• 한국동물학회지
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• 제39권2호
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• pp.173-181
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• 1996

The time-course process by which spermatozoa penetrates through the micropvle apparatus into the egg cytoplasm of rainbow trout, Oncorhvnchus mvkiss, was examined with transmission and scanning electron microscopy. In the unfertilized egg, the ess surface beneath the inner opening of the micropylar canal did not differ distinctly from the rest of the animal pole area. A spermatozoon attached to the micropvle opening 20 seconds after insemination. In the initial stases of penetration, the spermatozoon still within the micropvlar canal attached perpendicularly at its apical tip to the ess surface, then the sperm head was rapidly engulfed by the folded egg surface with its manly microvilli. A large fertilization cone with microvillus-free surface appeared on the esS surface sutra-rounding the penetrating spermatozoon. The head portion of the penetrating spermatozoon was completely wrapped by the ess surface with only the tail portion visible externally 30 seconds after insemination. The fertilization cone displayed the tail portion of the penetrating spermatozoon on the central portion of its surface 60 seconds after insemination. 150 seconds after insemination, breakdown of the cortical granules elevation were initiated at the animal pole, then completed at the vegetable pole area. The spermatozoon disappeared from the outer surface of the ess before the fertilization cone completely retracted 250 seconds after insemination. In result, the block to polvspermv to permit entry of a sin81e sperm is considered to be mechanical by the rnorpholoSical design of the micropvle and fertilization cone.

• 한국수정란이식학회지
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• 제14권1호
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• pp.9-15
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• 1999

This study was conducted to investigate the effect of $\alpha$-tocopherol and cysteamine with Whitten's medium in supporting the development on in vitro maturation(IVM), in vitro fertilization (IVF) and in culture(IVC) on porcine oocytes. When the immature oocytes were cultured of $\alpha$-tocopherol for 40h, the nuclear maturation rates were 39, 4, 52.5 and 54.1%, respectivley. The nuclear maturation rates of treat groups were signficantly (P<0.05) higher than those of non-treat groups. After matureation, the oocytes were inseminated in vitro in medium 199 with ejaculated spermatoza for examination of sperm penetration, polyspermy, male pronuclear(MPN) formation, and cleavage rate. Sperm penetration rates of treat higher than the control groups(P<0.05), and MPN formation rates were significantly(P<0.05) higher on treated groups (24.3~53.1%) than control groups(14.2~21.4%). After insemination, the cleavage rates at 120hr were groups higher than control groups(P<0.05).

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1996년도 추계 학술대회 및 정기총회
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• pp.52-52
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• 1996

• Journal of Animal Science and Technology
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• 제65권6호
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• pp.1205-1213
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• 2023

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

• Clinical and Experimental Reproductive Medicine
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• 제18권1호
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• pp.81-87
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• 1991

There studies were carried for evaluation of the efficiency of freezing of hamster oocytes for use in a human sperm penetration assay. The hamster oocytes fully equilibrated in various cryoprotectant agents and inseminated with human sperm. After insemination with hamster oocytes, there was no difference in penetrated rates. Cumulus free oocytes equilibrated in 1.5M various cryoprotective agents and slowely cooled to temperature $-30^{\circ}C$ before rapid cooling and storage in nitrozen tank. After rapid thawing, survival rates of frozen oocytes according to cryo-protective agents were examined and the human sperm penetration assay with zona free hamster oocytes was conducted. 1. Survival rates of oocytes after cryoprotectants exposure have no significant difference (range 88-91%) and peneration rate was 51.1%. 2. Recovery and survival rate of frozen-thawed oocytes were 85.1 and 66.8%. There was no significant difference on cryoprotective agents. 3. Penetration rates of the frozen-thawed and intact oocytes were 69.0 and 77.0%, respectively. 4. Hamster oocytes cryopreservation provides a convenient way of supplying and trans-porting hamster oocytes for the assessment of the fertilizing potential of human spermatozoa.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.57-66
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• 2002

Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.

