• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.257-262
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• 2009

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen-thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2-cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time-dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

• Development and Reproduction
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• v.21 no.2
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• pp.205-213
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• 2017

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and $100{\mu}M$) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with $50{\mu}M$ ALA were fertilized and cultured in IVC medium with ALA (25, 50 and $100{\mu}M$) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with $25{\mu}M$ ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by $50{\mu}M$ ALA treatment groups compared with control groups (p<0.05). Treatment of $25{\mu}M$ ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by $25{\mu}M$ ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.361-371
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• 2013

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

• Korean Journal of Agricultural Science
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• v.48 no.1
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• pp.163-170
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• 2021

Bacterial infections in the female reproductive tract negatively affect ovarian function, follicular development, and embryo development, leading to the eventual failure of fertilization. Moreover, bacterial lipopolysaccharides (LPS) can interfere with the immune system and reproductive system of the host animal. Therefore, this study examined the effect of LPS on the in vitro maturation (IVM) of pig oocytes. Oocytes were matured in TCM199 medium in the presence of varying concentrations of LPS (0 - 50 ㎍·mL^-1). The maturation rate, cortical granules (CGs) migration, and chromosome alignment were subsequently evaluated during the meiotic development of the oocytes. We observed a dose-dependent and significant decrease in the metaphase II (MII) rate with increasing concentrations of LPS (97.6% control [0 ㎍·mL^-1 LPS] vs. 10.4-74.9% LPS [1 - 50 ㎍·mL^-1], p < 0.05). In addition, compared to the control oocytes without LPS, higher levels of abnormal CGs distribution (18.1 - 50.0% LPS vs. 0% control), chromosome/spindle alignment (20.3 - 56.7% LPS vs. 0% control), and intracellular ROS generation were observed in oocytes matured with LPS (p < 0.05). Nitrite levels were also increased in the maturation medium derived from the oocytes matured with LPS (p < 0.05). These results indicate that LPS induces oxidative stress during IVM and affects oocyte maturation, including CGs migration and chromosome alignment of pig oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.3
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• pp.177-184
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• 2023

Objective: Reconstructed oocytes after polar body genome transfer constitute a potential therapeutic option for patients with a history of embryo fragmentation and advanced maternal age. However, the rescue of genetic material from the first polar body (PB1) through introduction into the donor cytoplasm is not yet ready for clinical application. Methods: Eighty-five oocytes were obtained following in vitro maturation (IVM) and divided into two groups: PB1 nuclear transfer (PB1NT; n=54) and control (n=31). Following enucleation and PB1 genomic transfer, PB1 fusion was assessed. Subsequently, all fused oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured in an incubator under a time-lapse monitoring system to evaluate fertilization, embryonic morphokinetic parameters, and cleavage patterns. Results: Following enucleation and fusion, 77.14% of oocytes survived, and 92.59% of polar bodies (PBs) fused. However, the normal fertilization rate was lower in the PB1NT group than in the control group (56.41% vs. 92%, p=0.002). No significant differences were observed in embryo kinetics between the groups, but a significant difference was detected in embryo developmental arrest after the four-cell stage, along with abnormal cleavage division in the PB1NT group. This was followed by significant between-group differences in the implantation potential rate and euploidy status. Most embryos in the PB1NT group had at least one abnormal cleavage division (93.3%, p=0.001). Conclusion: Fresh PB1NT oocytes successfully produced normal zygotes following PB fusion and ICSI in IVM oocytes. However, this was accompanied by low efficiency in developing into cleavage embryos, along with an increase in abnormal cleavage patterns.

• Development and Reproduction
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• v.12 no.2
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• pp.125-132
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• 2008

