bovine blastocysts after vitrification and thawing. In vitro cultured day 8 blastocysts after IVF were equilibrated 20% ethylene glycol (EG) for 3 min. and were vitrified using EFS40, which is consisted of 40% EG, 18% ficoll, 0.3M sucrose and 10% FBS added in mDPBS for 30 sec. before being plunged into $LN_2$. Also, survival in vitro was assessed by re-expansion and hatching or hatched at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) When the embryos were cultured for 8 day after IVF, 41.0% of the cleaved embryos developed to the blastocysts (early; 7.6%, expanded; 22.9%, hatching; 4.6% and hatched; 5.9%). 2) When the embryos were exposed or vitrified to the freezing solution, the re-expansion of vitrified embryos (73.3%) was significantly lower than that of control and exposed embryos (100, 97.0%) (p<0.05). But the formation rate of hatching or hatched blastocysts of vitrified embryos (66.7, 46.7%) at 48h after thawing was similar to that of exposed embryos (66.7, 39.4%) but not control (100, 100%) (p<0.01). However, in the total cell numbers of those developed hatched blastocysts, there were not significantly different among the treatment groups. 3) When the embryo survival rates by different developmental stage were examined, the re-expansion was not different among the groups $(64.5{\sim}75.6%)$. After warming 48 h, the hatching and hatched formation of early blastocysts (25.8, 9.7%) was significantly lower than those of expanded (69.7, 39.4%) and hatching blastocysts (53.3, 43.3%) (p<0.05). 4) In addition, when the expanded blastocysts at day 7, 8 and 9 were vitrified, the re-expansion of day 8 and 9 embryos was significantly lower than that of day 7 (day 7; 93.9%, day 8; 75.8% and day 9; 87.5%) (p<0.05). However, the rates of development to hatched blastocysts were no difference among the groups (day 7; 36.4%, day 8; 36.4% and day 9; 31.3%). These results suggested that in vitro produced expanded or hatching blastocysts can be efficiently cryopreserved by the two-step vitrification method using EFS40.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.123-128
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• 2005

This study examines the effects of kinds and concentrations of cryoprotectants on the survival rate of vitrification-thawed porcine oocytes, together with the effects on survival, in vitro fertilization and development of immature oocytes. 1. The developmental rate of oocytes to MII and diploid stage when the vitrification-thawed of recovered immature oocytes cultured for 0, 15, 30 and 40h were cultured for 0, 15, 30 and 40h were $56.7\%,\;53.3\%,\;63.3\%,\;65.0\%\;and\;23.3\%,\;18.3\%,\;10.0\%,\; 3.3\%$, respectively. The in vitro development to MII stage were lower than the control group $(78.2\%)$, but higher fo. diploid stage $(5.5\%)$. 2. When the vitrification of immature oocytes after being culture for 0, 15, 30 and 40 hours, the survival rate were $34.0\%,\;26.0\%,\;18.0\%\;and\;10.0\%$ respectively. This result was lower than that of the control group $(60.0\%)$. 3. When the fertilization of the vitrified immature oocytes after being culture for 0, 15, 30 and 40 hours, the in vitro fertilization rate were $60.0\%,\;54.0\%,\;48.0\%,\;38.0\%$, and developmental rates were $26.0\%,\;18.0\%,\;8.0\%,\;4.0\%$, respectively. This results were lower than the control group $(78.0\%\;and\;38.0\%)$. 4. When the fertilization of the immature oocytes after being culture for $0\~15$ hours vitrified with EDS and ETS, the fertilization and developmental rates were $50.0\%,\; 22.0\%$ and $46.0\%,\;18.0\%$, respectively. This results were lower than the control group $(74.0\%\;and\;38.0\%)$.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Animal Bioscience
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• v.34 no.4
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.12
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• pp.1681-1690
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• 2012

The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-${\alpha}$ (PFT-${\alpha}$), on preimplantation porcine in vitro fertilized (IVF) embryo development in culture. Treatment of PFT-${\alpha}$ was administered at both early (0 to 48 hpi), and later stages (48 to 168 hpi) of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3), was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-${\alpha}$, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-${\alpha}$ treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-${\alpha}$ administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-${\alpha}$ treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-${\alpha}$ may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-${\alpha}$ as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.173-179
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• 2004

This study was conducted to investigate the toxi-cological effects of different vitrification solution on development of immature porcine oocytes in vitro. Oocytes were exposed to EFS solution [40% ethylene glycol (EG) + 18% Ficoll + 0.3M sucrose], ES solution (5.5M EG + 1.0M sucrose) or GE solution [10% glycol (G) + 20% EG], and these oocytes were transferred to sucrose solution directly. Maturation rates were significantly (P<0.05) higher in the ES solution (44.5%) and control (57.6%) than in the EFS solution (38.8%) and GE solution (22.4%). No differences among three solution were found in fertilization rates. Cleavage rates was significantly (P<0.05) higher in the ES solution (47.1%) and control (65.9%) than in the EFS solution (21.9%) and GE solution (19.0%), but no difference among three solutions was found in the blastocyst formation rates. These results indicate that combination of EG and sucrose solutions had effects on development of immature porcine oocytes.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.141-148
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• 2014

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein $50{\mu}M$ for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and $50{\mu}M$ and genestein 1 and $50{\mu}M$ for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and $50{\mu}M$, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin $1{\mu}M$ for 3 hr incubation. In mitochondrial activities, addition of quercetin $50{\mu}M$ for 6 hr incubation increased mitochondrial activity but decreased at $100{\mu}M$ concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin ($1{\sim}10{\mu}M$), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin ($25{\sim}50{\mu}M$). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin $10{\mu}M$ (p<0.05). The developmental rates to morula or blastocysts at low ($0.01{\sim}1{\mu}M$) and high ($5{\sim}10{\mu}M$) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and $10{\mu}M$) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations ($0.01{\sim}10{\mu}M$).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.