• 한국수정란이식학회지
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• 제27권4호
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• pp.259-264
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• 2012

Solution of glycerol, ethylene glycol, sucrose, dextrose (GESD) and cryotop methods were carried out to investigate the survivability on vitrification of embryos. Embryos cultured in vitro were vitrified by GESD of 10 or 8 step and cryotop methods of 6 step, from cryopreservation step to frozen-thawed and culture step. Survival rate and ICM, TE cells of embryos were investigated after frozen-thawed 24 h. As a results, cryotop method was significantly (p<0.05) higher ($85.76{\pm}5.3$ vs. $66.71{\pm}2.4$, $44.80{\pm}2.1%$) than GESD 10 or 8 step methods on survivability. Also, In ICM cell number, cryotop method was significantly (p<0.05) higher to $45.67{\pm}4.7$ cells than GESD 8 step method. TE cell number was significantly (p<0.05) highest to $111.00{\pm}11.0$ cells in cryotop method. On the other hand, survival rate, TE and total cell number were all the significantly (p<0.05) high, except ICM in GESD 10 step method between GESD 10 step method and GESD 8 step method. In conclusion cryotop method was to be most effective, but it is considered necessary to study vitrification method for step-by-step freezing and thawing process.

• 한국수정란이식학회지
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• 제29권1호
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• 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.

• 한국가축번식학회지
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• 제17권3호
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• pp.233-242
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• 1993

The effects of in vitro maturation and sperm treatment condition on the in vitro fertilization (IVF) and developmental capacity of bovine oocytes were investigated and the development of embryos was compared under the 2 different co-culture system, with GC or BOEC. The cultured embryo to 16 cell or morula wre transferred into recipients or frozen by 2 different freezing method. The results obtained were summarized as follows; 1. In vitro maturation rates of vovine follicular oocytes cultrued in TCM199 with 10% FCS or ECS were 64.0% and 72.7%, but the case of addition of 10% FCS or ECS to TCM199 co-cultured with granulosa cells were 81.3% and 84.0%, respectively. IVM rate of three TCM199 added to granulosa cells was higher than that of media without granulosa cells. 2. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC and then fertilized in vitro by sperm treated with caffeine, embryo developments of bovine oocytes co-cultured with BOEC were 38.4% and 51.4%, respectively. But those of bovine oocytes co-cultured with GC were 52.2% by sperm treated with caffeine-heparin. 3. Cleavage rates of bovine oocytes cultured with 10% FCS alone and fertilized in vitro by sperm treated with caffeine-heparin was 33.0%. 4. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC, embryo developments of bovine ooctyes co-cultured with BOEC of GC were 46.0% and 50.2%, respectively. 5. When bovine follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developed co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developments of the bovine oocyte co-cultured with BOEC and GC were 41.8% and 60.1%. But with FCS 10% those of the bovine oocytes co-cultured with BOEC and GC were 42.0% and 48.4%, respectively. 7. When Holstein's follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments fo the bovine oocytes co-cultured with BOEC and GC were 50.0% and 57.7%, but with ECS 10% those of the bovine oocytes co-cultured with BOEC and GC were 52.2% and 56.5%, respectively. 8. The viability of frozen-thawed embryos ranged from 60~80% and those of frozen-thawed embryos from vitrification was lower than that from conventional metiod. 9. The selected fresh embryos were transferred nonsurgically to 7 recipients but did not result in pregnancy.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.134-134
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• 2003

Bovine embryos produced by in vitro maturation, feretilization and development was examined for presevation and transfer. The fertilization medium used BO medium with 5 mM/$m\ell$ caffeine and 10$\mu\textrm{g}$/$m\ell$ heparin and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 1$\times$$10^{6}$ cells/$m\ell$ motile sperm during fertilization in vitro. At 8~10 hrs after insemination, the oocytes were transferred into CR1aa medium and cultured for 7 days. Embryos were preserved by vitrification method for transfer. When the embryos of early, blastocyst and expanded blastocyst stages were frozen-thawed, the proportions of embryos with normal morphology 83.6, 88.1 and 85.2%. (중략)

• Animal Bioscience
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• 제34권6호
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• pp.985-992
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• 2021

Objective: Artificial insemination plays an important role in genetic improvement in the goat farming system. The aim of this study was to investigate the effect of cervical application of hyaluronan (HA) on the fertility in goats after cervical artificial insemination using frozen-thawed (F-T) semen. Methods: After oestrous synchronisation with progesterone sponges and pregnant mare serum gonadotropin injection, both nulli- and multi-parous goats, were randomly allocated to 2 groups, and were inseminated with 0.25 mL of F-T semen (150×106 spermatozoa) twice at 52 h and 56 h after sponge removal. Prior to the insemination, goats in Group 1 only were given topical cervical HA application at 48 h after sponge removal. Site of insemination was recorded as os-cervix or intra-cervix or intra-uterus. Pregnancy was tested ultrasonographically 42 days after insemination. The data on pregnancy rates and percentage of animals according to the site of semen deposition were compared by Chi-square analysis. Results: The overall pregnancy rate was significantly (p<0.004) higher in goats with prior application to the cervix with HA (63.3%) than without (36.0%). Same pattern was observed in the pregnancy rates of nulli- and multi-parous goats in both the groups. Percentage of nulliparous goats according to the site of insemination in the HA group did not differ between first and the second insemination. However, in multiparous goats the percentage of animals inseminated intra-cervically was significantly increased (p≤0.05) between the first and the second inseminations. Conclusion: The results suggest that significantly higher fertility rate in the "HA goats" compared to the "without HA" group was because of deeper insemination facilitated by topical cervical application of HA. The deeper insemination into the cervical canal increase the rate of fertilisation when the cervical artificial insemination is performed.

