• Journal of Embryo Transfer
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• v.12 no.1
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• pp.27-36
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• 1997

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 $\beta$-estradiol and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l$\times$ l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+$\beta$-estradiol, hCG+$\beta$-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.187-192
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• 2022

The repeated monthly or weekly subculture of plant callus is labor intensive and increases the risk of somaclonal variation from the parental callus line. The most effective method for preserving plant callus is cryopreservation, which involves storage in liquid nitrogen. However, this method cannot be applied to the callus of different plant species in the same manner, so it is difficult to develop a standardized cryopreservation method. In addition, the survival rate of the frozen callus after thawing and the regeneration rate after survival are uncertain. Therefore, it is necessary to develop a method to extend the subculture interval of plant callus in an active state. In this study, active plant calli of various species without freezing was incubated at 15℃ for 4 to 12 weeks without subculture. After 12 weeks, 8 lines of plant callus grew less than 2-fold when cultured at 25℃, but at least 2 times as much when cultured at 15℃. Moreover, total antioxidant activity did not differ significantly between plant callus recovered at 25℃ after culturing at 15℃ or at 25℃. These results show that the subculture interval can be extended at a temperature of 15℃ without need for modified medium composition or additional processes. In addition, positive results in all calli of several plant species are expected to reduce labor as well as somaclonal variation by increasing the subculture.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.133-145
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• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.245-252
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• 2002

This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5 $\mu\textrm{g}$/$m\ell$ Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 ${\mu}{\textrm}{m}$ ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into L$N_2$. Thawing was taken by 4-step procedures at 37$^{\circ}C$. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide and 2.5 $\mu\textrm{g}$/$m\ell$ of cytochalasin D added CRlaa medium for 1 h, and then in 10 $\mu\textrm{g}$/$m\ell$ of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.797-804
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• 2006

The present study was undertaken to investigate the post-thawed survivability of bovine embryo depending on different dose of ethylene glycol and sucrose. Ovaries were collected at local slaughterhouse and the cumulus-oocyte-complexes aspirated from ovaries were in vitro matured, fertilized and cultured at 39°C in an atmosphere of 5% CO2 incubator. For conventional slow-freezing, d 7 or 8 expanded blastocysts were collected. Embryos were equilibrated in 1.5 M and 1.8 M ethylene glycol(EG) with 0.1 M and 0.3 M sucrose in Dulbecco's phosphate-buffered saline(D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25ml-straw and placed directly into cooling chamber of programmable freezer precooled to 󰠏7°C, after 2 min, the straw was seeded, maintained at 󰠏7°C for 8 min, and then cooled to 󰠏35°C at 0.3°C/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 sec and exposed to 37°C water for 20 sec. Straws were then removed from 37°C water. Rates of blastocyst survive and hatching were evaluated at 24 to 72 h post-warming. No difference of the survivability was shown between 1.5 M and 1.8 M EG (71 and 70%, respectively). Addition of 0.1 M sucrose to 1.5 M and 1.8 M ethylene glycol in the freezing solution did not differ significantly embryo survival (74 and 77%, respectively), whereas survival rates was higher(89%) in freezing solution contained 0.3M sucrose to 1.8M EG compared with 0.3M sucrose to 1.5M EG group(71%). However, there was no difference in the overall total cell number between the two groups (122±1.8 vs 131±1.4, respectively). In conclusion, the results suggest that 0.3 M sucrose in 1.8 M EG may be optimal condition for freezing and thawing methods with in vitro produced embryos and may be applied to on-farm conditions for embryo transfer.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.251-258
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• 2020

This study investigated the effect of Zardaverine supplementation in freezing extender, on kinetic characteristics of post-thawed boar sperm. Cryopreservation of boar sperm is an important technique of assisted reproductive technology and genetic resource banking. Although this technique is particularly useful, freeze-thaw cycles associated with sperm cryopreservation significantly reduce sperm quality. Semen from mature Duroc boars were collected and cryopreserved in freezing extenders (LEY) treated with varying concentrations of Zardaverine (0, 20, 50, 75, 100 𝜇M). The time-dependent kinetic characteristics of post-thawed spermatozoa were determined after thawing by applying computer-assisted sperm analysis (CASA). We observed that the motility immediately after thawing was significantly higher in 20 𝜇M stocks than in control (0 𝜇M) and the other treatments (p<0.05). Curvilinear velocity (VCL) in 0 𝜇M and 20 𝜇M stocks were significantly higher than the other treatment groups, except 75 𝜇M (p<0.05). Higher average path velocity (VAP) was obtained at 20 𝜇M as compared to 100 𝜇M, whereas amplitude of head lateral displacement (ALH) was significantly higher at 20 𝜇M than 50 𝜇M and 100 𝜇M (p<0.05). No differences were obtained for Straight-line velocity (VSL) and Linearity (LIN). In conclusion, our results indicate that Zardaverine improves the motility, VCL, VAP, and ALH of post-thawed boar sperm.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.3
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• pp.203-212
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• 2008

