• 한국유기농업학회지
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• 제9권1호
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• pp.17-27
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• 2001

To clarify the proper and safe duration of maturation periods for sawdust-pig manure composts, leaf lettuces were applied with pig manure composts fermented for 15, 30, 45, 60, 75 days and 1 year (control) and cultivated in a plastic house with or without additional PE film tunnel. The changes in physiochemical properties of soil and leaf lettuce growth were measured. Shorter duration of maturation periods enhanced the generation of N $H_3$ gas from the composts and resulted in significant decrease in seed germination, growth and yield of leaf lettuce. Under with and without PE tunnel conditions the concentration of N $H_3$ from compost over 3.8 and 2.1 mg/100g composts, respectively induced growth reduction. In proportion to the increase of maturation duration saw-dust containing pig manure exhibited decrease in C/N ratio, N $H_4^{+}$-N, N $H_4^{+}$N $O_3^{-}$ ratio but increase in N $O_3^{-}$-N contents. In case of applying pig manure compost without PE film tunnel condition the minimum maturation period of pig manure composts for safe leaf growth was 60 days, while minimum 75 days of maturation was required when applied with PE film tunnel. tunnel.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.221-226
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• 2004

The present study was performed to investigate the effects of in vitro maturation (IVM) and in vitro fertilization (IVF) duration on the development of Korean Native Cattle embryos. The time of blastocyst formation and the quality of blastocysts based on cell numbers were examined. The cleavage rate increased with the length of IVF duration in the groups of 18-hr IVM, but was constant in the groups of 24-hr IVM. The development rate to the 8-cell stage was significantly higher in the IVM 18: IVF 20 group than in the IVM 24: IVF 24 group. The development rate to the blastocyst stage was highest in the IVM 18: IVF 20 group, significantly different from that of the IVM 18: IVF 16, IVM 24: IVF 20 and IVM 24: IVF 24 group. The time of blastocysts formation tended to be shorter when IVM and IVF duration were decreased. The number of inner cell mass, trophoblast and the total cells were significantly higher in the IVM 18: IVF 16 group than in the IVM 24: IVF 24 group (P<0.05). These results demonstrated that the IVM and IVF duration should be adequate for the efficient production of bovine embryos, and it might particularly be essential to determine the proper combination of IVM and IVF duration.

• 한국수정란이식학회지
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• 제29권3호
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• pp.201-206
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• 2014

The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.

• Plant Biotechnology Reports
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• 제2권2호
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• pp.153-161
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• 2008

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation ($57.22{\pm}2.02$), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation ($58.80{\pm}1.47$) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyl-adenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.

• 한국동물생명공학회지
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• 제34권2호
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• pp.117-122
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• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• 한국동물생명공학회지
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• 제34권3호
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• pp.205-211
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• 2019

In this study, we investigated the possibility of using mouse embryonic stem cell conditioned medium (ESCM) and embryonic stem cell medium (ESM) for in vitro maturation in the efficient in vitro production of blastocysts from porcine follicular oocyte. Depending on the concentration of supplement of ESCM added to the NCSU-23 solution did not affect 2-cell development rates and blastocysts development. However, in particular, the survival rate (10 days of culture) of blastocyst was significantly higher than that of the control group as the additive concentration (30%) increased (p < 0.05). The survival rate of blastocysts showed a similar tendency even with addition of ESM (30%) alone. On the other hand, the duration of the addition of these additives during IVM (0-44 h) was that the IVM I period (0-22 h) were more effective than the IVM II period (22-44 h). Thus, the effect of these additives is probably due to the combination of the various physiologically active substances of ESCM or the appropriate amino acids and vitamins of ESM. In particular, these additives were more effective during the first half (IVM I) of in vitro maturation. In summary, optimization of ESCM or ESM supplementation may improve in vitro maturation of porcine oocyte and affect developmental competency. Therefore, if more efficient methods of adding ESCM or ESM to basal culture medium can be developed during in vitro maturation of porcine follicle oocytes, high quality blastocysts will be developed from low porcine follicular oocyte compared to other domestic animals.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제47권4호
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• pp.306-311
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• 2020

Objective: The aim of this study was to determine whether co-administration of a gonadotropin-releasing hormone (GnRH) agonist and human chorionic gonadotropin (hCG) for final oocyte maturation improved mature oocyte cryopreservation outcomes in young women with decreased ovarian reserve (DOR) compared with hCG alone. Methods: Between January 2016 and August 2019, controlled ovarian stimulation (COS) cycles in women (aged ≤35 years, anti-Müllerian hormone [AMH] <1.2 ng/mL) who underwent elective oocyte cryopreservation for fertility preservation were retrospectively analyzed. Results: A total of 76 COS cycles were triggered with a GnRH agonist and hCG (the dual group) or hCG alone (the hCG group). The mean age and serum AMH levels were comparable between the two groups. The duration of stimulation, total dose of follicle-stimulating hormone used, and total number of oocytes retrieved were similar. However, the number of mature oocytes retrieved and the oocyte maturation rate were significantly higher in the dual group than in the hCG group (p=0.010 and p<0.001). After controlling for confounders, the dual-trigger method remained a significant factor related to the number of mature oocytes retrieved (p=0.016). Conclusion: We showed improved mature oocyte collection and maturation rate with the dual triggering of oocyte maturation in young women with DOR. A dual trigger appears to be more beneficial than hCG alone in terms of mature oocyte cryopreservation for young women with DOR.

