• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.77-83
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• 2011

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures [10%, (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.] which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into $LN_2$. One-step dilution in straw was done in $25^{\circ}C$ water for 1 min, by placing vertically in the state of plugged-end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.293-301
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• 1999

This study was to examine whether Hanwoo IVM/IVF/IVC blastocyst can be successfully survived in vitro/in vivo after vitrification and one-step dilution. For vitrification, blastocysts were serially exposed in glycerol (G) and ethylene glycol(EG) mixtures［10% (v Iv) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.］ which is diluted in 10% FBS added D-PBS. And then they were loaded in the straw, placed in cold nitrogen vapor for 3 min. and plunged into L$N_2$(－196$^{\circ}C$). One-step dilution within the straw was done in $25^{\circ}C$ and 36$^{\circ}C$ water for about 5 min. and 3 min., respectively. Recovered embryos after one-step dilution were cultured in cumulus cell mono-layered drop for 48 h or were transferred into recipient cows. When the embryo survival in vitro was assessed to re-expanded and hatched rates at 24 hand 48 h after one-step dilution, the results of vitrified group (85.4, 43.8%) was high, although these results were significantly lower than normal development (100.0, 63.3%) of control group, respectively (P＜0.001, P＜0.05). When in vitro survival of vitrified groups according to developmental stage was compared, the results of fast developed embryos (expanded blastocyst and early hatching blastocyst stage) were significantly higher than those of delayed developed one (early blastocyst stage) after one-step dilution (early hatching: 88.0, 48.0%: expanded: 81.1, 45.3%; early: 66.7, 14.3%) (P＜0.05). Also, in case of in vitro survival of vitrified groups according to embryo age (day 7, 8 and 9), when embryo age was younger, in vitro survival was significantly higher (day 7: 67.3, 34.5%; day 8: 76.9, 40.7%; day 9: 60.9, 23.9%)(P＜0.05). Finally, when in vivo development potential of vitrified and one-step diluted Hanwoo blastocysts was examined, 4 of 8 recipient (50%) cows became confirmed pregnant. These results demonstrated that our vitrification and one-step dilution technique can be applied easily and effectively on field trial without the equipment and embryological skills required for conservative dilution and transfer.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.201-201
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• 2004

In this study we examined whether vitrified Hanwoo (Korean cattle) IVM/IVF/IVC blastocysts can survive in vitro/in vivo by a quick one-step dilution method and these embryos result in live births. Blastocysts produced in vitro were vitrified by serial exposure to glycerol (G) and/or ethylene glycol (EG) mixtures of 10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 see. (omitted)

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.73-73
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• 2001

This study was to examine whether the vitrified, one-step diluted and direct transferred Hanwoo IVM/IVF/IVC blastocysts can be successfully survived in vivo and they were succeeded into the live birth. For vitrification, blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures [10% (v/v) G for 5 min, 10% G plus 20% EG (v/v) for 5 min, and 25% G plus 25% EG (v/v) for 30 sect] which is diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. One-step dilution within the straw was done in water bath of $25^{\circ}C$ for 1 min. Vitrified and one-step diluted embryos were directly transferred into 36 (natural or hormone induced synchronized) recipient cows in 6 areas of Kyungsang Buk-Do. Pregnancies were confirmed at first when recipient cows did not return to the subsequent estrus cycle, and later by manual palpation per rectum on day 45, 90 and then living calves were derived into parturition. Overall pregnancy was 33.3%(12/36), However, higher pregnancy was obtained when the recipients exhibited estrus one day earlier than the age of transferred embryos (53.3 vs 25.0-27.3%), irrespective of synchronization methods. Also, parous recipients became pregnant higher than nulliparous heifers, And, there were not different in pregnancy rates by the aspect of corpus luteum (CL) quality of recipients (good, 29.4; fair, 37.5; poor, 33.3%). One hundred eight of frozen-thawed Hanwoo blastocysts were directly transferred into 36 recipient cows. In 12 of pregnant cows, 3 cows were aborted and 9 cows were calved [single, 66.7% (6/9): twin, 33.3% (3/9)]. Total embryo implantation rate was 11.1% (12/108). However, 9 Hanwoo calves were lived. Therefore, these results demonstrate that direct transfer technique of vitrified and one-step diluted bovine blastocysts can be applied easily and effectively with the higher pregnancy rate on field trial without the equipment and embryological skills.

