• 한국수정란이식학회지
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• 제14권2호
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• pp.113-119
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• 1999

This study was performed to improve the development of the in vitro fertilized bovine embryos by the condition of in vitro maturation. COCs were matured in TCM 199 supplemented with 0.1% PVA, 10ng/ml EGF, Hormones (5$\mu\textrm{g}$/ml FSH, 10 IU hCG, 1 $\mu\textrm{g}$/ml estradiol 17-$\beta$) or granulsa cell+Hormones atmosphere 39$^{\circ}C$, 5% CO2, 95% air for 24hrs. Matured oocytes were fertilized with frozen-thawed semen capacitated with 5mM caffein in BO medium for 20 hrs. IVF embryos were cultured in TCM 199 containing with hormones(same as matured medium), 10% FBS and co-culture with bovine oviduct epitherial cells. Maturation rates of COCs were showed 73.8%, 78.5%, 83.2% and 87.6% respectively, and were significant differences between PVA, EGF, and Hormones, GC+Hormones(p<0.05). The cleavage rates of IVF embryos were revealed 72.5%, 78.4%, 82.3% and 84.2% and showed same tendency as maturation rates(p<0.05). The blastocysts matured by above maturation condition and cultured for 7~10 days after fertilization had 34.4, 43.6, 52.3 and 59.3 cells had no differences among the treatments. These results suggest that high molecules as a substitutes of serum and growth factor may induce nuclear resumption of COCs but we need more study to produce transferable IVF blastocysts by use of that agents.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.500-506
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• 2009

A modified Tris-buffered medium (mTBM) has been widely used as an insemination medium for porcine in vitro fertilization (IVF). We examined whether mTBM could be used for bovine IVF. Bovine cumulus-oocyte complexes (COCs) were cultured in a serum-free medium containing 30 ng/ml EGF for 22 h. After culture, COCs were inseminated with spermatozoa for 12 h in mTBM containing 5 mM caffeine and 10 g/ml heparin. The penetration of oocytes increased significantly (p<0.05) as the sperm concentration increased from 0.1 (30%) to 1-10 $(87-100%){\times}10^6$ cells/ml. This was significantly different from values obtained at 1 (87%) and 10 $(100%){\times}10^6$ cells/ml. However, when COCs were inseminated with spermatozoa from different bulls, the proportions (62-100%) of oocytes penetrated varied according to the bull. The proportion (18%) of oocytes penetrated was significantly (p<0.05) lower in a fertilization medium without caffeine and heparin but increased with the addition of caffeine and/or heparin to the medium, and the proportion (93-96%) of oocytes penetrated increased significantly (p<0.05) when the medium was supplemented with heparin and caffeine. In this medium, sperm penetration was first observed at 3 h after insemination. Irrespective of the presence of glucose in the fertilization medium, the proportion (93-97%) of oocytes penetrated and the proportion (83-84%) of embryos at the ${\geq}2$-cell stage cultured in a chemically defined medium were not significantly different. However, the proportion of embryos developing to the blastocyst stage was significantly (p<0.05) higher in the presence (11%) of glucose in the fertilization medium than in its absence (2%). In conclusion, the present study demonstrated that bovine oocytes penetrated in vitro in mTBM can develop to the blastocyst stage and mTBM may be used for the in vitro production of bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제32권8호
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• pp.1112-1121
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• 2019

Objective: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Methods: First, we developed an in vitro model to study the response of bovine cumulusoocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in $10{\mu}g/mL$ of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-${\kappa}B$); and the concentrations of interleukin (IL)-$1{\beta}$, tumor necrosis factor (TNF)-${\alpha}$, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Results: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-${\kappa}B$. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-$1{\beta}$, TNF-${\alpha}$, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. Conclusion: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.272-280
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• 2021

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.23-28
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• 2003

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into $30^{\circ}C$ water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p<0.05). The effect of room temperature ($25^{\circ}C$ for 10 min) and cold temperature ($4^{\circ}C$ for 10 min) on COCs were determined in this study. After IVF, the cleavage and blastocysts rate of oocytes exposed to room temperature and cold temperature in VS+5% PVP was significantly different (2 cell: 63.20% vs 37.97%, blastocysts: 18.40% vs 2.53%). The cleavage rates of frozen-thawed oocytes were 20.53% with PVP and 22.13% without PVP (p>0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation.

• 한국수정란이식학회지
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• 제21권2호
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• pp.157-162
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• 2006

소 난포란의 체외 성숙은 과립막 세포, 난자의 핵성숙을 촉진하는 미지의 혈청내의 물질뿐만 아니라, 호르몬이나 생리 활성 인자 등에 의해 촉진됨이 밝혀졌다. 이에 따라 체외 성숙 및 체외 발달에 사용되는 배양액의 조성도 복합 배양액에서 단순 배양액으로 전환을 시도하고 있으며, 체내의 조건에 보다 더 접근하고자 하는 시도들이 수행되고 있다. 본 연구는 한우 난포란의 성숙 시 FGF의 첨가가 체외 성숙율 및 체외 수정 후 배발달율에 미치는 영향에 대하여 조사하였다. 한우 난포란의 체외 성숙시 FGF를 0.1, 1, 10 ng/ml를 첨가하였을 때 24 시간째에 Metaphase II 도달율은 각각 72.7, 70.0, 75.0%로서 무첨가 대조군의 37.5% 에 유의적으로 높은 성숙율을 보였다(p<0.05). 그러나 FGF 첨가 농도간에는 차이가 인정되지 않았다. FGF로 체외 성숙된 난포란의 체외 수정 후 배발달율은 0, 0.1, 1, 10 ng/ml FGF 첨가군에서 각각 25.0, 9.5, 0, 2.9%를 보여, 무첨가 대조군에 비해 FGF 첨가군에서 낮았다(p<0.05). FGF로 체외 성숙된 난포란을 체외 수정 후 10% FBS-HPM 199, 0.8% BSA-HPM 199 및 0.1% PVA-HPM에 배양한 결과 12.4, 12.8, 8.5%의 배반포 발달율을 보였으며, 무혈청 배양액인 IVMD, IVD에 배양한 결과 38.4 및 34.8%의 배반포 발달율로 유의적인 차이를 나타냈다(p<0.05). 결론적으로 FGF는 한우 난포란의 체외 성숙시 유용한 물질이나, 한우 난자의 체외 발달에는 단독의 효과를 기대할 수 없었으며, 다른 생리 활성 인자들 간의 상호 관계에 대하여 더 많은 연구가 요구된다.

