• 한국가축번식학회지
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• 제22권2호
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• pp.171-176
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• 1998

본 연구는 체외에서 배양된 생쥐 완전탈출 배반포기배를 동결보존액 EFS35를 이용하여 초자화 동결하였을 때 체내발달의 적합성 여부를 조사하기 위해 실시하였다. 공시된 완전탈출 배반포기배($\theta$ 130$\mu\textrm{m}$)는 체내에서 생산된 전핵기 수정란을 5일 동안 체외배양하여 얻었으며, 10% ethylene glycol(EG)에 5분 노출한 후 EFS35(35% EG, 18% Ficoll, 0.3 M sucrose)에 30초 동안 노출하거나, 초자화 동결하였다. 융해 후, 재팽창이 이루어진 완전탈출 배반포기배는 가임신 3일된 대리모의 한쪽 또는 양쪽 자궁각(4~6개/자궁각)에 이식하였다. 대리모의 임신율과 착상율은 임신 15일재 외과적 해부로 판정하였다. 그 결과를 요약하면 다음과 같다. 융해 30분 후, 완전탈출 배반포기배의 체외생존율은 노출군(65.5%)과 동결군(54.5%)간에 유의한 차이는 없었다. 또한, 체내발달율을 조사하였던 바, 착상율에 있어서 동결군(41.0%)과 대조군(58.5%)간에 유의한 차는 없었지만, 정상산자율에서는 동결군(24.0%)의 결과가 대조군(58.3%)보다 유의하게 낮게 나타났다(p<0.05). 이러한 결과는 EFS35를 이용한 완전탈출 배반포기배의 초자화 동결은 정상산자율은 감소하였지만, 완전탈출 배반포기배의 이용 효율성을 넓히는데 이용될 수 있다는 것을 알 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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• pp.244-252
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• 2023

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.87-92
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• 2006

The possibility of producing transgenic embryos expressing the green fluorescence protein (GFP) gene have been evaluated after transfer of exogenous gene into the porcine zygote cytoplasm using the intracytoplasm sperm injection (ICSI) as gene delivery method. For DNA binding to sperm heads, 0.05% Triton X-100 or Lipofectin was used. After injection of the sperm bound to DNA by means of Lipofectin or Triton X-100 triturate, the blastocyst formation rates on day 6 were not significantly different from that of ICSI only group (18.8, 19.2 and 25.3%). In terms of GFP expression, more embryos were in GFP form in Triton X-100 group than in Lipofectin group (40.6 vs 36.4%), while percentage of non-mosaic embryos expressing the GFP gene in all blastomere was higher (P<0.05) in Lipofectin group than in Triton X-100 group (4.2 vs 0.9%). ICSI embryos derived from sperm treated with Lipofectin/DNA complex was transferred into 3 recipients and were collected by uterine flushing on days 5, 7 and 15 after embryo transfer, and then GFP expression was observed by a fluorescence microscopy. Over 26% of the collected embryos were normally expressed GFP gene. These results suggest that foreign gene transfer method with DNA bound sperm caused minimal damage to structure of oocytes that can result to full development of porcine embryos. This was confirmed in this study when the embryos that were transferred after ISCI of DNA bound sperm had a normal development and gene expression until preimplantation.

• 한국수정란이식학회지
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• 제28권3호
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• pp.243-250
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• 2013

Pyracantha is a genus of thorny evergreen large shrubs in the family of Rosaceae, with common names Firethorn or Pyracantha. It's extract has also been used in cosmetics as a skin-whitening agent and functioning through tyrosinase inhibition. Recent studies have shown that pyracantha extract possesses antioxidant activities and may significantly improve lipoprotein metabolism in rats. Although the mode of action of Pyracantha extract is not fully understood, a strong relationship was observed between antioxidant and apoptosis in some types of cells. Thus, the aim of this study was to evaluated the effect of pyracantha extract on blastocysts formation and their quality of the porcine parthenogenetic embryos. After parthenogenetic activation by chemicals, presumptive porcine parthenogenetic embryos were cultured in PZM-3 medium supplemented with extracts of pyracantha leaf, stalk and root for 6 day (1, 5 and $10{\mu}g/ml$, respectively). In our results, the frequency of blastocyst formation in pyracantha root extract ($5{\mu}g/ml$) treated group had increased that of other groups. Furthermore, blastocysts derived from pyracantha root extract ($5{\mu}g/ml$) treated group had increased the total cell numbers and reduced apoptotic index. Blastocyst development was significantly improved in the pyracantha root extract ($5{\mu}g/ml$) treated group when compared with the $H_2O_2$ treated group (p<0.05). Subsequent evaluation of the intracellular levels of ROS in pyracantha root extract ($5{\mu}g/ml$) treated groups under $H_2O_2$ induced oxidative stress were decreased (p<0.05). In conclusion, our results indicate that treatment of pyracantha root extract may improve in vitro development of porcine parthenogenetic embryos through its antioxidative and antiapoptotic effects.

