• Reproductive and Developmental Biology
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• 제37권3호
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• pp.97-102
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• 2013

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under $30^{\circ}C$ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in $0.3^{\circ}C$. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group ($43.3{\pm}4.7%$) was significantly (p<0.05) higher than other treatment groups (Tissue: $16.3{\pm}2.7%$ and Water: $27.5{\pm}3.1%$), dying sperm (SYBR+/PI+) in Air treatment group ($55.6{\pm}4.7%$) was significantly lower than other treatment groups (Tissue: $77.6{\pm}3.2%$ and Water: $67.6{\pm}3.3%$) (p<0.05). Acrosome reaction in Air treatment group ($0.2{\pm}0.1%$) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: $0.7{\pm}0.2%$ and Water: $0.5{\pm}0.1%$), the acrosome reaction in Air treatment group ($28.6{\pm}2.8%$) within all sperm also was significantly lower than other treatment groups (Tissue: $44.2{\pm}1.8%$ and Water: $36.2{\pm}2.0%$) (p<0.05). And mitochondrial intact in Air treatment group within live ($97.1{\pm}0.4%$) and all ($61.9{\pm}3.3%$) sperm were significantly higher than other treatment groups (Tissue: $85.2{\pm}3.3%$, Water: $87.8{\pm}2.9%$ within live sperm and Tissue: $49.28{\pm}3.7%$, Water: $42.0{\pm}3.1%$ within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.9-17
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• 2004

Objective: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. Methods: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG + 0.2 M sucrose or 1.5 M PROH + 0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. Results: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. Conclusion: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

• 한국산학기술학회논문지
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• 제21권4호
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• pp.490-496
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• 2020

가축유전자원으로서 동결정액을 생산하는 가장 쉬운 방법은 스티로폼박스를 이용한 간이동결법으로 알려져 있다. 본 연구에서는 스티로폼 동결박스를 제작하여 가축의 동결정액 생산에 활용하는 방법을 검토하였으며 칡소 동결정액 생산을 최적화 할 수 있는 방법을 제시하고자 박스의 크기, 액체질소와 노출된 정액 스트로와 거리, 노출시간 및 생산량을 검토하였다. 2가지 동결박스를 비교하여 자료를 확보하였으며 내부 크기는 세로×가로×높이가 23.5×30.5×22.5 cm와 25.5×46.5×26.5 cm로 측정되었다. 액체질소를 5cm 높이로 채우고 액체질소 위 2, 5 및 8cm 높이에서 동결하여 융해 후 생존성을 조사하였다. 칡소 정액을 동결할 경우, 액체질소와의 노출시간은 모두 10분이 적절하였으며 25.5×46.5×26.5 cm 크기의 상자가 높은 생존율을 보여주었다(60.4±5.3% 대 67.2±3.1%). 동결 상자의 최적화 공간은 정자 동결에 가장 중요한 요소로 판단되며 1회 동결 시 최대 생산 가능한 칡소 동결정액은 60개 이상으로 증가시킬 수 있었다. 이러한 정보를 활용하면, 축종에 따라 동결 정액 생산량 결정하고 목적에 맞는 용기를 활용하여 효율적인 동결정액 생산이 가능할 것으로 판단된다.

• Journal of Chest Surgery
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• 제37권8호
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• pp.623-631
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• 2004

배경: 대망의 가장 중요한 성질 중 하나인 혈관 형성 촉진 기능을 이용하여 초냉동 보관된 기관 이식편의 대망 내 이식이 기관 이식편의 생육성이나 혈관 형성에 미치는 영향을 연구하고자 한다. 대상 및 방법: 8주령의 Sprague Dawley rat 수컷의 초냉동 보관된 기관 이식편을 백서의 복강 내 대망에 이식하였다. 연구군은 냉동 보관기간과 대망 내 이식기간에 따라 4개의 군으로 분류하였다(n=52). 이식된 기관 이식편을 획득하여 기관 평활근 및 주변 결합 조직의 섬유화 및 염증 정도, 기관 연골의 석회화 정도, 기관 내 상피세포의 변화 및 연골간 간격에서의 혈관 형성 정도 등을 검사하였다. 결과: 기관 평활근의 염증 정도는 냉동 보관기간이나 대망 내 이식기간에 따라 유의한 차이를 보이지 않았고, 기관 연골의 석회화 정도는 냉동 보관기간과 상관없이 대부분 심하게 진행되어 있었다. 혈관 형성은 기관 이식편의 양끝뿐만 아니라 중간 부위에서도 충분히 이루어져 있었다. 결론: 초냉동 보관된 백서의 장분절 기관 이식편을 2주간 대망 내 이식을 시행한 결과 기관 이식편의 조직 생육성이 적절히 유지되면서 새로운 혈관이 형성되었다. 향후 동종 기관 이식 시 초냉동 보관된 기관 이식편을 대망 내 이식하여 새로운 혈관이 형성된 후 단계적으로 기관 이식을 시행하는 데 도움이 될 수 있다고 생각한다.

