• 한국가축번식학회지
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• 제11권1호
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• pp.73-84
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• 1987

This experiment was carried out to investigate the effects of hormone addition(FSH, HCG, estrogen and progesterone) and composition (BSA and FCS) of mKRB on the in vitro maturation and fertilizability of follicular oocytes of the Korean native cattle. The ovaries were removed at a slaughterhouse, returned to laboratory in a thermostat (30-35$^{\circ}C$) within 4 hr, and collected by aspirating normal follicles which had diameters of 1 to 6 mm. The oocytes with cumulus cells were cultured for 8, 16, 24 and 30 hr in a modified KRB solution containing BSA or FCS and hormones. The in vitro matured oocytes in mKRB containing FCS, FSH and steroids were transferred in the rabbit uterus for examination of their in vivo fertilizability with bovine sperm preincubated 4 to 6 hr in the rabbit uterus. 1. The mean number of oocytes collected per cattle was 6.5 from 1-3mm follicles, 1.3 from 4-6mm follicles, and total was 7.7. 2. The meiotic division at 16hr-cuture in the oocytes from 1-3mm follicles was slightly stimulated by the addition of FSH in mKRB + BSA solution compared with the control. At 30hr-culture, their maturation rates(%Met II) were also increased by FSH of 1 $\mu\textrm{g}$/ml(38.4%) and 5$\mu\textrm{g}$/ml(35.7%) as compared with the control (21.4%). The maturation rate at 30hr-culture in the oocytes from 4-6mm follicles was 53.8% and 57.1% by the FSH addition of 1$\mu\textrm{g}$/ml and 5$\mu\textrm{g}$/ml, respectively. These rates were similar with the control(57.1%), but higher than those of oocytes from 1-3mm follicles. 3. The meiotic division at 16hr-culture in the oocytes from 1-3mm follicles was stimulated by the HCG addition of 1IU/ml and 5IU/ml. However, the maturation rate at 30hr-culture was greatly decreased by the HCG addtion (26.6% and 13.3%) compared with the control(53.3%) and these rates (30.8%) in the oocytes from 4-6mm follicles were also lower than that fo the control(58.3%). 4. Low maturation rate (37.5%) of the oocytes cultured in mKRB containing BSA and 5IU/ml HCG was increased (55.0%) when 15% FCS with HCG was added to mKRB instead of BSA. 5. When 16hr-cultured oocytes in mKRB containing BSA and gonadotropins (5$\mu\textrm{g}$/ml FSH and 5IU/ml HCG) were transferred in the medium without gonadotropins and recultured for 16hr, the maturation rate of HCG-treated oocytes was greatly improved. 6. The maturation rates of oocytes were greatly affected by steroids. The combined addition of FCS+FSH+estrogen or +progesterone to mKRB increased the maturation rate compared with the combination of BSA+FSH or FCS+FSH in mKRB. 7. The fertilization rate, presence of pronuclei, was increased by the combination of FCS+FSH+p in mKRB as compared with that (5.6%) of BSA+FSH and the rates of FCS+FSH+steroids ranged from 12.5 to 17.6%.

• 한국가축번식학회지
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• 제22권1호
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• pp.95-99
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• 1998

Three experiments were conducted with follicular oocytes, to compare some somatic cells, growth factors and media for in vitro maturation of bovine oocytes. In the first experiment, the type of somatic cells had no effects on in vitro maturation of bovine follicular ooctyes. In the second experiment, oocytes were matured in TCM199 su, pp.emented with growth factors on IVM of bovine follicular oocytes, then all were co-cultured with cumulus cells. The proportion of used oocytes that developed to expanding blastocysts was 22.2%, 20.2%, 17.7%, 22.2%, 24.4% and 20.2% after maturation in TCM199 su, pp.emented with control, insulin, IGF-I, IGF-Ⅱ, FGF and EGF, respectively. In the third experiment, oocytes were matured in BO, Ham's F10 and TCM199, then all were fertilized in BO, and embryos cultured in BO, Ham's F10 and TCM199, respectively. Cleavage rates in BO were 90%, had higher than in Ham's F10(80%) or in TCM199(64%). But production of expanding blastocysts in TCM199(21%) or Ham's F10(20.6%), had higher than in BO(4.6%).

• 한국수정란이식학회지
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• 제16권3호
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• pp.173-182
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• 2001

본 연구는 돼지 난포란의 체외성숙배양액에 superoxide dismutase(SOD)를 첨가배양하여 체외성숙과 수성이후 배발달에 미치는 영향과 산소농도 및 SOD가 체외생산된 돼지초기수정란치 배발달에 미치는 영향을 규명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지 난포란을 체외성숙배양액인 NCSU-23에 SOD를 각각 0, 100,500 및 1,000uni1s/m1 첨가하여 성숙시킨 다음, 체외수정을 실시한 결과, 핵성숙률, 정자침입율, 다정자침입률 및 평균침입정자수는 처리구와 대조구간 유의적(P<0.05)인 차이가 인정되지 않았고, 웅성전핵형성률은 처리구(90.9∼81.2%)보다 오히려 대조구(95.0%)가 유의적으로 높은 결과를 나타냈다(P<0.05). 2. 체외수정 후, 배발달배양액인 NCSU-23에 7일 동안 배양한 결과, 배반포형성률과 총세포수는 처리구가 대조구보다 유의적(P>0.05) 으고 낮은 결과를 나타냈다. 3. 배발달배양액인 NCSU-23에 SOD를 각각 0, 100, 500 및 1,000uni1s/m1을 첨가하고 5% 및 20% 산소조건하에서 7일동안 배발달을 유기한 결과, 배양 7일째 산소조건에 따른 처리구간 배반포형성률 및 총세포수는 유의차가 없었으며, SOD농도별 배반포형성률은 100 units/ml 처리구에서 0, 500 및 1,000uni1s/m1 처리구 보다 유의적으로 높은 배반포형성률을 나타냈으나, 총세포수에서는 처리구(27.6∼37.3개)가 대조구(45.6개)보다 유의적(P<0.05) 로 낮은 결과를 나타내었다. 따라서 NCSU-23배양액에 SOD의 첨가는 돼지난포란의 체외성숙과 초기배발달에 영향을 미치지 않는 것으로 조사되었으며, 5% 및 20% 산소농도에 따른 영향도 없는 것으로 사료된다.

