• 한국수정란이식학회지
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• 제10권1호
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• pp.45-51
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• 1995

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 $\mu$g /nl of FSH, 10 $\mu$g /ml of LH and 1 $\mu$g /ml of estradiol-17$\beta$ at 39t in a 5% $CO_2$ incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1$\mu$rn) to 23 hours(58.4$\mu$m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.17-23
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• 2002

체외수정의 성공률에 있어서 배란되기 직전의 충분히 성숙된 난자를 채취하는 것이 중요하며, 최근에는 질 벽을 통하여 초음파를 이용하는 방법을 많이 사용하고 있다. 난자 채취 시술 시 정맥 마취제를 투여하는데 최근에는 수술회복이 빠르고 오심이나 구토 등의 부작용이 적은 Propofol(2,6-disoprooylphenol)과 Thiopental sodium을 사용한다. 이들 마취제가 마취 투여 시간과 양에 따라 난자의 성숙률과 수정률, 발생률에 어떠한 영향을 미치는지 조사하고자 하였다. 돼지에서 추출한 성숙 전 단계의 난자를 마취제인 Propofo1 및 Thiopental sodium의 다양한 농도와 시간에 노출시킨 후 난자 성숙를을 조사하였으며 돼지 정자와 체외수정시킨 후 수정률과 발생률을 관찰하였다. 또한, 수정 단계 없이 단독 발생하는 처녀생식에 대한 영향도 조사하였다. Propofol에 단시간 노출된 난자는 대조군과 큰 차이가 없었으나 고농도에서 장시간 노출 시 성숙률이 현저히 높게 나타났다. 그러나 수정률은 높은 농도에 장시간 노출할 경우 대조군보다 낮아졌는데, 이와 같은 일시적인 난자 성숙은 Propofol이 처녀생식 유발물질이기 때문으로 보여진다. Thiopental sodium은 저농도 단시간 처리 시 난자의 성숙률과 수정률을 모두 감소시켰다. 이상의 결과로 볼 때, 각 마취제의 최적농도와 투여시간을 결정하여 이러한 영향을 최소화시키는 것이 체외수정의 성공률과 밀접한 관계가 있는 것으로 생각된다.

• 대한수의학회지
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• 제36권4호
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• pp.929-939
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• 1996

The present study was undertaken to establish a relationship between bovine follicle size and oocyte diameter, compare the nuclear maturation competence of oocytes of different diameter groups and the nuclear maturation changes in Korean Native Cattle according to in vitro maturation period. To compare the relationship between follicle size and oocyte diameter, follicles were dissected, measured, and assigned to one of the following size categories($4{\geq}mm$, 3-4mm, 2-3mm, 1-2mm, and < 1mm), investigate the maturation competence in the different-sized oocytes, which were divided into three groups( < $110{\mu}m$, 110 - < $120{\mu}m$, and ${\geq}120{\mu}m$). Oocytes were cultured in the culture medium during 0, 6, 12, 18, and 24hrs, respectively, stained, and measured the nuclear maturation degree according to period. When compared the relationship between follicle size and intrafollicular oocyte diameter, oocyte diameters of three groups of ${\geq}3mm$ follicle-sized were significantly higher than < 3mm (p<0.01). After in vitro maturation, the rates reached to MI stage of < $110{\mu}m$ oocyte groups(25%) was higher than $110-120{\mu}m$ and ${\geq}120{\mu}m$ oocyte groups(11 and 10%) reached to the same stage(p<0.01), and the rates throughout MII stage of $110-120{\mu}m$ and ${\geq}120{\mu}m$ and < $110{\mu}m$(70 and 76%) groups were higher than < $110{\mu}m$(35%)(p<0.01). When nuclear maturation rates were measured according to period, < 6hr groups(7 and 10%) showed lower rates reached to MI than ${\geq}12hr$ groups(100%), 24hr groups(76%) revealed higher rates throughout MII than 18hr groups(40%). These results indicate that the preparation of oocyte for the production of in vitro fertilization embryos and nuclear transplantation ones could be adapted, as follicle increased up to appointed size there was a corresponding increase in oocyte diameter, and differences of nuclear maturation rate revealed according to oocyte diameter and maturation period.

• Clinical and Experimental Reproductive Medicine
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• 제32권4호
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• pp.331-336
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• 2005

Objective: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. Method: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was $31.8{\pm}3.1years$. Ovulation was triggered by urinary or recombinant hCG. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/mL), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female (< 34 or ${\geq}34years$). Results: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). Conclusion: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes.

• Journal of Animal Science and Technology
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• 제65권2호
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• pp.324-335
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• 2023

