• 한국가축번식학회지
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• 제25권3호
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• pp.259-267
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• 2001

본 연구는 저정자증 또는 불임축의 수태율 증진과 불임해결에 적용할 목적으로, 일차적으로 소 정자의 PVP, HA의 농도, 신선 및 동결정자, 정자의 농도, 활력, 정자의 수정능획득법 및 난자의 투명대 부착과 미부착별로 현미조작에 의해 난자의 세포질내 단일정자의 주입했을 때 수정율 및 체외발생율을 조사하였다. 1. 소 난자의 ICSI시 PVP 농도를 0.01, 0.02, 0.03, 0.05%별로 처리한 정자를 micropipette내에 흡입하여 세포질내 주입하였을 때 수정율과 분할율은 각각 72.7∼90.9% 및 38.5∼54.5%로서 0.02%농도에서 비교적 높은 수정율과 분할율을 나타냈다. 2. 소 난자의 ICSI시 HA농도를 0.01, 0.02%, 0.02%의 PVP + HA별로 micropipette내에 흡입하여 세포질내 주입하였을 때 수정율과 분할율은 각각 72.7%와 81.8% 및 45.5%와 54.5% 및 83.3%와 37.5%였다. 3. 소 난자에 신선 및 동결정자를 이용하여 ICSI법으로 수정시 수정율은 각각 93.3%, 86.1%였으며, 분할율은 60.0%, 46.7%로서 신선정자를 이용하였을 때가 동결정자를 이용했을 때보다 높은 수정율과 분할율을 나타냈다. 4. Heparin, BFF 및 His법으로 수정능획득 처리한 정자로 IVF 및 ICSI시 수정율은 각각 61.9%, 52.6%, 45.0% 및 85.7%, 78.9%, 65.0%였으며, 분할율은 각각 23.8%, 15.8%, 10.0% 및 61.9%, 52.6%, 50.0%로서, 수정능획득 처리에 있어서 heparin법이 다른 수정능획득 처리법에 높은 수정율과 분할율을 나타났다. 5. 투명대의 부착과 미부착별 난자로 IVF 및 ICSI 법으로 수정시 수정율은 각각 63.2%, 47.8%와 84.2%, 78.3%였으며 분할율은 각각 15.8%, 8.7%와 57.9%, 34.8%였다. 6. 소 체외성숙 난자에 IVF법과 ICSI법으로 수정시켰을 때 수정율은 각각 63.3%, 64.6%와 88.2∼90.0%였으며, 분할율은 각각 26.7%, 29.2%와 52.9%, 67.5%로서 ICSI법으로 수정시켰을 때 수정율과 분할율이 크게 향상되었다.

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

• 한국가축번식학회지
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• 제26권4호
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• pp.311-320
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• 2002

The objective of this study was to assess the developmental potential in vitro produced embryos frozen-thawed with the various containers, and also examined expression and localization of heat shock protein 70 at these embryos. For the vitrification, 2-cell, 8-cell and blastocyst stage embryos produced by in vitro fertilization (IVF) and nuclear transfer (NT) were exposed the ethylene glycol 5.5 M freezing solution (EC 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop, and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min. and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid and cryo-loop. However, survival rates by straw were relatively lower than other containers. The use of cryo-loop resulted in only survival of nuclear transferred embryos (43.7%). Also, there embryos after IVF or NT were analysed by semi-quantitive reverse transcription-polymerase chain reaction (RT- PCR) methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNh were higher thawed embryos than control embryos. Immunocytochemistry used to localize the hsp 70 protein in embryos. Two and 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some frozen-thawed embryos. However, in the control, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform In distribution. Therefore, this result suggests that the exploiting Hsp 70 in the early embryos may be role for protection of stress condition for increase viability of embryos within IVF, NT and there frozen-thawed embryos.

• 한국수정란이식학회지
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• 제23권2호
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• pp.93-100
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• 2008

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient macromolecule in the porcine in vitro production (IVP) technology. To choose the efficient macromolecules in the development of porcine embryos, the effects of 3 kinds of macromolecules (porcine serum; PS, porcine follicular fluid; pFF, and polyvinyl alcohol; PVA) supplemented in IVM media on the maturation, cleavage, and development rates to blastocyst of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were examined. The maturation rates of porcine oocytes in media supplemented with PS were significantly higher than those with pFF and PVA (92.4% vs. 85.4%, 77.1%; p<0.05). In the cleavage and development to blastocyst rates, supplement with PS or pFF in the IVM media was more effective than PA. However, there were no significant differences in cleavage and development to blastocyst between PS and pFF group. From the results of this study, it was demonstrated that PS was optimal macromolecule in the porcine IVM media.

