• 한국수정란이식학회지
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• 제31권3호
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• pp.299-305
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• 2016

Although cryopreservation of sperm is routinely used for clinical requirement, it has some problems, such as high generation of reactive oxygen species (ROS) and cold-shock. To reduce the detrimental damage in sperm, anti-oxidants were added to cryoprotectant for sperm. Curcumin is one of anti-oxidants, which are added in cryoprotectants. However, recent studies have demonstrated that curcumin decreases sperm viability and motility. This study was performed to identify the effect of curcumin on hydrogen peroxide ($H_2O_2$)-exposed bovine sperm, which were cryopreserved-thawed. In $H_2O_2$-exposed bovine sperm, reactive oxygen species (ROS) were significantly reduced by treatment with curcumin in a dose-dependent manner (p < 0.05). Among tested concentrations of curcumin (1 to $50{\mu}M$), 30 and $50{\mu}M$ curcumin showed anti-oxidant effect on $H_2O_2$-induced ROS generation. On the other hand, combination of 30 or $50{\mu}M$ curcumin with anti-oxidant $H_2O_2$ increased the percentage of apoptotic sperm compared to only $H_2O_2$ treatment. Sperm viability was also decreased in the combination of 30 or $50{\mu}M$ curcumin with $H_2O_2$ as judged by FDA/PI staining. $H_2O_2$-induced decrease in sperm progressive motility was recovered by treatment with $1{\mu}M$ curcumin. These results show that high concentration of curcumin has anti-oxidant effect, but it has also cytotoxic effect on bovine sperm. Sperm viability and motility might be more affected by cytotoxic signals of curcumin compared to antioxidant signals.

• 한국수정란이식학회지
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• 제27권3호
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• pp.155-162
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• 2012

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY ($69.00%{\pm}4.18$; EG and $63.00%{\pm}9.75$; 7% G) than 8% LDL ($57.00%{\pm}5.70$; EG and $52.00%{\pm}4.47$;G). Treatment of 4% LDL + 5% EY-EG ($66.85%{\pm}5.06$) has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG ($64.65%{\pm}6.10$) among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

• 한국발생생물학회지:발생과생식
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• 제11권3호
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• pp.219-226
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• 2007

본 연구는 생식선 키메라 생산효율을 높이기 위한 방법으로 busulfan 가온 주입법을 이용하여 효과적인 원시생식 세포의 이동능력을 검증하였다. 효율적인 생식선 키메라 닭 생산에서 중요한 요건 중 하나인 공여체 원시생식세포의 생존율을 측정한 실험에서는 시간이 지남에 따라 생존율에 변화를 보였으나, 평균 $70{\sim}80%$을 유지하고 있었으며, busulfan 처리 유무에 따른 공여체 원시생식세포 이동능력은 형광염색 후 주입한 실험에서 대조구가 4.8%인 반면 실험구는 23.5%을 나타냈다. 이식전 원시생식세포 배양 조건에 따라, 96시간과 118시간 배양 처리구에서 높은 이동능력을 보여 주었다. 원시생식세포의 형태학적, 생리학적 특징을 응용한 이식방법은 매우 효과적일 것이다. 그리고 본 연구에서는 생식반월의 발달단계 별 busulfan 처리 효과는 48시간이 가장 높은 53.4%였으며, 그러나 본 연구에서는 생식반월 유래 원시생식세포 이식은 48시간 이전, 혈관계가 발달하기 직전으로 가장 높은 효율을 보였다. 결론적으로 생식선 키메라 방법을 통한 형질전환 닭 생산 연구의 가장 큰 관건은 최대한 많은 수의 공여체 원시생식세포가 수용체의 저해작용 없이 안정적으로 수용체 gonad로 이동하여 분화하는 것으로, 본 연구 결과를 토대로 개선된 방법을 이용하면 높은 효율의 생식선 키메라 닭이 생산될 것으로 사료된다.

• 한국수정란이식학회지
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• 제25권4호
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• 한국수정란이식학회지
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• 제18권1호
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• pp.51-59
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• 2003