Melatonin (N-acetyl-5-methoxytryptamine), a major hormone of pineal gland in vertebrates, is known to be associated with regulation of the dynamic physiological functions in general and has some functions on reproduction in the ovarian follicles in particular. And its antioxidant properties as a scavenger are also reported. The aim of this study was to investigate the effect of melatonin on the in vitro maturation of mouse germinal vesicle (GV)-stage oocytes. Oocyte maturation, apoptosis, and mRNA expression of melatonin receptor were analyzed in the cumulus cell-enclosed oocytes (CEOs) cultured with melatonin for 18 h. The CEOs were obtained from 3 wk-old ICR female mice cultured in media with 0, 0.1 nM, 10 nM, or 1,000 nM melatonin for 18 h. And then the extrusion of the first polar body was assessed to evaluate the maturation rate. The apoptosis and mRNA expression of melatonin receptor (Mtnr1-a and Mtnr1-b) in cumulus cells of each group were measured by TUNEL assay, ELISA, and real time RT-PCR after in vitro maturation(IVM). The addition of melatonin in the IVM medium significantly improved nuclear maturation of the mouse GV oocytes and the highest maturation rate were obtained from the group treated with 1,000 nM melatonin. Apoptosis was not detected in IVM oocytes, but detected in cumulus cells. And cumulus cells treated with 1,000 nM melatonin exhibited significantly lower apoptosis. In the group treated with 1,000 nM melatonin, the expression of melatonin receptor mRNA was decreased in CEOs. In conclusion, melatonin has a potentially important role for regulating oocyte maturation and reduces the apoptosis of cumulus cells in vitro.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.257-265
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• 2017

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 ($0{\mu}M$) and treatment groups ($5{\mu}M$, $10{\mu}M$, $20{\mu}M$) on maturation rates. Intracellular GSH levels in oocyte treated with $5{\mu}M$ of FA significantly increased (P < 0.05), and $20{\mu}M$ 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 $10{\mu}M$ showed significantly higher blastocyst formation rates (28.3% vs 40.3%, respectively) after PA than the control group. Total cell numbers in blastocyst of $10{\mu}M$ 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.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.105-110
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection(ICSI) with epididymal spermatozoa. The ovaries were obtained from slaughtered small species dogs. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with epididymal spermatozoa. After ICSI, one group of oocytes was activated with 2.0 mM dimethylaminopurine or 7% ethanol for 5 min. and second group was not activated. The follicular oocytes were cultured in synthetic oviductal fluid(SOF) and TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. 1. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 48 hrs of incubation were significantly higher(p<0.05) after culture with 48 hrs(9/30, 30.0%) than that after culture with 24hrs(a/30, 26.7%). 2. Results of IVM showed that the percentage of oocytes reaching MII after 48 hrs of incubation were significantly higher(p<0.05) after culture with SOF media(10/30, 30.3%) than TCM-199 media (7/30, 23.3%). 3. The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(5/16, 25.0% vs 1/13, 5.0%). 4. The rates of development of cleavaged embryos to blastocyst obtained by ICSI treated sperm of fresh, epididymal and frozen-thawed epididymal were 8/18(44.43%), 5/16(31.3%), 2/14(14.3%), respectively. and these values of frozen-thawed epididymal sperm injection were lower than fresh sperm injection.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.161-166
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• 2004

This study was designed to evaluate the effects of nitric oxide scavenger (hemoglobin) and inhibitor (L-nitro-L-arginine methyl ester; L-NAME) with or without cumulus cell on the development of bovine IVM/IVF embryos. When CR/sub 1aa/ medium were supplemented with different dosage (lug/m, 5ug/m and 10ug/ml) of hemoglobin at 48hrs for in vitro culture, the proportion of embryos developing beyond morulae stage in 0, 1ug/ml and 5ug/ml with or without cumulus cell were 23.8%, 33.3 % and 26.8%, and 39.5%, 54.8% and 48.8%, respectively. There was a significantly difference the developmental rate of 1ug/ml hemoglobin intact cumulus cells to any other groups (P<0.05). On the other hand, when added to hemoglobin at 96 hrs, 1 ug/ml hemoglobin with cumulus cell group was significantly increased the percentage of developing into morulae and blastocysts to any other groups (P<0.05), and similar trend that of added at 48hrs. The overall means of the percentage of developing into morulae and blstocysts in 1ug/ml hemoglobin group was significantly increased than those of any other groups (P<0.05) and cumulus co-culture with hemoglobin was increased the in vitro developing rate of IVM/IVF embryos. In CR/sub 1aa/ medium treated with L-NAME 0, 10, 50 and 100mM, the developmental rate of morula plus blastocysts were 55.6%, 64.9%, 58.8% and 66.7%, respectively. The L-NAME did not affect the developmental rate and the cell numbers of blastocysts in all treated groups. These results indicate that hemoglobin and cumulus co-culture can increase the proportion of embryos that developed into morulae and blastocysts, but cell numbers of blastocysts were not affect in all groups.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.