• 한국발생생물학회지:발생과생식
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• 제8권2호
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• pp.119-122
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• 2004

소 체외성숙시 난포란에 영향을 주는 과립막세포와 난자의 핵성숙을 촉진하는 물질이 확인되지 않은 혈청내의 물질 뿐만 아니라 호르몬이나 생리활성인자 등에 의해 촉진됨이 밝혀졌다. 이에 따라 체외성숙 및 체외발달에 사용되는 배양액의 조성도 복합배양액에서 단순배양액으로 전환을 시도하고 있으며, 체내의 조건에 보다 더 접근하고자 하는 시도들이 수행되고있다. 본 연구는 한우 난포란의 체외성숙율 및 체외수정 후 배발달율을 향상시키기 위하여 TGF-${\beta}$의 첨가가 미치는 영향에 대하여 조사하였다. 한우 난포란의 체외성숙시 TGF-${\beta}$ 0.1, 1. 10ng/ml를 첨가하였을 때 24시간에 MetaphaseⅡ 도달율은 95.8%, 95.8%, 100%를 나타내었으며, 농도간의 유의적인 차이는 없었다. TGF-${\beta}$로 체외성숙된 난포란을 체외수정 후 TGF-${\beta}$가 체외성숙 배양액을 동일하게 체외발달을 유도하였으나, 0~0.8%의 배반포율을 보였으며, TGF-${\beta}$로 체외성숙된 난포란을 체외수정 후 TCM 199_10% FBS, 0.8% BSA, 0.1% PVA에 배양한 결과 12.4%, 12.8%, 8.5%의 배반포 발달율을 보였고, 무혈청 배양액인 IVMD, IVD에 배양한 결과 38.4%, 34.8%의 배반포 발달율을 보였으며, 유의적인 차이를 나타냈다(P<0.005). 결론적으로 TGF-${\beta}$는 한우 난포란의 체외성숙시 상당히 유용한 물질이나, 체외발달에는 만족할 수 없어, 이외의 생리활성 인자들간의 상호관계에 대하여 더 많은 요구가 요구된다.

• 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.

• 한국수정란이식학회지
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• 제15권3호
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• 한국수정란이식학회지
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• 제20권3호
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• pp.303-309
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• 2005

본 연구는 한우 난구 복합체의 체외성숙에서 OA가 미치는 영향에 대하여 조사하였다. 도축 한우암소의 난소로부터 난구 복합체를 채취하여 $0.1\%$ PVA-TCM199로 3회 이상 세정 후 $0.1\%$ PVA-TCM 199, 0.2 uM, 2 uM, 20 uM OA를 각각 첨가하여 $5\%\;CO_2,\;95\%$ 공기, $39^{\circ}C$에서 6, 12, 24시간 동안 체외성숙을 실시하였다. 또한 체외성숙시 cycloheximide(CX)와 OA와 체외성숙 효과를 확인하기 위하여 0.1M-PVA TCM199, CX 25 ug/mL, 동량의 CX를 6시간 처리한 후 2uM의 OA로 체외성숙을 실시하거나, 0.2 uM OA 단독으로 체외 성숙시켰다. 체외 성숙된 한우 난구 복합체의 핵형을 조사하기 위하여 $0.5\%$ hyaluronidase 용액으로 난구세포를 용해하고, 난자는 1:3 acetic acid, ethanol 용액에 30초간 고정하였으며, $3\%$ basic Fuchsin을 염색하여 핵형을 관찰하였다. 체외 성숙된 난자의 핵형 및 체외 발달율에 대한 통계분석은 3반복을 하여 얻어진 결과를 ANOVA test로 분석하였다. 한우 난구 복합체의 체외성숙율은 $0.1\%$ PVA-TCM199, 0.2 uM, 2 uM, 20 uM OA를 첨가시, 각각 72.0, 50.0, 70.9, $68.8\%$를 나타내어 유의적인 차이를 보였으며(p<0.05), CX와 OA가 한우 난구복합체에 미치는 영향을 조사한 결과, 0.1M-PVA, CX 25 ug/mL, 동량의 CX를 6시간 처리한 후 2uM의 OA로 체외성숙을 실시, 0.2 uM OA 단독처리시의 체외 성숙율은 각각 73.8, 7.2, 45.5, $73.7\%$를 나타냈어 극도의 유의적인 차이를 보였다(p<0.01). 미토콘드리아의 활성은 0.1M-PVA, CX 25 ug/mL, 동량의 CX를 6시간 처리한 후 2uM의 OA로 체외성숙을 실시, 0.2 uM OA 단독 처리시, 핵성숙기간 동안 증가하는 경 향을 보였고, CX 처리시 다른 처리와 비교하였을 때, 성숙 6시간에 1/3의 FI를 나타내었다. 이상의 결과를 미루어 OA는 한우 난구 복합체의 체외성숙에 중요한 조절물질이며, 핵성숙과정 중 미토콘드리아의 활성에도 중요한 역할을 하는 것으로 나타났다.