• 한국발생생물학회지:발생과생식
• /
• 제25권3호
• /
• pp.173-183
• /
• 2021

The incidence of infertility among individuals of reproductive age has been growing due to genetic and environmental factors, and considerable research efforts are focused on solving this issue. Ovarian development is an overly complex process in the body, involving the interaction between primordial germ cells and gonad somatic cells. However, follicles located in the center of the in vitro ovary are poorly formed owing to ovarian complexity, nutrient deficiency, and signaling deficiency. In the present study, we optimized methods for dissociating gonads and culture conditions for the in vitro generation of miniaturized ovaries. The gonads from embryos were dissociated into cell masses and cultured on a Transwell-COL membrane for 3-5 weeks. Approximately 12 follicles were present per in vitro ovary. We observed that miniaturized ovaries successfully matured to MII oocytes in vitro from 150 to 100 ㎛ gonad masses. This method will be useful for investigating follicle development and oocyte production.

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

• 식물조직배양학회지
• /
• 제22권5호
• /
• pp.273-276
• /
• 1995

네오레게리아(Neoregeria carorinae 'Tricolor')의 기내배양시 무늬소실을 줄일 수 있는 재료의 채취시기와 발근을 위하여 기내에서 처리된 오옥신이 온실에 이식된 식물체의 생육 및 개화에 미치는 영향에 관하여 실험한 결과, 화아분화 4주후(III단계)에 미숙화기를 배양한 것이 분화된 식물체 중 정상 식물체가 67%로 가장 많았고, 화아분화 직후 및 화아분화 5주후에 배양한 것은 모두 반입이 소실되었다. 미숙화기와 액아를 배양하여 나온 식물체중 정상식물체 획득율은 미숙화기 배양에서는 67%, 액아배양에서는 56.2%였다. 미숙화기를 배양하여 얻은 식물체가 액아를 배양하여 얻은 식물체보다 온실재배에서 생육이 월등이 좋았고, 개화율도 미숙화기를 배양하여 얻은 식물체는 27.8%이었으나 액아를 배양하여 얻은 식물체는 전혀 개화하지 않았다. IBA 0.5 mg/L가 첨가된 배지에서 발근시킨 식물체의 기외생육이 왕성하였으며, 기내에서 처리한 오옥신 종류와 농도는 온실에서 재배되고 있는 식물체의 변이에 영향을 미치지는 않았다.

• 한국가축번식학회지
• /
• 제7권1호
• /
• pp.24-29
• /
• 1983

These experiments were carried out to obtain basic information necessary of the success of in vitro culture of blastomeres separated from mouse embryo. Total 446 single blastomeres separated from 2-, 4- and 8-cell mouse embryos by protease treatment (0.5% in Whittingham's medium), were cultured under the gas phase of 5% CO2 in air at 37$^{\circ}C$. whittingham's medium was used for culture of blastomeres. The results obtained in these experiments were summerized as follows: 1. Of total 446 blastomeres cultured, 127(87.0%), 134(73.2%) and 77(65.8%) blastomeres separated repectively from 2-, 4- and 8-cell embryos were developed to morula or blastular stages. 2. The numbers of blastomeres, being separated from 2-. 4- and 8-cell embryos and developing to blastocysts containing inner cell mass, were 97(76.4%), 86(64.2%) and 33(42.9%) respectively. 3. After in vitro culture of the blastomeres, the incidence of trophoblastic vesicles increased with the development of the cell stage of embryo. In case of blastomeres separated from 8-cell embryos, 50.6% of blastomeres that developed to blastular stage was trophoblastic vesicles.

• 한국수정란이식학회지
• /
• 제11권2호
• /
• pp.125-135
• /
• 1996

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6$^{\circ}C$ to -35$^{\circ}C$ at -0.3$^{\circ}C$ or -O.6$^{\circ}C$ /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3$^{\circ}C$ /min and -0.6$^{\circ}C$ /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3$^{\circ}C$ /min(63.6%) than in -0.6$^{\circ}C$ /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3$^{\circ}C$ /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