• Reproductive and Developmental Biology
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• 제40권3호
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• pp.27-31
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• 2016

The aim of this study was to evaluate effect of ${\alpha}$-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at $37.5^{\circ}C$ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

• 한국수정란이식학회지
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• 제28권2호
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• pp.141-147
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• 2013

The purpose of this study was attempted to new methods in mammalian embryos vitrification. This method was affected to increase of the embryo vitrification efficiency and it would be applied to the field of embryo transfer to recipient by modified loading method of embryo into 0.25 ml plastic straw. The frozen mouse embryos were carried out warmed from two different cell stages (8-cell and blastocyst, respectively) by attachment of an embryo in the vitrification straw (aV) method. All groups were cultured in M-16 medium to determine the development and survivability for 24 h, respectively. Results shown that, the survivability of two different groups were significantly different (94.8% vs. 70.9%). Total cell number was not significantly different the non-frozen blastocyst ($99.7{\pm}12.4$) compared to the post-thaw blastocyst ($94.8{\pm}15.1$). From the 8-cell embryo, total cell number of frozen blastocysts were significantly lower than others groups ($74.7{\pm}14.6$, p<0.05). In the case of cell death analysis, the blastocysts from non-frozen and frozen-thawed 8-cell group were not different ($0.0{\pm}0.0$ vs. $1.9{\pm}3.1$, p>0.05). However, the apoptotic nuclei of blastocyst were significantly observed the frozen-thawed group ($5.4{\pm}4.4$) compared to non-frozen group (p<0.05). Therefore, this new method of embryos using in-straw dilution and direct transfer into other species would be more simple procedure of embryo transfer rather than step-wise dilution method and cryopreservation vessels, so we can be applied in animal as well as human embryo cryopreservation in further.

• 한국수정란이식학회지
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• 제7권2호
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• pp.81-88
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• 1992

Cryopreservation of mouse embryos biopsied at 4-cell stage was investigated by ultrarapid freezing. Four-cell embryos were obtained from ICR mice on 55h after hCG injection. Zona pellucida of the embryos were partially dissected with a cutting pipet, and then single blastomeres were biopsied from the embryos followed by incubation in $Ca^2$+ and $Mg^2$+-free M16 medium for 30min. Biopsied embryos cultured for lh or 15h were frozen by ultrarapid freezing method using 3M DMSO or 5M glycerol as a cryoprotectant, respectively. The developmental rate of biopsied embryos after ultrarapid freezing and thawing to blastocysts was 81 % in the group of biopsied embryos cultured for lb and 98% in the group of biopsied embryos cultured for 15h, respectively. When biopsied embryos after ultrarapid freezing and thawing were transferred to the uteri of pseudopregnant recipients, normal live young were born. These results suggest that this freezing method can efficiently cryopreserve biopsied mouse embryos.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.98-98
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• 2003

• 한국자원식물학회지
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• 제32권6호
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• pp.730-734
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• 2019

Cryopreservation is one of the ideal and suitable methods for long-term storage of plant germplasm. The plant contaminated with diseases and pathogens are decreased the multiplication rate, survival rate and high quality of plants after cryopreservation. The aim of this work was to improve a micropropagation method for lily in Korea, which is indigenous plant. In the last process of rinsing scales after surface-sterilization, we tried to control the diseases and pathogens lived within the tissue by rinsing in 0.03% sodium hypochlorite (NaClO) instead of sterile distilled water. Bulb scales of Lilium were cultured in vitro on MS medium supplemented with Plant Preservative Mixture (PPM). The results showed that L. tsingtauense accessions were observed ranged from 53.9 to 100% with a mean value of 76.8% and L. hansonii accessions were checked from 84.5 to 85.5% with a mean of 85% survival rate. The newly small bulb formed from bulb-scales was transferred to MS medium. We checked the presence of microorganisms and survival rate after 3 weeks in culture after examination of bacterial incidences. The results indicated that the non-contamination rate were shown ranged from 75.0 to 94.1% with mean value of 83.2% in L. tsingtauense species, and that L. hansonii were observed 85.1 to 91.7% with mean value of 88.4%. This study will provide a valuable basis for establishment of effective axenic cultures for in vitro micropropagation of Korean native lily species.

• 한국발생생물학회지:발생과생식
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• 제11권2호
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• pp.79-84
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• 2007

냉동보존이 진주조개(Pincata fucata martensii) 유생의 형태 및 구조에 미치는 영향을 알아보기 위하여 냉동전후 D형 및 각정기 유생을 광학 및 전자현미경으로 조사하였다. 동해방지제는 0.2 M sucrose를 첨가한 2.0 M $Me_2SO$를 사용하였다. 냉동후 유생은 일부 패각이 손상되긴 했지만 hinge와 prodissoconch가 뚜렷하게 나타났으며, 소포체, 지질 과립, 미토콘드리아, 핵 등을 포함한 세포내 소기관들이 고르게 분포되어 있었다. 또한, 섬모가 규칙적으로 배열되어 있었고, 섬모 아래 미토콘드리아와 지질과립이 위치해 있는 것이 관찰되었으나, 일부 해동된 유생에서 섬모의 불규칙적인 배열과 섬모환이 둥글게 뭉쳐져 있는 모습이 관찰되었다. 이러한 결과는 진주조개의 D형 유생과 각정기 유생이 냉동에 쉽게 영향을 받을 수 있다는 것을 보여준다. 따라서 냉동보존 시 세포의 손상을 감소시킬 수 있는 연구가 이루어져야 할 것으로 사료된다.