• 한국수정란이식학회지
• /
• 제32권4호
• /
• pp.297-304
• /
• 2017

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG ($45.3{\pm}6.2%$) compared to control ($25.6{\pm}3.4%$). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG ($89.3{\pm}1.9%$) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG ($94.6{\pm}1.1%$). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation ($27.8{\pm}4.9%$) compared to no treatment ($41.4{\pm}1.9%$) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM ($37.4{\pm}3.4%$ and $34.4{\pm}2.6%$, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

• Reproductive and Developmental Biology
• /
• 제28권1호
• /
• pp.29-36
• /
• 2004

본 연구는 체외에서 한우 난포란의 핵성숙과 그 후의 초기 배발달에 있어서 체외성숙 배지에 아미노산의 첨가가 난포란의 제1극체(PB) 출현율, 배발달율 그리고 배반포의 세포수에 미치는 영향을 검토하였다. 첨가하는 아미노산의 종류와 농도는 각각 MEM 배지의 non-essential amino acids (NEAA)와 BME 배지의 essential amino acids(EAA)는 1, 2, 4배 및 유청중의 lactalbumine hydrolysate(LAH)는 1, 5, 10 mg/$m\ell$이었다. 그 결과 NEAA와 EAA의 경우 PB 출현율은 1배 첨가군이 미첨가군보다 유의하게 높았지만(p＜0.05), 첨가량이 증가할수록 오히려 감소하였다. 그러나 배반포로의 배발달율은 모든 군에서 비슷한 경향이었다. 그리고 배반포의 총 세포수와 총 세포수중 TE 세포수는 2배 처리군이 가장 높았으며, ICM 세포수는 아미노산 첨가량이 증가할수록 많아졌다. 한편 LAH의 경우 PB 출현율은 5mg 첨가군이 가장높았으며, 배반포로의 발달율은 미첨가군과 1 mg 첨가군이 5 mg 첨가군과 10 mg 첨가군보다 각각 유의하게 높았다(p＜0.05). 그리고 배반포의 세포수는 NEAA와 EAA를 이용하였을 경우와 비슷한 경향이었다. 이상의 결과로부터 체외성숙용 배지에 아미노산의 첨가는 생산된 배반포의 품질을 향상시킬 수 있기 때문에 배반포의 체외생산에 이용할 수 있는 새로운 아미노산의 종류 및 농도를 탐색할 필요가 있다고 사료된다.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2002년도 국제심포지엄
• /
• pp.8-17
• /
• 2002

There have been intensive attempts to establish reliable methods far in vitro production (IVP) methods for of porcine embryos. Although a great deal of progress has been made, our current IVP systems still need to be improved. In this review, we focused on studies about in vitro maturation and fertilization (IVM-IVF) of porcine oocytes and their in vitro culture (IVC), especially on an excellent piglets production system using modified IVP system producing porcine blastocysts with high Quality.

• 농업과학연구
• /
• 제48권1호
• /
• pp.163-170
• /
• 2021

Bacterial infections in the female reproductive tract negatively affect ovarian function, follicular development, and embryo development, leading to the eventual failure of fertilization. Moreover, bacterial lipopolysaccharides (LPS) can interfere with the immune system and reproductive system of the host animal. Therefore, this study examined the effect of LPS on the in vitro maturation (IVM) of pig oocytes. Oocytes were matured in TCM199 medium in the presence of varying concentrations of LPS (0 - 50 ㎍·mL^-1). The maturation rate, cortical granules (CGs) migration, and chromosome alignment were subsequently evaluated during the meiotic development of the oocytes. We observed a dose-dependent and significant decrease in the metaphase II (MII) rate with increasing concentrations of LPS (97.6% control [0 ㎍·mL^-1 LPS] vs. 10.4-74.9% LPS [1 - 50 ㎍·mL^-1], p < 0.05). In addition, compared to the control oocytes without LPS, higher levels of abnormal CGs distribution (18.1 - 50.0% LPS vs. 0% control), chromosome/spindle alignment (20.3 - 56.7% LPS vs. 0% control), and intracellular ROS generation were observed in oocytes matured with LPS (p < 0.05). Nitrite levels were also increased in the maturation medium derived from the oocytes matured with LPS (p < 0.05). These results indicate that LPS induces oxidative stress during IVM and affects oocyte maturation, including CGs migration and chromosome alignment of pig oocytes.