• Journal of Plant Biotechnology
• /
• 제7권2호
• /
• pp.135-141
• /
• 2005

Protocols for in vitro conservation was developed for Coleus forskohlii. Plants maintained both in field served as explant source. Shoot tips and single node cuttings were used to optimize protocols for in vitro multiplication. MS basal medium supplemented with $0.54\;{\mu}M$ naphthalene acetic acid (NAA) and $8.87\;{\mu}M$ benzy-ladenine (BA) induced multiple shoots in shoot tips and nodes. Shoot multiplication was amplified with a gradual decrease of BA concentration, leading to its final omission after 4 months. Concomitant rooting on multiplication media enabled successful establishment extra vitrum. For in vitro conservation studies, experiments were carried out with 2-3 week maintained in vitro plants under standard and reduced culture conditions (SCC, RCC). In vitro plants could be successfully conserved in full strength MS medium (FMS) under SCC for 6 months without subculture with full potential to regenerate, producing viable shoots and nodes. The root production remained unaffected due to conservation, showing high rooting activity in mannitol and low temperature treatments. Preset low temperature (15 and $10^{\circ}C$) and reduction in media constituents does not appear to favour conservation, although the former accomplished conservation levels equal to (FMS) under SCC.

• Reproductive and Developmental Biology
• /
• 제33권3호
• /
• pp.125-131
• /
• 2009

The aim of this study is to investigate the effect of porcine epididymal fluid (pEF) on in vitro-maturation and subsequent fertilization of porcine follicular oocytes. Porcine cumulus-oocytes complexes retrieved from antral follicles were cultured in tissue culture medium (TCM)-l99 supplemented with pEF of different concentrations. At 48 h after culture, development of oocytes to germinal vesicle (GV) breakdown, metaphase I, anaphase-telophase I, and metaphase II were examined Significant (p<0.05) increase in the proportion of oocytes developed to MII stage was observed in oocytes cultured in pEF-containing TCM-l99 than in oocytes cultured in pEF-free TCM-199 (46.2% vs 16.7%), which was a dose-dependent manner. Subsequently, the proportion of monospermic fertilization were significantly (p<0.05) increased in oocytes cultured in the TCM supplemented with pEF than those cultured in pEF-free TCM-199 (51.0% vs 24.1%). In the second series of experiment, the percentage of MII oocytes was significantly (p<0.05) increased after exposure of oocytes to pEF during the first 22 h period of culture than after exposure of oocytes to pEF during the next 24 h of culture, while no significant difference in the percentage of monospermy was observed. The results of this study demonstrate that pEF contains at least enhancing component(s) for nuclear maturation.

• Asian-Australasian Journal of Animal Sciences
• /
• 제1권3호
• /
• pp.153-156
• /
• 1988

Bovine blastocysts were obtained by in-vitro culture of embryos derived from in-vitro fertilization of oocytes matured in-vitro. These blastocysts and blastocysts from inseminated donors were bisected by a simple method (without a holding pipette) using a microblade operated by a micromanipulator. A pair of demi-embryos was transferred nonsurgically into each uterine horn of a recipient cow 6 or 8 days after estrus. Pregnancy resulted from the third transfer. Ultrasound examination done 52 days after estrus (46 days after transfer) confirmed the presence of at least one fetus in the each uterine horn. This is the first report to show the viability of bisected bovine blastocysts obtained from in-vitro culture of embryos derived from in-vitro fertilization of oocytes matured in-vitro. In addition, a simple method to bisect bovine embryos is described.

• 한국수정란이식학회지
• /
• 제15권1호
• /
• pp.67-75
• /
• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.63-63
• /
• 2003

This study was carried out to investigate the effects of liquid boar sperm stored at 4$^{\circ}C$ on sperm motility, normal acrosome, and in-vitro fertilization and culture of pig oocytes matured in-vitro. The sperm-rich fraction (30~60 ml) of ejaculate was collected into an insulated vacuum bottle. Semen was slowly cooled to room temperature (20~23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800$\times$g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent to provide 1.0$\times$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$ and preserved for 5 days to examine sperm motility and normal acrosome. The medium used for oocyte maturation was modified tissue culture medium (TCM) 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 6 h in 500 ${mu}ell$ mTBM fertilization media with 0.2, 1, 5 and 10$\times$10$^{6}$ /ml sperm concentration, respectively. At 6 h after IVF, oocytes were transferred into 500 ${mu}ell$ Hepes-buffered NCSU-23 culture medium for further culture of 6, 48 and 144 h. There were significant differences in sperm motility and normal acrosome among preservation days and incubation times, respectively. The rates of sperm penetration and polyspermy were higher in 5 and 10$\times$10$^{6}$ sperm/ml than in 0.2 and 1$\times$10$^{6}$ sperm/ml. Male pronuclear formation was lower in 0.2$\times$10$^{6}$ sperm/ml than in 1, 5 and 10$\times$10$^{6}$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. The rate of blastocysts from the cleaved oocytes (2~4 cell stage) was highest in 1$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend 1$\times$10$^{6}$ ml sperm concentration for in-vitro fertilization of pig oocytes.