• Reproductive and Developmental Biology
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• 제34권3호
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• pp.271-274
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• 2010

This study was conducted to investigate the survival rate of frozen-thawed spermatozoa in equine by glycerol concentration and freezing speed Two stallions (1 Thoroughbred-13 year old and 1 Arab-7 year old) bred in Korea Racing authority was examined for 1 times in a couple of weeks. Semen was collected by condom method standing heated mare and were centrifuged 650 g for 15 min. and isolated the seminal plasma. Thick fraction of semen was diluted EDTA-Lactose-egg yolk diluents to 1:1 and contained in 0.5 ml straw as $6{\sim}14{\times}10^7\;cells/ml$. Final concentrations of glycerol were 3, 5 and 7% in cryopreseved diluents and added 4 times for 2 hours equilibration. For the freezing, equilibrated straws were located 3 or 5 em above $LN_2$ gas for 5 or 10 min. Survival rates of pre-frozen sperm were $65.0{\pm}13.2%$, $68.3{\pm}10.4%$, $66.7{\pm}11.5%$ and post-frozen were $53.3{\pm}23.1%$, $45.0{\pm}15.0%$, $50.0{\pm}18.0%$ in 3, 5, 7% glycerol concentration, respectively. There was no difference between glycerol concentrations. Survival rates of frozen-thawed sperm on freezing speed were $36.7{\pm}10.4%$, $40.0{\pm}7.1%$, $30.0{\pm}13.2%$ at 3 cm-5 min and $33.3{\pm}11.5%$, $31.7{\pm}2.9%$, $21.7{\pm}10.4%$ at 3 cm-10 min in 3, 5, 7% glycerol concentration, respectively. Survival rates of frozen-thawed sperm on freezing speed were $43.3{\pm}15.3%$, $32.0{\pm}17.9%$, $22.3{\pm}15.7%$ at 5cm-5 min and were $47.5{\pm}15.0%$, $43.3{\pm}12.6%$, $48.3{\pm}15.3%$ at 5cm-10 min in 3, 5, 7% glycerol concentration, respectively. There were significantly different between groups (p<0.05). These results suggest that glycerol concentration did not affect cryopreservation of stallion semen within 3~7% but freezing speed affects. In our experiment, the best cryopreservation condition was at 5 cm above $LN_2$ gas for 10 min for pre-freezing and 7% of glycerol concentration. These results lead to commercial AI with frozen-thawed stallion semen.

• 한국양식학회지
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• 제17권1호
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• pp.46-50
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• 2004

In the present study, attempts were made to cryopreserve sea urchin, Anthocidaris crassispina sperm in liquid nitrogen, to evaluate the effects of various cryoprotectants and freezing rates on motility, survival rate and fertilization rate of the post-thawing sperm, and the ultrastructural changes of sperm after cryopreservation were observed. The highest values of sperm motility (motility index: 3.3$\pm$0.37) and survival rate (72$\pm$3.5%) were obtained with 15% dimethyl sulfoxide (DMSO), and these values were significantly higher than those of sperm preserved with glycerol. Comparisons of motilities and survival rates between treatments of difference freezing rates showed that there was no difference between procedures (a) 5$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.3$\pm$0.31 ; survival late 70$\pm$2.7%) and (b) 3$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.1$\pm$0.29; survival rate 69$\pm$3.7%), while the results of (c) 1$0^{\circ}C$/min to -8$0^{\circ}C$ were significantly lower than the others (motility index: 2.2$\pm$0.33 ; survival rate 42$\pm$4.6%). There was no significant difference in fertilization rate between fresh sperm and sperm preserved with 15% DMSO as cryoprotectant and freezing rate (3$0^{\circ}C$/min to -8$0^{\circ}C$). Some ultrastructural changes of sperm, such as the detachment of plasma membrane, the destruction of mitochondria, and the flagellum rolling up head, were observed after cryopreservation. Morphological normality of the sperm in 15% DMSO frozen at the ratio of 5$0^{\circ}C$/min to -8$0^{\circ}C$ was better than the others.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.278-278
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• 2004

The purpose of this study was to establish the convenient freezing method for more cheap and simple. Semen quality was evaluated the motility, viability, abnormality, acrosome intactness and membrane integrity. And there were also examined the developmental rates of IVM/IVF embryos using frozen-thawed boar semen in each treatment group. (omitted)

• 한국패류학회지
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• 제26권4호
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• pp.255-260
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• 2010

피조개, Scapharca broughtonii (Schrenck) 정자의 동결보존 위해 동결높이에 따른 예비동결 (0, 2, 4, 6 cm), 희석제 (150 mM NaCl, 300 mM glucose, Ringer's solution)와 동해방지제(glycerol, dimethylsulfoxide (DMSO), dimethylacetamide (DMA), methaol) 에 따른 효과를 정자의 운동성과 생존율로 비교하였다. 피조개 정자 동결보존에 있어 예비동결은 액체질소표면으로부터 2 cm가 가장 효과적이었으며, 희석제로는 Ringer's solution이 동해방지제로는 DMSO 10% 농도가 가장 적합하였다.