• Reproductive and Developmental Biology
• /
• 제31권3호
• /
• pp.169-174
• /
• 2007

개 난자의 체외성숙율을 높이기 위하여 다양한 방법들이 시도되고 있지만 여전히 그 효율성은 낮다. 본 연구는 개 난자의 체외성숙 시, 성선 자극 호르몬인 황체형성호르몬(LH)과 난포자극호르몬(FSH), 상피세포성장인자(Epidermal growth factor, EGF) 그리고 시스테인(cysteine)을 각각 첨가하여 72시간 동안 체외성숙시킨 후 핵성숙율(GV: germinal vesicle, GVBD: germinal vesicle break down, MI: metaphase I, MII: metaphase II, UK: unknown stage)을 확인하였다. LH와 FSH를 첨가하였을 때 첨가하지 않은 군과 GV, MI 및 MII율에는 유의적인 차이는 없었다. 하지만 GVBD율은 첨가군이 유의적으로(p<0.05) 높았다. 성선 자극 호르몬을 첨가한 배지에 10ng/ml의 EGF를 첨가하였을 때 MII율이 첨가하지 않은 군보다 유의적으로(p<0.05) 높았다(4.54% vs. 7.06%). cysteine을 첨가하였을 경우, 핵성숙율에 유의적인 차이는 보이지 않았지만 전반적으로 핵성숙율이 향상된 것을 확인할 수 있었다. 따라서 개 난자의 체외성숙 시, $10 {\mu}g/ml$의 LH와 FSH, 10ng/ml의 EGF 그리고 0.57mM의 cysteine을 첨가하는 것이 핵성숙율을 향상시키는 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
• /
• 제48권4호
• /
• pp.352-361
• /
• 2021

Objective: The study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. Methods: In total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5. Results: In total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advent of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). Conclusion: Cilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

• Reproductive and Developmental Biology
• /
• 제32권3호
• /
• pp.147-152
• /
• 2008

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at $56^{\circ}C$ and $97^{\circ}C$, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and $56^{\circ}C$ group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

• 한국수정란이식학회지
• /
• 제28권2호
• /
• pp.127-132
• /
• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• 한국수정란이식학회지
• /
• 제10권3호
• /
• pp.219-227
• /
• 1995

This study was investigated to test in vitro-maturation rate of bovine follicular oocytes freezability of in vitro-matured bovine follicular oocytes with different stock solution in Glycerol and Propanediol, freezability of in vitro-rnatured bovine follicular oocytes on cryoprotectants, the viability of in vitro-rnatured bovine follicular oocytes by morphologically normal and FDA staining method. 1. The maturation rates of bovine follicular oocytes classified as grade A, B and C was 88, 63 and 21%, respectively. 2. Freezability of in vitro-matured bovine follicular oocytes on stock solution, TCM-199+5% FCS and m-PBS + 5% FCS was 61%(n=105), 48%(n=62) in $_1$M Glycerol and freeability of in vitro-matured bovine follicular oocytes on stock solution, TCM-199 +5% FCS and m-PBS + 5% FCS was 68%(n=112), 42%(n=57) in 1~2 Propanediol. The results indicate that freezability of in vitro-matured bovine follicular oocytes with different stock solution is important. 3. Freezability of in vitro-matured bovine follicular oocytes on cryoprotectants was Glycerol and PROH was 56%(n=167), 57%(n=169). The results indicate that PROH was superior to Glycerol. 4. The rates of morphologically normal IVM oocytes after thawing of cryopreserved oocytes with Glycerol and PROH were 39%(n=$_1$8), 65%(n=39), respectively. The results indicate that PROH was superior to Glycerol. 5. The fluorescent light intensity after thawing of cryopreserved oocytes classified with Positive, Partial-I, Partial-II, Negative with Glycerol and PROH. The results of FDA-positive 24%, 42%, Partial-I 17%, 10%, Partial- H 20%, 12%, FDA-negative 39%, 37%, and Partial-I, II, respectively.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2004년도 춘계학술발표대회
• /
• pp.170-170
• /
• 2004

First of all, in vitro production (IVP) of porcine embryos is an important as initial step to improve bio-technical applications such as transgenesis and cloning for xenotransplantation. In recent years, considerable progress has been achieved in the IVP embryos using advanced methods for in vitro maturation (IVM) and fertilization (IVF). (omitted)