• Journal of Practical Agriculture & Fisheries Research
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• v.12 no.1
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• pp.21-27
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• 2010

The objective of this study was to determine the effects of "Green-one" organic nutrient on mycelial growth and fruiting of P. ostreatus. The dilution concentrations of "Green-one" was treated as follows. There was control, 100, 200, 400 concentrations. That treatments were treated with step of each mycelial growth step. The best of growth steps was mycelial scratching step. At that time, DPI(Day required for primordial formation after inoculation) was shortened for 1 day. Valid germination stipe are 15 pieces, 3 pieces more than control. Stipe length and stipe diameter was long each 4mm, 3mm more than control. Pileus size is shortened than control. Yields per one bottle(g/850cc) was 146g increased 6.5% than control 137g/850cc.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2004.07a
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• pp.27-27
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• 2004

• 대한생식의학회:학술대회논문집
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• 1999.11a
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• pp.79-79
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• 1999

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.89-95
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• 2000

This study was to establish an effective dilution technique in a vitrification of bovine blastocysts for the field trial. For vitrification, blastocysts were exposed in glycerol (G) and ethylene glycol (EG) mixture in m-DPBS supplemented with 10% FBS. Blastocysts were first exposed to 10% (v/v) G for 5 min, and subsequently were transferred to 10% G plus 20% EG (v/v) for 5 min. Finally, embryos were transferred to 25% G plus 25% EG (v/v) for 30 see and were placed in nitrogen vapor for 3 min, and then were plunged into L$N_2$. At thawing, the straw containing blastocysts was placed in air for 10 sec, and then plunged into a water bath at $25^{\circ}C$ until all ice had disappeared. They were placed in $25^{\circ}C$ and 36$^{\circ}C$ water according to treatment group for different time. Also, in vitro survival was assessed by the re-expansion and hatched rates at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) In the survival rates of vitrified bovine blastocysts according to different dilution time at thawing, the data of 1 min group (86.6, 56.6%) were higher than those of other treatment groups (2 min; 93.5, 35.4%, 2.5 min; 76.9, 30.7%, 3 min; 88.8, 36.1% and 3.5 min; 83.7, 8.1%). 2) When the in vitro survival of vitrified groups according to different developmental stage was examined at 48 h after thawing using 1 min dilution method, the hatching rates of fast developed embryos (expanded blastocyst: 81.3%: early hatching blastocyst: 86.2%) were higher than that of delayed developed one (early blastocyst: 46.6%). 3) In addition, when the in vitro survival of vitrified groups according to different embryo age was compared, the hatched rates at 48 h after thawing of Day 7 (66.6%) and Day 8 embryos (60.0%) were significantly higher than that of Day 9 embryos (22.7%) (P＜0.05). These results demonstrate that vitrified bovine IVM/IVF/IVC blastocysts can be successfully survived in vitro using one-step dilution (1 min) method.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.4
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• pp.233-239
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• 2003

For the quantitative analysis of an unknown sample a calibration curve should be obtained, as analytical instruments give relative, rather than absolute measurements. Therefore, researchers should make standard samples with various known concentrations, measure each standard and the unknown sample, and then determine the concentration of the unknown by comparing the measured value to those of the standards. These procedures are tedious and time-consuming. Therefore, we developed a polymer based microfluidic device from polydimethylsiloxane, which integrates serial dilution and capillary electrophoresis functions in a single device. The integrated microchip can provide a one-step analytical tool, and thus replace the complex experimental procedures. Two plastic syringes, one containing a buffer solution and the other a standard solution, were connected to two inlet holes on a microchip, and pushed by a hydrodynamic force. The standard sample is serially diluted to various concentrations through the microfluidic networks. The diluted samples are sequentially introduced through microchannels by electro-osmotic force, and their laser-induced fluorescence signals measured by capillary electrophoresis. We demonstrate the integrated microchip performance by measuring the fluorescence signals of fluorescein at various concentrations. The calibration curve obtained from the electropherograms showed the expected linearity.

• Journal of Embryo Transfer
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• v.6 no.1
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• pp.13-24
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• 1991

The objective of this experiment was to study some possibilities to simplify freezing, thawing and transfer procedure of one-step straw method comparing with the conventional methods using bovine embryos. The previous work are also designed to investigate the thawing effect by development stage and its quality using the embryos. Results obtained were summarized as follows: 1. A total of 87 embryos from 14 donor cows were frozen-thawed and an average of frozen embryo/donor was 6.2. 2. The survival rates of morula stage(65.4%) were higher than those of blastocyst stage(57.l%) and vice versa in rate of morphological recovery (80% vs 95.4%). However. no significant difference was denoted between them. 3. In difference between the groups of good quality and poor quality. good quality was resulted in a significantly higher embryo survival rate(75%) and recovery rates(95%) than poor quality(P<0.0l). 4. In effects of non-permeable sugar dilution in added to l.0M glycerol. higher survival rates were orderd in sucrose. lactose, raffinose and xylose. But lactose-raffinose, sucrose-trehalose and xylose in added to 2.OM glycerol. 5. The highest survival rates were obtained by direct plunge into the liquid nitrogen with 3.OM concentration both of glycerol and trehalose. 6. The survival rates in vitro condition of one-step and direct plunge methods(75%-87.5%) were significantly higher than those of multiple steps (21.4-52.6%) in in vitro (P<0.0l). However, the results of single-step were critical in comparing to other steps of final pregnant conformation.