• 한국가축번식학회지
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• 제9권2호
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• pp.140-147
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• 1985

Seventy four Holstein heifers were randomly assigned over three trials to PRID-7+PG-6 and Synchromate B-9 regimens to synchronize estrus cycle for embryo transfer. Sexual behaviors; moounting, standing, orientation, chin-resting, sniffing, licking, rubbing and butting, vaginal swelling and mucus discharge were observed between 06-08, 12-14 and 18-20 h on 1st day and 00-02, 06-08, 12-14 and 18-20 h on 2nd day after removal of hormones. Synchromate-B treatment (81.6%) showed higher synchronized estrus rate than PRID treatment (77.8%) during observation period. Standing estrus was observed within 74 h after PG injection in PRID and within 52 h after removal of implant in Synchromate-B. About 68% of heifers in PRID and 74% of heifers in Synchromate-B showed standing estrus between 0-14 h on 2nd day after removal of the hormones. Synchromate-B resulted in a tighter synchrony of standing estrus than PRID. Incidence of average mounting and standing per head during observation period was 22.3 and 16.6 in PRID and 28.1 and 13.6 in Synchromate-B. The PRID showed peak in active mounting at 18-20 h on 1st day, however, the Synchromate-B showed at 0-2 h on 2nd day after removal of hormone. Active standing was shown between 18 h on 1st day to 20h on 2nd day in PRID, however, between 0-14 h on 2nd day after removal of hormone is Synchromate-B. There was slight difference in pattern of active mounting and standing during estrus between PRID and Synchromate-B. Conception rate of synchronized heifers transferred with fresh and frozen embryos by non-surgical and surgical methods was higher in synchromate-B (62.5%) than in PRID (38.5%). Chin-resting showed highest incidence among 6 sexual behavioral components in the both treatments. Synchromate-B showed higher incidence of chin-resting (16.6) than PRID (10.7). Synchromate-B group showed also higher incidence of orientation, sniffing and butting than PRID group. Synchromate-B resulted in more active sexual behaviors than PRID. The pattern of incidence of chin-resting, licking and butting was almost symmetrical in PRID with their peak values at 6-8 h on 2nd day, however in Synchromate-B chin-resting and sniffing was symmetrical with their peak values at 12-14 h on 2nd day after removal of hormone. There was tendency to increase vaginal swelling according to time passage of synchronized estrus in the both treatments. Incidence of mucus discharge in Synchromate-B was slightly higher than in PRID. Twenty to 40% was false negative in conception rate by tall painting before re-estrus day as judged by rectal palpation.

• 한국가축번식학회지
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• 제22권3호
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• pp.287-292
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• 1998

본 실험은 1.48$\mu\textrm{m}$ diode laser의 인간배에 대한 적용 가능성 여부를 조사하기 위한 예비실험으로, 체외생산된 생쥐배에 1.48$\mu\textrm{m}$ diode laser를 이용한 zona pellucida (ZP) drilling 처리가 배의 부화와 체내발달에 효과적인지를 조사하고자 실시하였다. 그 결과는 다음과 같다. 발달단계가 상이한(4-세포기배, 배반포기배) 생쥐배에 laser ZP drilling 처리한 후 72시간 (배반포기배) 또는 120시간 (4-세포기배) 동안 배양하였던 바, laser ZP drilling 처리를 받은 배반포기배의 부화율(81.8%)이 대조군 (54. 2%)이나 laser ZP drilling 처리를 받은 4-세포기배 (45.5%) 보다 유의하게 높게 나타났다(p<0.05). 또한, laser ZP drilling된 배반포기배를 가임신이 유도된 대리모에 이식하였던 바, 처리군 (48.7%)의 착상율이 대조군 (43.6%)보다 약간 높게 나타났다. 한편, 임신된 대리모 일부는 분만을 유도하였던 바, 태어난 모든 새끼는 처리군에 관계없이 정상적인 염색체수 (n=40), 정상적인 성장과 생식기능을 나타내었다. 이러한 결과는 1.48$\mu\textrm{m}$ diode laser를 이용한 ZP drilling이 생쥐배의 부화를 증진시키고 정상적인 임신을 유도할 수 있어 인간배에 대한 적용 간으성을 시사한다고 하겠다.