본 실험은 개의 인공수정에 사용할 정자의 보존에 있어서, 개 정액 동결시 동결속도와 응해온도에 따른 정자의 생존율, 운동성 그리고 intact acrosome의 비율을 조사하였던 바 결과는 다음과 같다. 1. 본 실험에서 실험견의 사출된 평균 신선정액의 농도는 3.44$\times$$10^{8}$ /ml로 정상범위에 들었으며, 정자의 형태학적 판정에서 정상적인 정자의 농도는 평균 59.45$\pm$3.45%로 상대적인 기형율은 약 30~40% 정도 나타났으며, 이는 정상적인 상태의 개 정액이라 할 수 있다. 2 개 정액의 동결속도는 동결하는 높이가 6, 10 및 17 cm 일 때 각각 최저온도는 -11$0^{\circ}C$, 7$0^{\circ}C$, -35$^{\circ}C$로 감소하는 경향을 보였으며 이때 최저온도로 감소하는데 소요되는 시간은 각각 6분, 8분 20초 그리고 12분 50초로 결과적으로 분당 동결속도는 각각 19$^{\circ}C$/min, 8.9$^{\circ}C$/min 그리고 3$^{\circ}C$/min로 나타났다. 3. 정액을 동결속도와 융해온도에 따른 정자의 생존율, 운동성 그리고 intact acrosome의 비율은 동결속도가 3$^{\circ}C$/min일 때 가장 높았으며, 융해 온도는 37$^{\circ}C$일 때 효율성이 가장 높은 것으로 나타났다.4. 동결의 방법에 있어서는 액체질소의 표면으로부터 17 cm 높이에서 동결하는 분당 -3$^{\circ}C$의 동결속도에서 동결하여 37$^{\circ}C$에서 2분간 융해하는 방법이 가장 좋은 결과를 보였으며, 생존성과 운동성은 문제없이 사용할 수 있을 것으로 판단되나, 첨체의 intact한 비율은 저조한 결과를 나타내었다.

• 한국수정란이식학회지
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• 제30권4호
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• pp.305-313
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• 2015

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level.

• 한국수정란이식학회지
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• 제4권1호
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• pp.35-40
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• 1989

The effects of cryoprotectants (glycerol, DMSO and ethylene glycol) and the concentrations (0, 0, 25, 0.5and 1.0 M) of sucrose in the diluent on the is vitro survival of mouse morulae froaen rapidly in liquid nitrogenvapour were examined. When the embryos were equilibrated in 1.5 M cryoprotectants +0.25 M sucrose in one-step or in 3.0 M cryoprotectants +0.25 sucrose in two-step and diluted with 0, 0.25, 0.5, or 1.0 M sucrose solution after thawing, high survival rates were obtained in ethylene glycol (48.0% to 88.2 %) or in glycerol (35.0 % to 77.8 %). These results show that 1.5 M ethylene glycol is a highly efficient cryoprotective agent for the rapid freezing of mouse morula embryos and 0.5 M sucrose was optimal concentration in the diluent after thawing.

• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.253-259
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• 2000

Objective: To verify the effect of Matrigel, a ECM complex from Engelbreth-Holm-Swarm (EHS) mouse sarcoma on the preimplantation development and apoptosis of mouse fertilized eggs. Method: Late pronucleus stage eggs were cultured through the blastocyst stage in the presence of Matrigel (0.5%, v/v). Characteristics of apoptosis and cell number assesed by Hoecst staining and TUNEL labeling at the blastocyst stage, respectively. Results: Morphological development, number of cells per embryo was significantly increased but rate and number of TUNEL positive nuclei of the embryo were decreased in the presence of Matrigel. Conclusion: This result suggested that at low concentration of Matrigel improves both viability and morphological development in the preimplantation mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.35-42
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• 1997

The present study was designed to define the role of prostaglandin in the development and hatching of mouse embryo. The effects of indomethacin, an inhibitor of prostaglandin synthesis, on the development and hatching of morula and blastocyst were examined. In early morula stage, embryos were degenerated significantly at 100 ${\mu}M$ and 200 ${\mu}M$ indomethacin. However, the viability of embryos was not influenced by concentration in any other embryonic stages. In all embryonic stages, the hatching was suppressed with concentration dependent manner, but expansion was not suppressed. Particularly, in 84h embryos post hCG injection, the hatching was suppressed significantly compared with post hCG 72h or 96h embryos. When embryos were treated with 100 ${\mu}M$ indomethacin for a specific time (12h) in according to the development stage, the hatching was suppressed all groups. These suppressional effect was decreased as embryonic development stage was progressed. However, the expansion was not affected in all treatment group. This study suggests that hatching-related metabolic substances are synthesized from morula stage and intraembryonic signaling mediated prostaglandin was important for development and hatching of mouse embryo.

• 한국수정란이식학회지
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• 제15권1호
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• pp.95-102
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• 2000

Techniques for manipulation of spermatozoa and oocytes have been widely used for in vitro production(IVP) of Hanwoo. This study was conducted to examine the effects of theophylline and heparin on frozen-thawed Hanwoo sperm for enhancing the efficiency of IVP technique. Oocytes were inseminated with forzen bull semen treated with either theophylline or heparin for examining the effect of each substance on fertilization and subsequent development. More (P<0.05) oocytes formed pronucleus and develop to the morula and blastocyst stages after inseminated with sperm treated with heparin than after inseminated with sperm treated with theophylline. The pregnancy rate after embryo transfer was higher after heparin treatment than after theophylline treatment, but did not differ significantly. There was no significant difference of offspring delivery between two groups. In conculsion, theophylline and heparin can be used for enhancing the efficiency of IVP system for Hanwoo. Considering characteristics of these substance, theophylline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.