• 한국수정란이식학회지
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• 제6권1호
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• pp.25-32
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• 1991

The present study was performed to investigate the effects of co-culture with granulosa cells on in vitro fertilization and cleavage of early bovine embryo development. Bovine oocytes were matured for 20-24 hrs in vitro with granulosa cells or without and then fertilized in vitro using frozen-thawed spermatozoa treated with BO-caffeine, BO-BSA(2OmM heparin added). At l8hrs after insemination, oocytes were fixed and examined or further cultured in TCM 199 for 48hrs. The fertilization rates between the control(70.4%) and the groups of co-cultured with granulosa cell(2.5$\times$106 cells/ml; 71.6%, 5.0$\times$ 106/ml; 71.9%, l.0$\times$ 107/ml; 71.1%) did not differ significantly. The cleavage rates in the groups co-cultured with granulosa cell(2.5$\times$ 106 cells/mi; 43.6%, 5.0$\times$ 106/ml; 46.8%. l.0$\times$ 107/ml; 45.0%)were significantly higher than that of without granulosa cell, respectively(P<0.05). However there were no significant differences between the groups co-cultured with granulosa cells. The result indicated that co-culture with granulosa cell was effective means to cleavage of bovine follicular oocytes but did not affect the in vitro fertilization.

• 한국수정란이식학회지
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• 제25권3호
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• pp.195-199
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• 2010

This studies were conducted to investigate the survival rate of frozen-thawed spermatozoa of Jindo Dog by monosaccharide and freezing rates. Experimental animals were prepared 12 males within 1~8 year's old and collected once in a couple of weeks by digital manuplation methods. Collected semen was diluted 1:1 with Tris-egg yolk extender and added 4, 6 or 8% of glycerol and none, 4 mM glucose or 4 mM fructose as cryoprotectant and was equilibrated for 2 hrs in $4^{\circ}C$. In monosaccharide groups, the freezing rate was 5 cm-5 min. above $LN_2$. The survival rates without monosaccharide were $50.7{\pm}19.0%$, $58.6{\pm}18.0%$, $40.0{\pm}10.0%$ in 4, 6 or 8% glycerol, respectively. In addition of glucose, the survival rates were $43.1{\pm}14.7%$, $38.1{\pm}16.5%$, $33.3{\pm}4.0%$ in 4, 6 or 8% glycerol, respectively and in fructose, were $47.9{\pm}21.1%$, $61.3{\pm}6.2%$, $34.3{\pm}12.6%$ in 4, 6 or 8% glycerol, respectively. There showed significantly different between glycerol groups and monosaccharides groups (p<0.05). The survival rates of freezing rate in 5 cm-5 min. group was $64.5{\pm}15.8%$, $51.9{\pm}27.6%$, $29.7{\pm}24.8%$ and in 10 cm-10 min. group was $62.5{\pm}20.3%$, $64.9{\pm}23.6%$, $34.5{\pm}27.4%$ in 4, 6 or 8% glycerol, respectively. There were significantly different between freezing rates (p<0.05). These results suggest that the addition of fructose with 6%-glycerol and slow freezing improve the survival of frozen-thawed sperm in Jindo Dog.

• 한국수정란이식학회지
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• 제29권2호
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• pp.163-169
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• 2014

The objective of this study was to evaluate the characteristics of Korean Native Cattle sperm frozen-thawed with L-cysteine and/or catalase. The semen from bulls was collected by the artificial vagina method, and Triladyl containing 20% egg-yolk and/or L-cysteine (L), catalase (C) and L-cysteine + catalase was added to the diluted semen for cryopreservation. The results showed that sperm viability was significantly higher in the L-cysteine + catalase ($69.49{\pm}3.16%$) group than in the control ($60.5{\pm}3.94%$) group (p<0.05). Acrosome damage was significantly lower in the L-cysteine ($17.12{\pm}1.08%$) group than in the control ($21.46{\pm}1.14%$), catalase ($20.54{\pm}0.76%$), and L-cysteine + catalase ($19.29{\pm}0.65%$) groups (p<0.05). In addition, the level of intact mitochondria in the spermatozoa was significantly higher in the L-cysteine ($58.65{\pm}1.39%$) group than in the control ($50.63{\pm}2.37%$) group (p<0.05). The hydrogen peroxide level in the frozen-thawed sperm was significantly lower in the L-cysteine ($3.74{\pm}1.66%$), catalase ($4.65{\pm}1.87%$), and L-cysteine + catalase ($8.11{\pm}2.15%$) groups than in the control ($13.22{\pm}1.6%$) group (p<0.05). The glutathione level was significantly higher in the L-cysteine ($1.33{\pm}0.03%$) group than in the control ($1.08{\pm}0.06%$), catalase ($1.05{\pm}0.02%$) and L-cysteine + catalase ($1.11{\pm}0.03%$) groups (p<0.05). In conclusion, L-cysteine and catalase could protect the membrane of Korean Native Cattle sperm from damage during sperm cryopreservation. Especially, L-cysteine was more effective for keeping acrosomes and mitochondria intactness during sperm cryopreservation.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.5-5
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• 2001

This lecture will begin by tracing some of the history behind techniques that we nowadays take for granted in the practice of embryo transfer, and in the application of the technique to various animal biotechnologies. It will be argued that an appreciation of such history can teach us a great deal about how we need to study and teach the subject, and about the best ways to conduct and finance the research that is essential to further progress. Examples in support of this argument will be taken from the changes that have occurred in the way embryos, particularly bovine embryos, have been collected, maintained in vitro, subjected to a variety of manipulations (sexing, division to produce identical animals, combination into chimeras, transfection with foreign genes), frozen and thawed, and transferred over the past 50 years. (omitted)