• Clinical and Experimental Reproductive Medicine
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• 제23권1호
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• pp.33-39
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• 1996

본 연구는 돼지 미성숙난포란의 체외배양시 EGF가 핵성숙의 GVBD와 M II 에 미치는 효과를 조사하였다. 실험 1에서는 EGF를 첨가하였을 때, 난포란의 배양경과 시간 (0, 16, 24, 42시간)에 따른 핵성숙도를 조사하였던바, EGF 10ng/ml이 첨가된 군이 무첨가된 군에 비해서 24시간 이후에 GVBD가 유의하게 높았다 (p < 0.001). 실험 2에서는 난포란의 배양시 EGF 노출시간에 따른 난포란의 핵성숙의 효과를 조사하였던바, 배양 초기 (0-24시간)와 배양 전시간 (0-42시간) 동안 EGF를 배양액내에 첨가한 군의 경우 최종 성숙단계인 M II까지의 핵성숙율은 72.8과 84.8%로써 배양 후기 (24-42시간)에 EGF가 첨가된 군의 53.5%와 배양 전시간 (0-42시간) 동안 무첨가한 군의 26.1%보다 유의하게 높았다 (p < 0.001). 그리고 실험 3에서는 난구세포 부착 난포란과 난구세포가 제거된 난포란에 있어서 EGF의 효과를 조사하였던바, EGF가 첨가된 난구세포 부착 난포란 군의 경우 핵성숙율 (M II)은 84.6%로써 EGF가 첨가된 난구세포 제거군의 53.0%와 무첨가 난구세포 부착군의 27.6%, 난구세포 제거군의 44.2%보다 유의하게 높았다 (p < 0.001). 따라서 이상의 결과로 미루어보아 EGF 단독만으로도 돼지 미성숙난포란의 체외성숙의 핵성숙에 있어서 GVBD와 M II 을 유도할 수 있다고 사료된다.

• 한국수정란이식학회지
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• 제33권1호
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• pp.1-11
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• 2018

Cellular cyclic adenosine-3' 5'-monophosphate (cAMP) modulator is known as meiotic inhibitor and can delays spontaneous maturation in IVM experiment. Among many cAMP modulators, the role of Pituitary adenylate cyclase activating polypeptide (PACAP) on IVM isn't known. The purpose of this study is to improve the maturation of oocytes derived from follicles ${\leq}3mm$ in diameter through PACAP as meiotic inhibitor during pre-in vitro maturation (pre-IVM). First, we checked PACAP and its receptors in cumulus cells and, to establish the optimal phase and concentration of PACAP for pre-IVM, we conducted chromatin configuration assessments. As a result, the rate of GV (Germinal Vesicle) according to duration of pre-IVM was significantly decreased 12 h and 18 h after IVM (87.1 and 84.1%, respectively) compared to 0 h (99.4%). When COC was cultured for 18 h, the GV rate in the $1{\mu}M$ of PACAP treatment group (82.1%) was significantly higher than any other PACAP treatment groups (60.5, 64.1, 74.4 and 69.9 %, respectively). So, we divided into four groups as follows; MF (the conventional IVM group, obtained from follicle from 3 to 6 mm in diameter), SF (the conventional IVM group, obtained from follicle ${\leq}3mm$ in diameter), Pre-SF(-)PACAP (IVM group including 18 h pre-IVM without $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter) and Pre-SF(+)PACAP (IVM group including 18 h pre-IVM with $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter). To examine the effect of PACAP during pre-IVM, we investigated analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. In cumulus cells, PACAP receptors, ADCYAP1R1 and VIPR1 were detected but were not detected in oocytes. After IVM, the Pre-SF(+)PACAP had the highest Metaphase II rate (91.7%) among all groups (P<0.05). The GSH levels in the MF and Pre-SF(+)PACAP were significantly higher than in the other groups (P<0.05) and ROS levels was no significant difference among all groups. In conclusion, these results indicated that even though the oocytes were derived from SF, pre-IVM application of PACAP improved meiotic and cytoplasmic maturation by regulating intracellular oxidative stress.