Korea, located in East Asia in the northern hemisphere, is experiencing severe climate changes. Specifically, the heat stress caused by global warming is negatively affecting the dairy sector, including milk production and reproductive performance, as the major dairy cattle Holstein-Friesian is particularly susceptible to heat stress. Here, we collected artificial insemination and pregnancy data of the Holstein and the Jersey cows from a dairy farm from 2014 to 2021 and analyzed the association between the conception rate and the temperature-humidity index, calculated using the data from the closest official weather station. As the temperature-humidity index threshold increased, the conception rate gradually decreased. However, this decrease was steeper in the Holstein breed than in the Jersey one at a temperature-humidity index threshold of 75. To evaluate the effects of heat stress on the oocyte quality, we examined the nuclear and cytoplasmic maturation of Holstein (n = 158, obtained from six animals) and Jersey oocytes (n = 123, obtained from six animals), obtained by ovum pick-up. There were no differences in the nuclear maturation between the different conditions (heat stress: 40.5℃, non- heat stress: 37.5℃) or breeds, although the Holstein oocytes seemed to have a lower metaphase II development (p = 0.0521) after in vitro maturation under heat stress conditions. However, we found that the Holstein metaphase II oocytes exposed to heat stress presented more reactive oxygen species and a peripheral distribution of the mitochondria, compared to those of the Jersey cattle. Here, we show that weather information from local meteorological stations can be used to calculate the temperature-humidity index threshold at which heat stress influences the conception rate, and that the Jersey cows are more tolerant to heat stress in terms of their conception rate at a temperature-humidity index over 75. The lower fertility of the Holstein cows is likely attributed to impaired cytoplasmic maturation induced by heat stress. Thus, the Jersey cows can be a good breed for the sustainability of dairy farms for addressing climate changes in South Korea, as they are more resistant to hyperthermia.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2001년도 제41차 추계학술대회
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• pp.100-100
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• 2001

• Reproductive and Developmental Biology
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• 제31권1호
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• pp.7-13
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• 2007

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gona-dotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in sserum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp.2:0, 0.5, 1.0 or 10 IU) or both (Exp. 3:1 IU FSH +1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with $1.9\;{\mu}M$. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.221-225
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• 2011

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide ($H_2O_2$), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decrease with addition of $H_2O_2$, and were significantly (p<0.05) lower in medium with 0.1 mM $H_2O_2$ than control group. Also, the rate of degenerated oocytes was increased in as $H_2O_2$ concentration in eased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When $H_2O_2$ concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM $H_2O_2$ and (3) control medium with 1.0 mM $H_2O_2$ along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. $H_2O_2$ decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity, against oxidative stress caused by $H_2O_2$. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM $H_2O_2$ alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

• Asian-Australasian Journal of Animal Sciences
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• 제21권10호
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• pp.1404-1410
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• 2008

This study was performed to clarify whether the variation of stress related heat shock protein 70 (HSP70) (GenBank X68213) gene was associated with the nuclear morphological change of in vitro maturation and in vitro capacitation in oocytes of pig ovaries obtained at the slaughterhouse. The nucleic acid substitution of C to G at the 483rd position was found out in HSP70 K1 (290-512) from X68213. The ovaries were categorized into CC, CG, and GG genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (BsiHKA I). After the second in vitro maturation of immature fresh oocytes, the relation of nuclear morphological change in oocytes with the genotype of HSP70 K1 gene was such that the MII ratios of the genotype GG and CG (46.93% and 42.20%, respectively) were significantly higher than that of the CC genotype (10.71%) (p<0.05). With respect to in vitro maturation of frozen-thawed oocytes by an open pulled straw (OPS) method, the percentage of oocytes matured to MII stage of the CG genotype showed a higher trend than CC and GG genotypes. After the in vitro maturation of immature fresh oocytes and frozen-thawed oocytes by the OPS method, the relation of the pronuclei change in oocytes matured in vitro with HSP70 genotype was assessed, and the result showed that the enlarged sperm heads (ESH) of matured fresh oocytes and frozen-thawed oocytes were 80.0% and 60.0% in the CC genotype, respectively. The CC genotype group had a significantly higher rate of ESH than the CG and the GG genotype group (p<0.05). The ratios of polyspermic invasion were not different among HSP70 of the three genotypes. It was considered that the rate of in vitro maturation of fertilized oocytes was expected to differ according to genotype of the stress related gene.

• 한국패류학회지
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• 제25권2호
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• pp.113-119
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• 2009

본 연구는 2008년 3월 24일에 전남 영광군에서 채취한 말백합 어미 1,200 마리를 확보하여 수온조절 (20, 25, $30^{\circ}C$ 및 자연해수) 에 의한 인위적인 성 성숙을 유도하였으며, 성숙 유도된 어미로부터 생존율, 비만도, 생식소 및 성숙 소요일수를 조사하였다. 말백합 어미의 연체부지수는 사육초기에는 다소 낮아지는 경향이 있었으나, 5월 중순부터 증가하는 경향이 있었으며, 시험 종료 시에는 연체부 지수가 급격히 증가하였으며, 각부용적지수도 유사한 경향을 나타내었다. 말백합의 생식소 발달단계는 비활성기, 초기 활성기, 후기활성기, 완숙기, 방출 및 퇴화기의 5 단계로 구분할 수 있는데, 시험 개시일인 3월 25일경은 전기 활성기였으며, 시험 종료일인 6월 30일에는 수온별로 다양한 생식주기를 나타내었다. 시험 종료 시 성숙 유도율은 $30^{\circ}C$ 실험구에서 46.0%로 가장 높았고, $15^{\circ}C$ 실험구 35.0%, 자연수온 18.0% 및 $20^{\circ}C$ 실험구 12.0%로 순으로 나타났으며, 시험 기간 중 생존율은 전 구간이 90% 이상으로 나타났다. 사육수온을 20, 25, $30^{\circ}C$ 및 자연수온으로 성숙 시험한 결과, 암컷의 경우 $30^{\circ}C$ 실험구가 성숙이 가장 빨랐고, 수온이 낮을수록 늦은 경향이었으며, 수컷도 비슷한 경향을 나타내었다. 말백합의 수온별 어미성숙은 $20^{\circ}C$ 보다는 수온이 높은 실험구인 25, $30^{\circ}C$에서 성숙 유도율이 높게 나타났다.