• 한국발생생물학회지:발생과생식
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• 제10권4호
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• pp.239-245
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• 2006

본 연구는 소의 미성숙 난포란의 체외성숙시 ${\beta}-mercaptoethanol({\beta}-ME)$의 첨가가 체외성숙, 체외수정 후 웅성전핵의 형성 및 세포질 내의 GSH 수준에 미치는 영향을 알아보고자 실시하였다. 체외성숙시 $25\;{\mu}M$과 $50\;{\mu}M$의 ${\beta}-ME$를 첨가한 경우 대조구에 비하여 성숙율이 증가하는 것으로 나타났으며(p<0.05), 모든 실험구에 있어서 12시간 체외성숙보다 24시간 체외성숙에서 높은 성숙율을 나타냈다(p<0.05). 체외수정 후 웅성전핵 형성에 있어서는 $25\;{\mu}M$와 $50\;{\mu}M$ 농도의 ${\beta}-ME$ 첨가구에서 대조구에 비하여 높게 나타났으나(p<0.05), $25\;{\mu}M$과 $50\;{\mu}M$ 농도구와의 유의적인 차이는 없었다. GSH의 수준은 체외성숙 후 $50\;{\mu}M$의 ${\beta}-ME$ 첨가구가 다른 처리구에 비교하여 높게 나타났으며(p<0.05), 체외수정 후 웅성전핵이 형성된 다음 세포질 내 GSH 수준 역시 $50\;{\mu}M$의 ${\beta}-ME$ 첨가구에서 가장 높은 결과를 나타냈다(p<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.225-234
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• 2004

Objectives: To evaluate the efficacy of GnRH antagonist cetrorelix in women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) and to determine changes in serum hormone concentrations during cetrorelix administration. Methods: We performed a clinical trial on 30 patients undergoing COH with highly purified follicular stimulating hormone (HP-FSH) and gonadotropin releasing hormone antagonist (GnRHant), cetrorelix. FSH was administrated from day 2 or 3 of cycle with fixed dose and adjusted according to individual response. 0.25 mg of cetrorelix was injected daily subcutaneously from stimulation day 5 until the day of hCG administration. Daily ultrasound monitoring was performed for growing follicles and serum levels of luteinizing hormone (LH), estradiol ($E_2$) and progesterone were measured daily during cetrorelix administration. Up to 4 embryos were transferred. Results: Mean age of enrolled patients was $32.0{\pm}3.4$ years (mean $\pm$ S.D.). All of 30 patients underwent oocyte pick-up, and embryo transfer was done in 28 patients. The total and mean numbers of received oocytes were 196 and $6.5{\pm}4.7$, the number of fertilized eggs was 111, and the fertilization rate was 56.6%. Total duration of FSH administration was $9.2{\pm}2.2$ days and mean of $24.3{\pm}7.7$ ampules of HP-FSH was administered. Total duration of cetrorelix administration was $5.7{\pm}1.9$ days. Serum LH and progesterone levels were maintained in the range of $1.4{\sim}2.9\;mIU/mL$ and $0.3{\sim}0.6\;ng/mL$, which respectively reflected effective prevention of premature LH surge. Clinical pregnancies were achieved in 9 patients, and overall clinical pregnancy rate was 30.0% per oocyte retrieval, and 32.1% per embryo transfer. Conclusion: GnRH antagonist is safe and convenient for COH for IVF-ET and effective with optimal pregnancy rate.

• Reproductive and Developmental Biology
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• 제38권3호
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• pp.115-121
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• 2014

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, $0.1{\mu}g/mL$, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by $0.01{\mu}g/mL$ compared with other groups (p< 0.05). Moreover, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supplementation of Humulus japonicus extract in IVF medium may have important implications for improving early embryonic development in bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제27권10호
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• pp.1417-1425
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• 2014

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.

• Clinical and Experimental Reproductive Medicine
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• 제15권2호
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• pp.149-155
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• 1988

To increase fertilization rate in vitro, separation of viable spermatozoa from the seminal plasma and its other components may be a useful procedure. Ejaculates from healthy men, whose semen analysis findings were normal in 19, and abnormal in 10, were filtered using the glass-wool filtration technique to yield a concentrated, viable sperm samples for IVF, and the usefulness and safety of this method were evaluated. The recovery rate of motile sperm in abnormal groups was 46.2% and 54.5% in normal group. The % motility was increased significantly compared with original sample after filtration, and the grade motility was improved, too. The sperm population with normal morphology was also increased significantly in both group. Using transmission electron microscopy, the ultrastructural integrity of acrosomal segment was examined in order to evaluate the potentially hazardous effect of glass-wool filtration to sperm head, however, sperm population with normal ultrastructure was increased compared with that of original ejaculate after separation. The filtered sperm was then processed for IVF, as the fertilizing capacity is the ultimate parameter of the sperm function. In abnormal group, the fertilization rate(41.5 %) and the ET rate per stimulated cycle were much lower than that of mormal group(69.6%). However, the cleavage rate and the number of embryos transfered per ET cycle were comparable with those of nomal group. The results suggest that the glass-wool filtration of sperm, particularly in oligo-asthenozoospsrmia, may be useful and safe method in the preparation of sperm for IVF.

• 한국수정란이식학회지
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• 제12권2호
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• pp.181-188
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• 1997

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7$\beta$. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% $CO_2$ in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.