• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.201-201
• /
• 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 Korean Society of Developmental Biology Conference
• /
• 2004.07a
• /
• pp.27-27
• /
• 2004

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

• 대한생식의학회:학술대회논문집
• /
• 2004.06a
• /
• pp.60.2-61
• /
• 2004

• Korean Journal of Animal Reproduction
• /
• v.22 no.4
• /
• pp.349-357
• /
• 1998

This study was to investigate whether the viability of Hanwoo IVM/IVF/IVC blastocysts was maintained after vitrification and thawing. In vitro produced Hanwoo blastocysts were vitrified by two-step method: equilibrated in EG20 for 3 min, and then exposed in EFS40 ［40% ethylene glycol (EG), 18% ficoll and 10.26% sucrose in mDPBS containing 10% FBS ］and vitrified in L$N_2$for 30 - 45 sec. After thawing, in vitro survival was assessed as the re-expanded and hatched rates at 24 hand 48 h, respectively. The results obtained in these experiments were summarized as follows: From the 12 replicates, 52.5% of Hanwoo blastocysts were produced in vitro at day 7 after IVF. When the effects of freezing solution to the embryo survival were examined, there is no significant toxicity in exposure (100.0, 73.8%) compared to that af control group (100.0, 87.0%). However, when embryos were vitrified, high survival (86.2, 55.4%) was obtained although it was significantly lower than those of exposure and control group (p＜0.05). When the in vitro survival of vitrified embryos according to developmental stage and culture day were examined, it showed that more advanced embryo stage exhibited a significantly higher survival rate irrespective of culture day (p＜0.05). Also, even in the same development stage, the in vitro survival of day 7 embryos (re-expanded: 75.0~87.5%, hatched: 21.4~66.7%) was higher than those of day 8 embryos(re-expanded: 58.6~78.3%, hatched: 10.3~52.2%). Therefore, these results suggested that in vitro produced Hanwoo blastocysts can be successfully cryopreserved by simple two-step vitrification method using EFS40 freezing solution, particularly at the expanded and early hatching blastocyst stage regardless of embryo culture duration (day 7 or day 8 after IVF).

• Journal of Embryo Transfer
• /
• v.14 no.2
• /
• pp.121-129
• /
• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• 대한생식의학회:학술대회논문집
• /
• 2001.06a
• /
• pp.70-71
• /
• 2001

• Korean Journal of Animal Reproduction
• /
• v.23 no.4
• /
• pp.281-291
• /
• 1999

The objective of this study was to optimize the freezing/thawing method of in vitro produced Hanwoo blastocysts. Day 7 blastocysts after IVF were vitrified using EFS40 (40% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS added m-DPBS) as a freezing solution and electron microscope (EM) grid (V-G) or straw (V-S) as an embryo container. In both method, freezing/thawing were treated by 2-step, treatment time was required in V-G method and V-S method, for 2 min / 3 min and 3.5 min / 10 min, respectively. Embryo survival was assessed as re-expanded and hatched rates at 24 h and 48 h after warming, respectively. The results obtained in these experiments were summarized as follows: when the effect of exposure in vitrification solution and chilling injury from freezing procedure on in vitro produced expanded blastocysts were examined, at 24 h after warming, embryo survival in exposure group (100.0%) was not different compared to that in control group (100.0%), although those results were significantly different with two vitrified groups (V-G: 87.8, V-S: 77.8%) (P＜0.001). However, at 48 h after warming, hatched rates of V-G group (67.8%) were significantly higher than those of V-S group (53.3%) (P＜0.05). In addition, this hatched rate in V-G group was not different with that in exposure group (73.3%). When the effects of embryo developmental stage (early, expanded and early hatching blastocysts) and embryo container (EM grid and straw) to the in vitro survival of vitrified-warmed day 7 Hanwoo blastocysts were simultaneously examined, fast developed embryos were indicated the better resistance to freezing than delayed developed one, irrespective of embryo containers (early; 57.1 ＆ 24.4%, expanded; 84.7 ＆ 60.6%, early hatching; 91.7 ＆ 80.0%) (P＜0.001). Especially, in expanded and early hatching blastocysts, embryo survival of V-G group (67.8, 95.0%) was significantly higher than those of V-S group (53.0, 65.0%) at 48 h post warming, respectively (P＜0.05, P＜0.001). Therefore, this study indicates that Hanwoo blastocysts can be cryopreserved more simple, efficient and successful by vitrification method using EM grid.

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