• 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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• 제23권3호
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• pp.229-237
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• 1999

본 연구는 돼지 동결-융해 정자의 전배양시 aseorbie acid (Ase)와 ferrous sulfate (Fe$^{2＋}$)가 정자의 수정능력획득, 첨체반응 및 난자내 침입능력에 미치는 영향을 검토하였다. 정자의 전배양시 0~1.0 mM의 Fe$^{2＋}$의 첨가는 비전배양에 비해 높은 첨체반응 (P＜0.05) 및 정자침입율을 얻었다. 이와 같은 결과는 0~0.5mM의 Ase 첨가 시 첨체반응율에서는 같은 결과를 나타냈지만 정자침입율은 오히려 정자의 전배양 보다는 비전배양시 높은 비율을 나타냈다. 한편, Fe$^{2＋}$가 함유 되어있는 배양액내에서 2시간동안 정자의 전배양시 0.1 mM Asc의 첨가는 0.5 mM Ase의 첨가에 비해 유의적으로 높은 첨체반응율을 나타냈으나 (P＜0.05), Ase의 농도사이에서 정자침입율에는 차이가 없었다. 또한, Ase가 함유된 배양액내에서 정자의 전배양시 0.1 mM Fe$^{2＋}$를 첨가했을 때 첨체반응율은 Fe$^{2＋}$ 무첨가시 유의적으로 높았으나 (P＜0.05), 오히려 가장 낮은 정자침입율을 나타냈다. 이와 같은 결과는 체외에서 돼지정자의 처리시 Fe$^{2＋}$ 또는 Ase의 첨가와 정자의 전배양에 의해 첨체반응과 정자침입에 효과적인 작용을 하는 것으로 생각된다.

• 한국가축번식학회지
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• 제21권3호
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• pp.239-246
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• 1997

본 연구는 돼지 난포란의 체외수정시 정소상체 미부정자의 형태학적 정상정자 비율에 따른 수정능의 차이를 조사하기 위하여 실시하였다. 그 결과는 다음과 같다. 1. 정소상체 미부로부터 회수된 전체의 정자중 형태학적 정상정자의 비율에 따른 ( 10%, 10~30%, 50%) 정자침입율과 다정자침입, 전핵형성율 그리고 난자에 침입한 평균 정자수를 조사하였던바, 50%의 정자침입율과 다정자침입은 82.4%와 87.4%로 10% (29.7%, 22.6%)와 10~30% (20.3%, 37.0%)보다 유의하게 높게 나타났다 (p<0.01). 또한, 공시된 난자의 전핵형성율도 50%이상의 형태학적 정상정자를 가진 실험군에서 유의하게 높게 나타났다(p<0.01). 2. 50% 이상의 정상정자를 가진 정소상체 미부정자를 100% (5$\times$105 cells/ml)로 조정하여 수정시킨 후 그 결과를 동결 융해된 사출정자와 비교하였던 바, 다정자침입과 전핵형성율은 정소상체 미부정자 (86.7%, 35.1%)와 동결 융해된 사출정자 (86.0%, 39.4%)간에 차이를 나타내지 않았으나, 정자침입율은 정소상체 미부정자가 79.7%로 동결 융해된 사출정자의 95.5%에 비해 유의하게 낮게 나타났다(p<0.01) 3. 또한, 정소상체 미부정자군에서 난자와 정자의 비율 (1:6000, 1:6650, 1:7700, 1:10000)에 따라 정자침입율과 다정자침입 그리고 전핵형성의 차이를 조사하였던 바, 수정시 난자당 정자의 수가 증가할수록 정자침입율, 다정자침입 그리고 난자에 침입한 평균 정자 수는 함께 증가하는 것으로 나타났다. 그러나, 전핵형성율은 난자당 정자의 수가 1:6000과 1:6650에서 높게 나타났다. 이상의 결과는, 돼지 난포란의 체외수정시 정소상체 미부정자를 사용했을 때, 50% 이상의 형태학적 정상정자를 가진 정자를 사용하면 동결 융해된 정자와 유사한 수정율을 얻을 수 있었으며, 정자의 형태학적 평가는 좀더 효율적인 수정능획득을 위해 선행되어져야 한다는 것을 시사한다고 하겠다.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.27-34
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• 2006

This study was conducted to compare the reproduction ability of the wild type boar and recombinant human erythropoietin (hEPO) transgenic boar semen. Ejaculated boar semen was analyzed by flow cytometry, Elisa and IVF methods. In experiment 1, flow cytometric analysis showed that the live sperm ratio of transgenic boar sperm significantly lower (P<0.05) than that of wild type boar after incubation at 20, 22, 24 and 26 hr. In experiment 2, the presence and levels of various cytokines (IL-6, IL-10 and $TNF-{\alpha}$) to related animal reproduction in the seminal and blood plasma were examined using specific enzyme immunoassay. There was no significant difference between both groups. In experiment 3, the fertilizing capacity and developmental ability of both boar sperm were compared. The transgenic boar sperm had a significantly low capacity of penetration, sperm-zona binding, embryo development, and blastocyst formation compared to wild type sperm (P<0.05). These results suggest that transgenic boar sperm harboring hEPO gene has low sperm viability than wild type boar, and it is a reason to decrease of fertility and litter size.