• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.307-311
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• 2006

The purpose of this study was to determine effects of oxytocin and $interleukin-1{\alpha}$ on in vitro development of bovine embryo cultured with endometrial epithelial and stromal cells isolated from bovine uterus. The expressions of COX-2 mRNA in bovine endometrium were also studied. When embryos were cultured with epithelial cells, the rate of blastocysts was significantly (p<0.05) higher in embryos treated with oxytocin than that of control group. The rate of hatched blastocysts was also significantly (p<0.05) higher in embryos treated with oxytocin than those of two control groups. On the other hand, when the embryos were cultured with stromal cells, the rate of blastocysts were significantly (p<0.05) higher than those of groups treated with $IL-1{\alpha}$, oxytocin and control with stromal cells than that of control group without stromal cells. The rate of blastocysts hatched were also significantly (p<0.05) higher in group treated with $IL-1{\alpha}$ than those of control group without stromal cells and oxytocin group. In another experiment, COX-2 gene was expressed in embryo group treated with oxytocin during the co-culture of embryos with epithelial cells. In contrast, COX-2 mRNA was expressed in group treated with $IL-1{\alpha}$ when the embryos were cultured with stromal cell. This result shows that oxytocin and $IL-1{\alpha}$ were stimulate embryo development in vitro when embryos were cultured with epithelial and stromal cells, and can affect the development of bovine embryos in the uterus.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.3
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• pp.183-192
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• 2022

Lipopolysaccharide (LPS) is an endotoxin factor present in the cell wall of Gram-negative bacteria and induces various immune responses to infection. Recent studies have reported that LPS induces cellular stress in various cells including oocytes and embryos. Melatonin (N-acetyl-5-methoxytryptamine) is a regulatory hormone of circadian rhythm and a powerful antioxidant. It has been known that melatonin has an effective function in scavenging oxygen free radicals and has been used as an antioxidant to reduce the cytotoxic effects induced by LPS. However, the effect of melatonin on LPS treated early embryonic development has not yet been confirmed. In this study, we cultured mouse embryos in medium supplemented with LPS or/and melatonin up to the blastocyst stage in vitro and then evaluated the developmental rate. As a result of the LPS-treatment, the rate of blastocyst development was significantly reduced compared to the control group in all the LPS groups. Next, in the melatonin only treated group, there was no statistical difference in embryonic development and no toxic effects were observed. And then we found that the treatment of melatonin improved the rates of compaction and blastocyst development of LPS-treated embryos. In addition, we showed that melatonin treatment decreased ROS levels compared to the LPS only treated group. In conclusion, we demonstrated the protective effect of melatonin on the embryonic developmental rate reduced by LPS. These results suggest a direction to improve reproduction loss that may occur due to LPS exposure and bacterial infection through the using of melatonin during in vitro culture.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.143-153
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• 1996

To improve the efficiency of nuclear transplantation in the rabbit, this study were evaluated the influence of celly cycle of donor nuclei on the in vitro developmental potential in the nuclear transplant embryos. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G1 phase of 32-cell stage. Synchronization of the cell cylce of blastomeres were induced, first, using an microtubules polymerization inhibitor, 0.5$\mu\textrm{g}$/ml colcemid for 10h to arrest blastomeres in metaphase, and secondly, using a DNA synthesis inhibitor, 0.1$\mu\textrm{g}$/ml aphidicolin for 1.5 to 2h to cleave to 32-cell stage and arrest them in G1 phase. The separated G1 phase blastomeres of 32-cell stage were injectied into enucleated recipient cytoplasms by micromanipulation. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. The nuclear transplant embryos were co-cultured for 120h. In vitro cultured embryos were monitored every 24h to assess for development rate. After in vitro cultue for 120h, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye for counting the number of blastomeres under a fluorescence microscopy. The cleavage rate of blastomeres from 16-cell stage stage rabbit embryos treated with colcemid for 10h or aphidicolin for 6h following colcemid for 10h were not significantly different. The electrofusion rate was similar by high in S and G1 phase donor nuclei as 80.6 and 79.1%, respectively. However, the nuclear transplant embryos using G1 phase donor nuclei were developed to blastocyst at high rate(60.3%) than those using S phase donor nuclei(26.0%). Moreover, the mean blastocyst stage were increased significantly(P<0.05) with the G1 phase donor nuclei(176.6 cells and 1.50 cycles), as compared with the S phase donor nuclei(136.6 cells and 1.42 cycles). These results show that the blastomeres of G1 phase were more successful as donor nuclei in the nuclear transplant procedure, compared with S phase.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.77-82
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• 2004

