• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.59-63
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• 2004

1. The concentrations of Ang. II were 7.20.91 ${\times}$ $10^3$ , 3.80.34 ${\times}$ $10^3$, 3.50.30 ${\times}$ $10^3$, 2.80.22 ${\times}$ $10^3$ pg/ml in bovine follicular fluids from 1∼3 mm, 3∼5 mm, 5∼7 mm and 8∼10 m follicles, respectively. The concentrations of Ang. II decreased in follicular fluids from large follicles. 2. When oocytes were cultured in media containing various concentrations of Ang. II, a higher proportion of oocytes developed to MII stage in medium with 100 ng/ml (79.5%) Ang II compare to that without Ang. II (58.8%). When oocytes from different sizes of follicles were separately cultured in media containing 100 ng/ml Ang. II, maturation rates were higher in oocytes from small and medium follicles those from controls. 3. GSH content in oocytes cultured for 24 hrs in TCM-199 medium containing 10 and 100 ng/ml of Ang. II was also higher than that of oocytes cultured in medium containing 0 or 10 ng/ml Ang. II. When oocytes were cultured in media containing 0, 10, 100, 1,000 ng/ml of Ang. II, the concentrations of GSH were 5.1M, 5.5M, 7.2M, 8.7M, respectively. 4. When oocytes were cultured in media containing various concentrations of 10, 100, 1,000 ng/ml Ang. II, in vitro maturation and developmental rates were 84.0%, 90.0%, 78.0% and 28.0%, 36.0%, 20.0%, respectively. When oocytes were cultured with an addition of Ang. II in media, in vitro maturation rates higher than that of their controls (76.0%).

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.29-33
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• 2002

This study was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 24 hrs post onset of maturation, the oocytes were cultured $3{\sim}13{\mu}M\;Ca^{2+}$ for 5 min., $5-8{\mu}g/ml$ cytoclacin for 6 hrs, 0.5~2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs alone or combination. The activated oocytes were cultured in TCM-199 media at 5% $CO_2$, 95% air, $38^{\circ}C$. The cleavage rate after 48 hrs culture of oocytes treated with $3-13{\mu}M\;Ca^{2+}$, $5-8{\mu}g/ml$ cytoclacin and 0.5~2.0 mM DMAP for 5 min., 6 hrs and 3 hrs were 9.6%~20.0%, 0.0%~7.3% and 9.4%~21.8%, 0.0%~7.3% and 9.1%~21.8% and 0.0%~7.3%, respectively. When oocyte were treated with $10{\mu}M\;Ca^{2+}$, $10{\mu}g/ml$ cytoclacin and 2.0 mM DMAP the blastocyst formation rate was significantly higher than other group. The cleavage rate after 48 hrs culture of oocytes treated with $Ca^{2+}$ + cytoclacin, $Ca^{2+}$ + DMAP, cytoclacin + DMAP were 75.9%~93.5% and 9.7%~19.0%, respectively. When oocytes were treated with $Ca^{2+}$ followed by DMAP, the blastocyst formation rate was significantly higher than other group(p<0.05). When necleus transferred embryos co-cultured with bovine serum albumin(BSA), epithemal growth factor(EGF) and calf serum(CS), the developmental rate to blastocyst were higher than control group.

• 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.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.757-762
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• 1993

The ovaries of Korean Native cows or heifers were obtained from an abattoir and kept on 20 to $25^{\circ}C$ and transported to laboratory within 2 hrs. The follicular oocystes were collected from 2~6mm follicles in diameter and classified into 3 grades by the morphology of cumulus cells attached. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with $23{\mu}g/ml$ FSH, $10{\mu}g/ml$ LH, $1{\mu}g/ml$ estradio-17 ${\beta}$ and granulosa cells at $39^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by incubation for 12 hrs. of epididymal spermatozoa pretreated with heparin, and then the zygotes were co-cultured in vitro(IVC) with oviductal epithelial cells for 7 to 9 days. Assessment of maturation revealed that 93.0%(147/158) of grade I oocytes had expanded of cumulus cells, which was higher(p<0.05) than the 79.4%(85/107) of grade II oocytes. Compared to epididymal sperm(32.9%), the insemination with frozen and thawed sperm resulted in slightly lower(20.5%), but not significant, development to morulae and blastocysts from grade I oocytes. Co-culture of bovine IVF embryos with oviductal epithelial cells improved the development to transferable embryos significantly(38.1%), compared to co-culture with granulosa cells(20.0%). When VF bovine embryos were vitrified at blastocyst, the post-thaw survival rate was obtained higher resulf for 1 min. equilibration time(82.6%) or 2 min.(73.9%) than 3 min.(18.2%) in EFS solution.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.969-976
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• 2009

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.385-393
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• 1998

