• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.299-306
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• 2021

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito-TEMPO (0.1 µM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozen-thawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

• Korean Journal of Animal Reproduction
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• v.9 no.1
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• pp.36-39
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• 1985

This experiment was carried out to investigate the morphology of bovine embryos thawed after deep freezing at -196$^{\circ}C$ and the development of frozen-thawed embryos after in vitro culture in Ham's F-10 medium with 10% NBCS. The results obtained were summarized as follows: 1. The propotion of embryos which a, pp.ared mophologically normal was averaged 77.5% (79/102). 2. The morphologically normal rate of frozen-thawed blastocyst (78.6%) was higher than that of morula (76.7%), but there was no significant difference. 3. Normal development was observed in 20 of 68 embryos cultured for 24-72hr in medium and overall survival rate was 29.4%. 4. Survival rate fo blastocyst (33.3%) was higher than that of morula (25.7%).

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.54-54
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.54-54
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• 2005

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.29-34
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• 2001

To investigate the effect of co-transfer of trophoblastic vesicle (TV) with frozen-thawed in vitro Produced (IVP) bovine embryo on pregnancy rate, IVP blastocysts were transferred to synchronized recipients. Elongated blastocysts were recovered at Day 13 to 15, and dissected more than 4 pieces to removed the embryonic disc. Throphoblastic fragments were cultured for 48 hours to make throphoblastic vesicles (TVs). TVs were cryopreserved in ethylene glycol or vitrification solution and frozen-thawed TVs were co-transferred to recipients with frozen-thawed IVP embryos. 1 The recovery rate of elongated blastocyst on Day 13 to 15 was 22.5% (18/80) and the size of recovered elongated blastocysts was 0.2∼5.0mm. 2. Eighteen elongated blastocysts were dissected into 88 pieces and 61.4% of those pieces were formed to TV (54/88) 3. The viability of frozen-thawed TV in ethylene glycol was higher than in vitrified solution (92.8% vs. 68.8%) 4. The pregnancy rate in co-transfer with frozen-thawed TV and IVP blastocyst was better than transfer only IVP blastocysts (50.0% vs. 23.1%).

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.9
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• pp.1242-1248
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• 2005

This study was designed to investigate the in vitro developmental ability and apoptosis of bovine embryos nucleartransferred (NT) with frozen-thawed or cooled donor cells. Cultured adult bovine ear cells were used as donor cells after sub-culturing to confluence (CC), cooling to 4$^{\circ}C$ for 48 h, or freezing-thawing (FT). Apoptotic cells in blastocysts were evaluated for apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Fusion, cleavage and blastocyst rates were 69.0 (167/242), 68.8 (115/167), and 29.9 (50/167) with CC cells, 70.4 (88/125), 69.3 (61/88), and 29.6 (26/88) with cooled cells and 66.1 (117/177), 70.1 (82/117), and 13.7 (16/117) with FT cells, respectively. Blastocyst rates of NT embryos derived from FT cells were significantly lower than those from CC or cooled cells (p<0.05). In addition, NT blastocysts produced by using FT cells showed significantly higher apoptosis rates (6.4${\pm}$4.0%) than those produced by CC (2.8${\pm}$1.7%) or cooled (2.3${\pm}$1.3%) cells. However, cooling of donor cells had no significant adverse effect on blastocyst rate as well as apoptosis rate. Therefore, our results suggest that cooled cells may be used as an alternative to freshly cultured confluent culture cells, as donor cells, for the production of Somatic nuclear cloned cattle.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.163-169
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.1
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• pp.7-12
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• 2001

Factors Affecting the Survival of Frozen Thawed Bovine In Vitro Produced Blastocysts. The effect of some factors on the post-thaw survival of a total of 240 in vitro produced bovine blastocysts was investigated using logistic regression analysis. The explanatory variables tested were: type of culture medium before freezing (TCM 199 supplemented with BSA, BSAITS (BSA+insulin+transferrin+selenium), ECS (estrous cow serum) with or without BOEC (bovine oviductal epithelial cells), age of the blastocyst (Day 7, Day 8+9), morphological appearance before freezing (distinct=Q1 or indistinct=Q2 inner cell mass) and type of cryoprotectant (glycerol, 1.0 M or ethylene glycol, 1.6 M). The survival after thawing based on the post-thaw quality and the development after co-culture with BOEC for 24 and 48 hours. Day 7 blastocysts had an almost three times better chance of survival than Day 8+9 blastocysts. Q1, Day 8+9 blastocysts had higher odds to survive after 48 hours in culture than Q2 blastocysts (p<0.05). Blastocysts produced in BSAITS medium had the best chances of survival; however, the odds were not always significant. Blastocysts frozen in glycerol had a better post-thaw quality rating than those frozen in ethylene glycol; however, the difference in post-thaw development at culture was not significant. The relationship between post-thaw quality and post-thaw development at culture was significant (p<0.05). The developmental stage and/or age of the embryo and culture medium where development up to blastocyst takes place affect the post-thaw survival of the bovine embryos.

• Journal of Embryo Transfer
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• v.12 no.1
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• pp.67-74
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• 1997

This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5$^{\circ}C$ in 2% $CO_2$ in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2$0^{\circ}C$ water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100$\mu$M $\beta$-mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF$_2$$\alpha$(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.55-59
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• 2002

The objective of this study is to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, a group of oocytes was activated with 7% ethanol fur 5 min, and the other group was not activated. The oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~30 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The percentage of oocytes reaching M II after 24 hrs and 30 hrs of incubation were significantly higher(p<0.05) after culture with TCM-199 media(80.0% and 88.3%) than M I(8.3% and 6.7%). The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(22/46, 47.8% vs 10/39, 25.6%). The rates of embryos development to blastocyst obtained by ICSI treated sperm of flesh, epididymal and frozen-thawed epididymal were 24/45(53.3%), 15/40(37.5%), 11/43(25.6%), respectively and these values of fresh sperm injection were higher than frozen-thawed epididymal sperm. We also concluded that embryos can be produced with ICSI of in vitro matured oocytes by ICSI using frozen-thawed epididymal semen.