• Reproductive and Developmental Biology
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• 제32권2호
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• pp.111-115
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• 2008

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH ($67.4{\pm}1.8\;{\mu}m$) was significantly (p<0.05) smaller than that of the fresh preantral follicles ($69.1{\pm}2.3\;{\mu}m$). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

• 한국가축번식학회지
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• 제10권2호
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• pp.175-181
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• 1986

These experiments were carried out to clarify the effects various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. As cryoprotectant, glycerol, DMSO and methanol were used and the procedures of adding them in medium were practiced by one-step or six-step adding method. Morphologically normal mouse embryos developed to blastocyst by in vitro culture after freezing and thawing were transferred to pseudopregnant recipients by surgical procedures. The results obtained in these experiments were summarized as follows: 1. The survival rates of the frozen-thawed 8-cell embryos, morulas and blastocysts following one-step addition of glycerol were 83.6, 80.3 adn 70.3%, respectively, while following six-step addition of glycerol, 69.2, 56.3 and 66.7% respectively. 2. When glycerol, DMSO and methanol were used as cryoprotectant under the same condition of freezing and thawing, the survival rates of frozen-thawed embryos were 74.0, 76.1 and 37.6%, respectively. 3. The implantation rate of embryos transferred to pseudopregnant recipients after freezing and thawing was 49.2%.

• 한국패류학회지
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• 제24권1호
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• pp.51-57
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• 2008

With the purpose to estimate the possibility of short-term storage and cryopreservation for sperm of Charonia sauliae, which is a potential preparation for its artificial reproduction and further research, in this study, protocols for short-term storage and cryopreservation of trumpet shell sperm was optimized. The effects of different immobilizing solutions, dilution ratios were estimated for short-term storage. And the effects of different cryoprotectant extenders and freezing rates were estimated for cryopreservation in terms of motility and survival of sperm. The results indicated that the artificial sea water of 350 mOsmol/kg is a better immobilizing solution and sperm which was diluted at a ratio of 1:1 (v/v) had higher motility and survival rate during short-term storage. The effect of 5% dimethyl sulfoxide was significantly better than those of other cryoprotectant extenders. And a freezing rate of $-20^{\circ}C\;min^{-1}$ showed better effect than other freezing rates. In conclusion, this study optimized some key factors of the short-term and cryopreservation of C. sauliae sperm, which can provide valuable data for germ-plasm conservation and artificial propagation of C. sauliae.

• 한국수정란이식학회지
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• 제7권1호
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• pp.13-19
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• 1992

This study were carried out to investigate the effective concentration of cryoprotective agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen porcine embryos. The porcine embryos foflowing dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined by FDA test. The results are sunnnarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen4hawing in the freezing medium with a various concentration of glycerol, DMSO and propanediol added 0.25M sucrose were higher survival rate than those of sucrose concentration of 0.50M. 2. The survival rates of porcine embryos after ultrarapid ftozen4hawing in the freezing medium added 0.25M and 0.SOM sucrose were higher survival rate than those of sucrose concentration of 0.75M and 1.00M. 3. The temperature thawed at 2$0^{\circ}C$ and 3$0^{\circ}C$ resulted in a significantly higher embryos survival rate after 72 hrs in culture than did at 35$^{\circ}C$. 4. The equilibration time on the survival rate of porcine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time(10~20 min.).

• Journal of Animal Science and Technology
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• 제55권5호
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• pp.417-425
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• 2013

동결 닭 PGCs의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 PGCs의 동결보존기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보 하는 것과, 생식계열 키메라의 제작효율을 높이는 것이 반드시 필요하다. 닭 PGCs는 배양 5.5일령의 닭 원시생식선으로부터 채취하고, ACS 방법에 의해서 순수 닭 PGCs를 분리했다. 닭 PGCs의 동결보존실험결과 다음의 4종류의 동결방법을 각각 비교 검토했다. 1. 플라스틱 스트로에 의한 완만동결법 (SF), 동결보호물질은 2M 에틸렌 글리콜 (EG), 2. 스트로에 의한 급속동결법 (RF), 8M EG + 7% PVP, 3. 동결용 Cryotube에 의한 SF, 2M EG, 4. 튜브에 의한 SF, 10% DMSO. 동결 및 융해 후의 PGCs의 생존율은 각각 76.4%, 70.6%, 80.5%, 78.1%로 관찰되었다.

• 한국가축번식학회지
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• 제13권3호
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• pp.141-148
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• 1989

The effects of seeding method and optimum time for freezing embryos according to the developmental stages on embryo survival rates after rapid freezing were determined using the FDA-test. The summarized results are as follows : 1. In the rapid freezing of embryos, the sucrose added medium together with Co-seeding or non-seeding showed the FDA scores of 4.67 and 4.20, respectively, but, raffinose addition obtained FDA scores of 4.27 and 3.97. 2. The developmental stage of embryos at freezing was most critical on the survival of embryos after thawing. Higher FDA scores were obtained in the order of blastocyst stage(4.94), morula stage(3.82) and ealy stage(2.65) in sucrose added medium. The same trend was observed in the raffinose added medium with an order or 4.91, 4.47 and 2.32. 3. Microscopic study of embryo before freezing and post-thawing indicated that the embryo showed shrinkage within 5 minutes after the embryo was transfer to the freezing medium. When thawed embryo was tranfered to the dilution medium, swelling of the embryo was observed and there after it reshrank indicating the removal of cryoprotectant from the embryo. The size of the embryo recovered to the original state when it was moved into a PBS-solution.