This study was conducted to investigate the effect of cysteine (CySH) and glutathione (GSH) on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows: 1. When the immature oocytes were cultured at 0, 0.04, 0.14, 0.6 and 1.2 mM of cysteine (CySH) for 36h, the germinal vesicle breakdown (GVBD) rates were 90.8, 89.9, 90.5, 92.0 and 91.3%, respectively, and the maturation rates of the oocytes with metaphase-II were 56.1, 50.7, 41.9, 49.0 and 61.5%, respectively. Especially, the maturation rates of 0.14 and 0.6 mM treated groups were significantly lower than those of control (non-treated) group (P＜0.05). After 44h of culture in the same treatments of CySH, the GVBD rates of porcine immature oocytes were 90.0, 91.8, 89.8, 90.5 and 89.6%, respectively, and the maturation rates were 80.2, 76.3, 69.4, 66.7 and 72.6%, respectively. Especially, the maturation rates of 0.14 and 0.6 mM treated groups were significantly lower than those of control group (P＜0.05). 2. When the immature oocytes were cultured at 0, 0.05, 0.1, 0.5 and 1.0 mM of glutathione (GSH) for 36h, the GVBD rates of porcine immature oocytes were 91.0, 90.9, 89.5, 92.0 and 91.1%, respectively, and the maturation rates were 59.0, 48.5, 47.8, 38.6 and 37.5%, respectively. All treated groups of GSH showed lower maturation rates than the control group (P＜0.05). After 44h of culture in the same treatments of GSH, the GVBD rates of porcine immature oocytes were 91.8, 94.1, 89.1, 91.3 and 91.1%, respectively, and the maturation rates were 84.6, 57.1, 69.6, 71.3 and 64.3% respectively. All treated groups of GSH showed lower maturation rates than the control group (P＜0.05).

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.375-383
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• 1998

This study was conducted to investigate the effect of $\beta$-mercaptoethanol ($\beta$-ME) and cysteamine on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows : 1. When the immature oocytes were cultured at 0, 10, 50, 100 and 200 $\mu$M of $\beta$-ME for 36h, the GVBD rates were 91.6, 92.5, 91.8, 91.9 and 92.7%, respectively, and the maturation rates of the oocytes with metaphase-II were 49.6, 41.7, 32.5, 34.1 and 35.4%, respectively. Thus, lower maturation rate was shown in $\beta$-ME treated groups of 50, 100 and 200 $\mu$M as compared to control (non-treated) group (P＜0.05). After 44h of culture in the same treatments of $\beta$-ME, the GVBD rates of porcine oocytes were 91.8, 90.4, 92.5, 91.2 and 93.9%, respectively, and the maturation rates were 71.9, 58.8, 56.7, 62.2 and 56.5% respectively. All treated groups of $\beta$-ME showed lower maturation rates than the control group (P＜0.05) 2. When the immature oocytes were cultured at 0, 10, 50, 100 and 200 $\mu$M of cysteamine for 36h, the GVBD rates were 90.6, 86.3, 88.2, 87.2 and 90.0%, respectively, and the maturation rates were 53.8, 45.1, 54.4, 57.5 and 63.3% respectively. Especially, the maturation rate of 200 $\mu$M treated group was significantly higher than those of control group (P＜0.05). After 44h of culture in the same treatments of cysteamine, the GVBD rates of porcine immature oocytes were 89.5, 93.1, 85.1, 89.8 and 91.3%, respectively, and the maturation rates of the oocytes with metaphase- II were 84.2, 77.6, 66.0, 67.8 and 78.3%, respectively. Especially, the maturation rates of 50 and 100 $\mu$M treated groups were significantly lower than those of control group (P＜0.05).

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.213-221
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• 2001

This study was conducted to examine in vitro developmental ability of porcine embryos after somatic cell nuclear transfer. The porcine ear fell was cultured in vitro for confluency in serum-starvation condition(TCM-199 + 0.5% FBS) far 3~6 days of cell confluency. The zona pellucida of IVM oocytes were partially drilled using laser system. Single somatic cell was individually transferred into enucleated oocytes. And the reconstructed embryos were electrically fused(single DC 1.9kv/cm, 30$\mu$ sec) with 0.3M mannitol. After electrofusion, embryos were activated(single AC 5v/mm, 5sec) and cultured in HCSU-23 medium containing 10% FBS at 39$^{\circ}C$, 5% $CO_2$ in air for 6 to 8 days. The fusion rate of donor cells was 45.6, 36.8 and 46.1% in 3~4, 5~6 days of serum starvation and non serum starvation(N-S), and were 52.7. 53.0 and 51.7% in 1~2. 5~6 and 13~14 passages of donor cell culture, respectively. No significant difference was found in the fusion rate of donor cells by the duration of serum starvation treatment or the number of donor cell passages. By the size of donor cells, however, the fusion rate was significantly higher(P<0.05) for reconstructed embryos derived from 25r $\mu$m $\geq$ site of donor cells (65.3%) than that of 25~30$\mu$ m(42.5%) or 30$\mu$ m(45.5%)$\leq$ cells. The cleavage rate was significantly (P<0.05) higher in 3~4 darts of serum starvation treatment(67.1%) than that in N-S (50.7%) or 5~6 days of starvation(57.1%). The activation rate by the size of donor cells in fused oocytes was 56.5, 68.8 and 58.5%, respectively, and was not significant.