• 한국수정란이식학회지
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• 제9권1호
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

• Asian-Australasian Journal of Animal Sciences
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• 제31권6호
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• pp.827-834
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• 2018

Objective: The aim of this study was to compare the effects of $36.5^{\circ}C$ and $38.5^{\circ}C$ incubation temperatures on the maturation of bovine oocytes and developmental competence of embryos. Methods: In experiment 1, oocytes were maturated in bicarbonate-buffered TCM-199 for 22 hours in a humidified atmosphere of 5% $CO_2$ in the air at either $36.5^{\circ}C$ or $38.5^{\circ}C$ and nuclear maturation status were determined. In experiment 2, in vitro fertilized oocytes were allocated randomly into synthetic oviductal fluid medium with or without a mixture of 1 mM L-glutathione reduced and 1,500 IU superoxide dismutase and cultured in a humidified atmosphere of 5% $CO_2$, 5% $O_2$, and 90% $N_2$ in the air at $38.5^{\circ}C$ for 8 days. Results: There were no significant differences between incubation temperatures in terms of oocyte maturation parameters such as cumulus expansion, first polar body extrusion and nuclear maturation. Incubation temperatures during in vitro maturation had no effects on developmental competence of embryos, but supplementation of antioxidants increased (p<0.05) developmental competence of the embryos. Blastocysts from oocytes matured at $38.5^{\circ}C$ had comparatively higher inner cell mass, but low overall and trophectoderm cell numbers (p<0.05). Conclusion: The results of present study showed that maturation of bovine oocytes at $36.5^{\circ}C$ may provide a suitable thermal environment for nuclear maturation and subsequent embryo development.

• 한국가축번식학회지
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• 제22권1호
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• pp.73-80
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• 1998

본 연구는 돼지난자의 체외성숙에 미치는 transforming growth factor $\beta$(TGF $\beta$)와 난수세포의 역할에 대하여 검토하였다. 난자의 체외성숙시 TGF $\beta$를 여러 농도에서 첨가한 경우 metaphase II로 성숙한 난자의 비율은 52~69%로 유의적인 차이는 나타내지 않았다. 성숙배양 24시간에서 난구세포의 유무에 관계없이 TGF $\beta$가 무첨가된 배양액 내에서는 성숙된 난자가 관찰되지 않았으나 TGF $\beta$첨가(5 및 4%)의 경우 성숙난자가 관찰되었다. 그러나, 성숙배양 48시간후 난자의 성숙율은 난구세포가 부착되어 있는 경우 TGF $\beta$첨가(70%)가 무첨가(52%)에 비해 높았으며, 난구세포를 세포를 제거한 경우 (35 및 26%)에 비해 유의적으로 높은 성숙율을 나타냈다(P<0.05). 한편, 나구세포가 부착된 난자의 성숙배양시 TGF $\beta$의 첨가시기에 의한 성숙율(54~71%)에는 큰 차이가 없었으나, 난구세포를 제거한 경우 전반기(59%) 또는 후반기(57%) 24시간 동안 TGF $\beta$를 첨가한 경우 48시간 동안 계속하여 첨가(27%) 또는 무첨가(38%)에 비하여 유의적으로 높은 성숙을 나타냈다(P<0.05). 이와 같은 결과는 난구세포가 돼지난자의 체외성숙시 필수적이지만, TGF $\beta$는 난구세포를 제거한 경우 난자의 성숙에 계속적인 역할을 하지 않는 것으로 추측된다.

• 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.

• 한국수정란이식학회지
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• 제14권3호
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• pp.177-184
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• 1999

This study was conducted to find out the effect of follicle size and oocyte type on in vitro maturation of poricine follicular oocytes. TCM-HEPEAS medium was used to basic medium, and the oocyte matured in vitro was stained with the Rapid staining method. The results obtained were summarized as follows; 1. The number of follicles an ovary was 20.5. The number of A-and B-typed oocytes an ovary was 2.34. The proportion of A-and b-types oocytes was 40% of the recovery oocytes. 2. Cumulus expanison indexes(CEI) by the follicle size were 1.62∼2.34(<2mm), 1.27∼2.28(2∼5mm) and 1.46∼2.75(>5mm). It was no differ to maturation rate by the follicle size. 3. The degree of oocyte maturation based on oocyte type did not differ for B-and C-typed oocyted but the index of oocyte type A was higher than that of b-and C-typed oocytes. 4. When follicluar oocytes were cultured for 42 hours, the proportion of the Met-II(second metaphase) stage were 22.5% (degree 1), 35.4%(degree 2) and 65.5% (degree 3).

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.223-227
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• 2005

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture $43.8\%$ of oocytes cultured in only TCM 199 remained at GV stage whereas $56.2\%$ of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than $90\%$ of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.53-53
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• 2002

This study was carried out to verify the incidence of oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups (vitrified respectively at 10, 14 and 20 hrs after the onset of maturation). (omitted)

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.72-72
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• 2002

Oocytes maturation, characterized by germinal vesicle (GV) breakdown, formation of the first meiotic spindle, expulsion of the first polar body and arrest in metaphase of second meiotic division (MII), occurs in preovulatory follicles in response to the surge of gonadotropin and leads to an ovulated oocyte in vivo. However, meiotic resumption in vitro occurs spontaneously following removal of cumulus-oocytes complexes (COCs) from the follicle. (omitted)

• 한국수정란이식학회지
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• 제9권3호
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• pp.261-268
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• 1994

Immatured bovine follicular oocytes added with serum, hormones, granulosa cells and bovine oviduct epithelium cells were fertilized in vitro after in vitro maturation. In vitro maturation and early development capacity were examined and IVF-derived embryos were transferred and to recipients and effects of sperm treatment on in vitro capacitation were investigated. The rate of in vitro maturation was improved when they were co-culutred with granulosa cells in the TCM199 medium added with 10% FCS and hormones. The percentage of acrosome reaction was not differed between sperm treatments and sperm of above 25% under-went AR during 30 min preincubation with caffeine and heparin. The cleavage rate of oocytes in vitro fertilized in TCM199 medium added with 10% FCS and hormones, GC or BOEG higher than that in medium with 10% FCS and GC. But the rate was not significantly different between GC and BOEG The cleavage of rate oocytes cultured in medium containing serum, hormones and BOEG was 80.2% and more embryos were developed to Blastocyst (17.3%). The selected embryos were transferred to 9 recipients by surgical or nonsurgical method but did not result in pregnancy.

• 한국가축번식학회지
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• 제13권2호
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• pp.105-112
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• 1989

These experiments were carried out to obtain the basic information for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2-6mm of diameter. Bovine oocytes were matured in vitro for 24-26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM199 supplemented with hormones, pyruvate, FBS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2-3 hours in BO solution containing bovine serum albumin(5mg/ml) and caffein(2.5mM). Insemination was made by introducing about 10-15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after inseminatin the eggs were transferred to TCM 199 supplemented with FBS(10%) for in vitro development. The results obtained in these experiments were summarized as follows : 1. The maturation rate of oocytes following incubation for 24-26 hours was 78.4%(228/291). 2. Of total 250 oocytes, 172 embryos extruded 2nd polar body following in vitro culture with spermatozoa for 20 hours, and the rates of embryos developed to 2-, 4-, 8-, 16-cells and morula or early blastocyst were 64.0, 39.2, 22.0, 15.2 and 11.2%, respectively. 3. The time needed for development to 2-, 4-. 8-, 16-cell stage and morula was 42.5$\pm$5.4, 58.0$\pm$9.2, 74.4$\pm$11.5, 96.1$\pm$13.4 and 119.0$\pm$18.2 hours, respectively.