Objective: During cryopreservation process, cold shock and cryo-injury affect the fertilizing capacity of the sperm by damaging cell membranes with loss of functional integrity. A longstanding concept for preventing the cryo-damage is to stabilize the plasma membrane by incorporating cholesterol. This study was to determine the effects of cholesterol in freezing media on the motility and functional integrity of human sperm after cryopreservation. Methods: Control group (non-cholesterol treated) and different concentrations of cholesterol-treated sperm (14 healthy males) were frozen and thawed. After freezing and thawing of sperm, the quality of sperm was evaluated by sperm analysis, acrosome reaction test and sperm chromatin structure assay. Results: When human sperm were incubated in sperm freezing medium (SFM) containing $0.5{\mu}g$ cholesterol and then freezing/thawing, the motility of sperm have significantly improved compared to those untreated cholesterol ($33.46{\pm}1.48%$ vs. $30.10{\pm}1.07%$, p<0.05). The rate of calcium ionophore-induced acrosome reactions in post-thawed sperm was significantly higher than that ($53.60{\pm}1.60%$ vs. $47.40{\pm}1.86%$, p<0.05) in SFM containing cholesterol. Sperm chromatin structure assay revealed that DNA damage to the sperm in the cholesterol-treated group was lower than that of non-treated group. Conclusion: These results suggest that increased cholesterol content of sperm plasma membrane by supplementation of cholesterol in SFM improves sperm motility, capacitation status, and DNA integrity. Therefore, addition of cholesterol into SFM could be a useful for protecting human sperm from cold shock and cryo-injury during cryopreservation.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.207-216
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• 2013

This study was conducted to establish the method for preserving PGCs that enables long-term storage in liquid nitrogen for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of freeze-thaw treatment on viability of PGCs in chickens. PGCs were collected separately from a germinal gonad of an early embryo of 5.5~6 day (stage 28) of Isa brown, Korean Oge (KO), White Leghorn and Commercial breeds. PGCs separated from a germinal gonad of an early embryo of 5.5~6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The PGCs were then purified using magnetic activated cell sorting (MACS) method. The viability of PGCs after thawing was $87.4{\pm}0.4%$ and $89.4{\pm}0.2%$ with the 10% EG treatments with no significant difference between the Isa brown and Commercial breeds. The viability of PGCs after freeze- thawing was significantly higher for Isa brown ($87.4{\pm}0.4%$) and Commercial breeds ($89.4{\pm}0.2%$) than Korean Oge (KO) ($77.6{\pm}1.1%$) and White Leghorn ($76.2{\pm}0.9%$)(p<0.05) using 10% EG cryoprotectant. This study established a method for pre- serving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at agermplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Korean Journal of Soil Science and Fertilizer
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• v.32 no.1
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• pp.31-38
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• 1999

Rainfall factor. R, and soil factor, K were estimated to use the Revised Universal Soil Loss Equation (RUSLE) to predict the amount of soil erosion from a land on slope in Kangwon-do, Korea. The average of R factor was 405 with a range from 251 to 601. The R factor differed among regions. The R factor at Taegwalryung, in the highland region, was 409 and those at Inje and Hongchon, in the mid mountainous regions, ranged from 310 to 493. The R factors at Wonju and Chuncheon, in the plain regions, ranged from 505 to 601. The R factors at Sokcho, Kangnung and Samchok, in the east coastal region, which ranged from 251 to 368, were lowee than those in the western part of the Taebaeg Mountains. The R factor during the winter including the effect of winter freezing and thawing was 12 to 30% of the annual average value in the east coastal and highland regions, while that in the western part of Taebaeg Mountains was lower than 7%. The average of K factor in the surface soil was 0.21 with a range from 0.06 to 0.42. The K factors of Odae and Weoljeong serieses were the lowest, while that of Imog was the highest. The average of K factor in the subsoil was 0.28 with a range from 0.07 to 0.45. The K factor of the subsoil was 1.3 times higher than that of top soil. The average of K factor in he soil including the effect of the gravel covering and percolation was 0.18 with a range from 0.03 to 0.33. In contrast. the K factor excluding the effect of the gravel covering was lower than this. The average of K factor in the frozen subsoil was 0.33, which was 1.6 times higher than that of the non frozen subsoil.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.