• 한국수정란이식학회지
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• 제26권4호
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• pp.265-270
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• 2011

Procymidone is a fungicide with anti-androgenic properties widely used to protect fruits from fungal infection, which induces an excessive reactive oxygen species production in male reproductive organs. In this study, to clarify whether procymidone affect the cellular antioxidant system of prostate at onset of puberty, gene expression patterns of the representative antioxidant enzymes such as cytoplasmic glutathione peroxidase (GPx1), phospholipid hydroperoxide GPx (PHGPx), selenoprotein P (SePP), cytoplasmic copper/zinc superoxide dismutase (SOD1), and manganese SOD (SOD2) were investigated in the rat ventral prostates exposed to procymidone using real-time RT-PCR analyses. Seven-week-old Sprague-Dawley rats castrated at 6 weeks old were treated with procymidone (25, 50, or 100 mg/kg per day) orally for 7 consecutive days after testosterone propionate (0.4 mg/kg per day) administration by subcutaneous injection. As compared to normal control animals, GPx1 mRNA expression in prostates significantly increased by the administration with TP and/or procymidone. However, PHGPx and SOD1 mRNA levels significanatly decreased by over 25 mg/kg of procymidone treatment and SePP and SOD2 mRNA levels was significanatly reduced by over 50 mg/kg of procymidone treatment. These findings indicate that procymidone may affect the antioxidant system of prostatic cells in up-regulation mode of GPx1, but in down-regulation modes of PHGPx, SePP, SOD1, and SOD2, suggesting that procymidone may affect differently the cellular antioxidant system of prostate according to the exposure doses.

• 농업과학연구
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• 제37권3호
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• pp.393-398
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• 2010

Early pregnancy diagnosis of bovine is an essential component for efficient reproductive plan in farms because long term of non-pregnancy results in economic losses by failure of offspring production and low milk yield in dairy cattle. The major steroid hormones related with reproduction are known to be progesterone and estrogen in bovine pregnancy. To evaluate detection level of hormones in milk, plasma and milk progestrone and estrogen of Holstein cows was analyzed during artificial insemination (AI) and embryo transfer (ET). Progesterone concentration at 21 days postestrus was significantly different in plasma and milk between pregnant and non-pregnant cows. Estrogen concentration at estrus was higher in pregnant recipients than that in non-pregnant recipients. To analyze correlation between hormone levels and conception rates in Holstein, the conception and return rates were checked following AI, and the returned cows were on the track of pregnancy after consecutive AI. Pregnant cows following first AI were considered as high conception group while pregnant cows following third AI were rated as low conception group. Proportion of high and low conception groups in this study was 78.2% and 9.1%, respectively. Hormone analysis indicated that high conception group had higher estrogen level during estrus than low conception group ($26.45{\pm}3.32$ vs $19.017{\pm}2.97$). Progesterone level was not different between high and low conception groups during estrus but increased significantly after 21 days postestrus (21 day: $4.95{\pm}1.12$ vs $0.95{\pm}0.23$, 35 day: $12.47{\pm}3.82$ vs $2.41{\pm}1.21$). In conclusion, the pattern of progesterone and estrogen secretion in Holstein milk samples could be a good candidate for early pregnancy detection and selection of recipients during ET.

• 한국수정란이식학회지
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• 제22권1호
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• pp.33-37
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• 2007

2년령의 암컷 비글개에서 교배 후 7일째에 혈액장액성 질 분비물과 외음부 종창의 주증을 보여 이를 진단하기 위해서 질 도말 검사, 초음파 검사를 실시하였다. 초음파 검사 소견상 양측 난소에 10mm 이상의 낭종성 구조물이 다수 관찰되어 양측성 다포성 난소낭종으로 진단하였다. 또한 질 도말 검사에서는 각화 상피세포가 90% 이상이었으며, 수컷의 허용 반응을 확인한 결과 수컷의 허용은 물론 정상적인 교배가 이루어졌다. 이상의 소견을 종합해 본 결과 본 증례는 양측성 다포성 난포낭종으로 진단하였으며 GnRH $50{\mu}g$으로 치료를 실시하였다. 치료후 2주째에 낭종의 크기는 현저하게 감소되었으며, 난포낭종으로 진단되기 전 최후 교배일로부터 68일째에 1두의 거대 태아를 분만하였다. 한편, 난포낭종 발생 시기와 임신이 이루어진 시기가 어느 시기인지를 판정하기 위하여 혈장 progesterone 농도 및 estrogen 농도를 측정한 결과, 난포낭종의 진단시 혈장 estrogen 농도는 78.0 pg/ml 이었고 난포낭종의 진단 전 최후 교배시 progesterone 농도는 6.87ng/ml, 난포낭종의 진단 시 progesterone 농도는 66.93ng/ml이었다. 이상의 결과를 미루어 볼 때 본 증례의 비글견에서 난포낭종이 발생한 시기는 발정 휴지기였으며, 난포낭종의 진단 전 최후 교배 시 임신이 이루어졌던 것으로 확인되었다.