• 한국수정란이식학회지
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• 제32권4호
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• pp.267-274
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• 2017

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.

• 한국가축번식학회지
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• 제21권2호
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• pp.117-122
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• 1997

The aim of this study was to investigate the effect of the follicular culture from which the oocytes originate on their subsequent in vitro maturation ability. Ovarian follicles were isolated and cultured according to size(1~2mm, 2~6mm and 6~8mm) for 42~44 h. The rates of germinal vesicle breakdown(GVBD) in each groups were 87%(65/75), 82%(80/97) and 89%(47/53), but the oocytes maturation were su, pp.essed at anaphase-I stage. In spite of the adding porcine follicular fluid and/or hormones in maturation medium, maturation ability of oocytes from follicle cultured for 21~22 h were inhibited. When oocytes from follicle cultured for 4 h at various temperature were incubated for 38~40 h, the rates of oocytes maturation from follicle cultured at 2$0^{\circ}C$(51%, 26/51) and 39$^{\circ}C$(54%, 26/48) were significant higher(P<0.05) than group cultured at 4$^{\circ}C$(33%, 19/58). On the other hand, the GVBD were stared 2 h after culture of follicle of oocytes. To summairze, oocytes maturation by follicular culture were inhibited at anaphase-I stage in porcine. When the follicle cultured for 4 h, maturation were completed to metaphase-II stage. However, rates of GVBD in oocytes from follicular culture were higher than oocytes cultured in medium.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.147-152
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• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.13-13
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• 2001

This study was undertaken to evaluate the effects of catalase using xanthine (X) - xanthine oxidase (XO) system on in vitro maturation and fertilization in pig. When follicular oocytes were cultured in maturation medium with X and/or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obrained in culture with X-XO system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, hut were not different in medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with, none (P<0.05), XO and X＋XO. On the other hand, when sperm were treated with none, X, XO and X＋XO, lipid peroxidation were higher in medium without that than with catalase. However, the changes in sperm penetration and lipid peroxidation showed opposite patterns. The sperm suspensions were also treated with X and/or XO for assay of sulfhydryl (-SH) group content. Under the above all conditions, sperm-SH group were higher detected In medium with that than without catalase. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were higher than in medium with X, XO and X＋XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO system may be caused stimulating in vitro maturation and fertilization in pig. This work was supported by grant No. 2000-1-22200-001-3 from the Basic Research Program of the Korea Science & Engineering Foundation.

• Asian-Australasian Journal of Animal Sciences
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• 제14권10호
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• pp.1360-1366
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• 2001

The aims of this study were first to evaluate the effects of different levels (20, 40 and 100%) and sources (follicular size: large, >7 mm; medium, >5-7 mm; small, 3-5 mm) of porcine follicular fluid (pFF) on the in vitro maturation (IVM) of porcine oocytes, and the effects of fertilization treatments and different culture conditions on development of fertilized oocytes were also investigated. No differences in the maturation (63.6-76.6%) and cleavage (24.8-34.3%) rates were observed among the 20,40 and 100% pFF groups (p>0.05). The cleavage rates of oocytes cultured and fertilized in 40% and 100% pFF maturation media were significantly higher than those fertilized in m199-NBCS (51.0-61.2% vs. 12.8-31.8%. p<0.05), regardless of sources of the pFF. When oocytes were fertilized in m199-NBCS followed by culture in rabbit oviducts for 4 days, the cleavage rate in 40% pFF group was better than that in 100% pFF group (46.9% vs. 32.5%, p<0.05). Two oocytes recovered from the oviducts in the 40% pFF group developed to blastocysts after IVC. However, none developed to blastocysts when fertilized in the IVM medium after being transferred to rabbit oviducts. In conclusion, addition of pFF accompanied with gonadotropins (FSH, LH) in IVM medium enhanced maturation and cleavage rates of porcine oocytes. Direct addition of sperm suspension to IVM medium may be an alternative to simplify the fertilization procedures and to reduce the mechanical lesion during manipulation. Furthermore, rabbit oviducts provide a better environment for the in vitro fertilized oocyte developing to the morula and blastocyst stages.

• Clinical and Experimental Reproductive Medicine
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• 제44권2호
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• pp.79-84
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• 2017

Objective: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. Methods: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged $31{\pm}4.63years$ during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n = 100), cleavage medium (II, n = 100), blastocyst medium (III, n = 100), and Sage IVM medium (IV, n = 100) and cultured for 24 to 48 hours at $37^{\circ}C$. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. Results: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). Conclusion: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.