The purpose 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 antioxidants(aesculetin, taurine and melatonin) in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation in NCSU 23 mediumand matured oocytes were inseminated with frozen semen. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of antioxidants in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. Aesculetin were added to NCSU 23 medium at concentration of 1 ug, 5 ug, and 10 ug, when treated with 10 ug(35.7%) of aesucletin at the rate of embryos of the morula plus blatocsyts were higher than those of any other groups (30.2%, 29.5% and 29.2%)(P<0.05). The developmental rates beyond morula stage of porcine embryos in NCSU 23 medium supplemented with taurine 0, 2.5 and 5.0 mM were 26.1%, 26.9% and 31.7%, respectively. The addition of 5.0 mM taurine was higher the developmental rate beyond morula stage than in any other groups. 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%, respectively. The developmental rate of morula and blascytocys treated with 1nM melatonin was higher than in any other groups(P<0.05). Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10nM were 41.0, 42.6, 39.6 and 33.0, respectively. These results indicate that aesculetin, taurine and melation can increase the developmental rate beyond the morulae and blastocysts in porcine embryos.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.77-82
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• 2007

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for $30{\mu}sec$; ES), 2) double electric stimulations (1.5 kV/cm for $30{\mu}sec$, followed by 1.0 kV/cm for $50{\mu}sec$ after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6-7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.3
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• pp.344-353
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• 2018

Objective: cAMP and mature promoting factor (MPF) play critical roles during the maturation of mammalian oocytes. The aim of this study was to produce the offspring from denuded oocytes (DOs) in mice by regulating cAMP and MPF. Methods: In this study, we used DOs at the germinal vesicle (GV) stage in mice and regulated levels of cAMP and MPF in DOs by adding Forskolin and PD166285 during in vitro maturation without follicle stimulating hormone and luteinizing hormone, respectively. Results: Combined use of $50{\mu}M$ Forskolin for 3 h and $2.5{\mu}M$ PD166285 for additional 21 h enhanced the developmental competence of DOs, maturation rate of DOs was $76.71%{\pm}4.11%$, blastocyst rate was $18.33%{\pm}4.44%$ after parthenogenetic activation (PA). The DOs could successfully be fertilized with sperm in vitro, cleavage rate was $17.02%{\pm}5.82%$ and blastocyst rate was $5.65%{\pm}3.10%$. Besides, 2-cell in vitro fertilization embryos from DOs produced 4 normal live offspring (4/34). Conclusion: The results confirmed that the combination of Forskolin and PD166285 can induce DOs to complete meiosis process and produce normal offspring.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.205-210
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• 2018

This study was performed to investigate the effect of types of oil (OVOIL vs. OIL) on B6D2F1 mice oogenesis. In this study, B6D2F1 F1 mice were used in order to maximize oogenesis. The expansion rate of cumulus cells ($82.0%{\pm}0.2$ vs. $78.0%{\pm}0.1$), in vitro fertilization rate ($92.0%{\pm}0.1$ vs. $88.0%{\pm}0.1$), developmental rate ($91.0%{\pm}0.1$ vs. $87.0%{\pm}0.2$), blastocysts formation rate ($56.0%{\pm}0.1$ vs. $57.0%{\pm}0.1$), and zona hatched rate($41.4%{\pm}0.2$ vs. $24.0%{\pm}0.1$) were not different between groups (NS; P>0.05). However, there was a significant difference in maturation rate; the OVOIL group showed higher maturation rate compared to that of the OIL group ($96.0%{\pm}0.1$ vs. $87.0%{\pm}0.1$; P<0.05). In the blastocysts cell numbers, the total cell numbers ($83.9{\pm}26.1$ vs. $56.9{\pm}23.9$), ICM cell numbers ($15.7{\pm}8.8$ vs. $6.3{\pm}3.5$), TE cell numbers ($68.3{\pm}25.7$ vs. $50.7{\pm}24.1$), % ICM ($21.6%{\pm}0.1$ vs. $12.7%{\pm}0.1$), and the ratio of ICM:TE ($1:6.2{\pm}6.5$ vs. $1:10.3{\pm}7.0$) were significantly higher in the OVOIL group than the OIL group (P<0.05). These results suggested that it is expected to achieve the more developmental ability of B6D2F1 mice depending on the type of oil (OVOIL vs. OIL). In addition, the results can provide essential information for culture condition on B6D2F1 mice. Henceforth, thus, it is expected that these results herein might be used for in vitro culture of human embryos.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.139-147
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• 2018

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.85-91
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• 2011

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.