• Journal of Embryo Transfer
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• v.10 no.3
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• pp.229-236
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• 1995

The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized(IVM /IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements. A total of 1,386 IVM /IVF oocytes were used to compare a simple defined medium(KSOM) with more complex culture conditions used successfully for culture of bovine embryos but do not permit study of specific requirements. All experiments were extensively replicated factorials. In Experiment 1, KSOM was superior to Menezo B$_2$ medium in producing morulae plus blastocysts from IVM /IVF oocytes(33 vs 20%, P<0.()5). The yield of morulae plus blastocysts with KSOM was 22% and with BRLC added was 30%. In Experiment 2, (a 2x2 factorial of KSOM with or without BRLC and 0, 1 ng /ml of platelet derived growth factor, PDGF) more morulae plus blastocysts (40%) were produced in KSOM-BRLC co-culture containing 1 ng /ml PDGF than in the control KSOM(12%). In Experiment 3, there was no dose response when 0, 1 and 5 ng /ml of PDGF were added. The results with simple defined KSOM medium are sufficiently promising to indicate that specific requirements of the embryo may be examined in future studies with KSOM as a base.

• Korean Journal of Animal Reproduction
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• v.8 no.1
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• pp.29-35
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• 1984

These experiments were carried out to obtain basic information necessary for in vitro culture of aggregated mouse embryos. Inbred ICR mice were used to obtain embryos. The zona pellucida was removed by placing the embryos in Whittingham's medium containing 0.5% protease for about 5-10minutes at 37$^{\circ}C$. Total 263 pairs of 2-, 4- and 8-cell zona free mouse embryos were subjected to aggregation by physical pressure and cultured in Whittingham's medium under the gas phase of 5% CO2 in air at 37$^{\circ}C$ for 24 to 60 hours. The results obtained in these experiments were summarized as follows: 1. Time needed for fusion of 2-, 4- and 8-cell embryos were 0-3, 0-3 and 0-3 hours, respectively and average time needed for in vitro development of 2-, 4- and 8-cell embryos after aggregation to morula and blastocyst were 42, 30 and 13.5 hours, and 51, 39 and 27 hours, respectively. 2. Of total 263 pairs of naked embryos, 227 were firmly aggregated together and the rats of aggregation in 2-, 4- and 8-cell embryos were 71.8, 88.3 and 97.0%, respectively. 3. The rates of aggregated pairs which obtained from 2-, 4- and 8-cell embryos developed to morula were 96.7, 95.6 and 96.9%, respectively, and embryos developed to blastocysts were 88.5, 89.7 and 90.8%, respectively. 4. Conspicuous differences in size of volume and inner cell masses between single and double blastocysts were observed. Although a single blastocolic cavity was formed in most double blastocysts, several formed two distinct cavities from the very beginning.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.357-363
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• 1994

To investigate the effect of extracellular matrix proteins on the in vitro development of blastomeres isolated from 2, 4, and 8-cell embryos(termed 1/2, 1/4 and 1/8 blastomeres, respectively) of ICR strain mice, those were cultured in fibronectin, gelatin, or collagen precoated culture dishes containing 1.5ml of NaHCO3-BMOC-3 medium at 37$^{\circ}C$ for 72 hrs, under the atmosphere at 5% CO2. and 95% air. Fibronectin, gelatin, or collagen significantly(P<0.01) increased and blastocyst formation rate compared with controls in 1/2(65.3, 59.2, 60.7% vs. 21.6%), 1/4(63.7, 53.4, 57.1% vs. 26.3%), and 1/8 blastomeres(61.1, 52.3, 53.7% vs. 19.1%). Both the nuclear number(P<0.05) and diameter of blastocysts(P<0.01) developed from balstomeres were significantly affected by the origin of blastomeres. The nuclear number of blastocysts developed from 1/2, 1/4, and 1/8 blastomeres ranged 29.3$\pm$1.6, 24.5$\pm$1.3, and 20..$\pm$1.2, respectively. And the diameter of those blastocysts was 88.3$\pm$2.4, 57.6$\pm$2.1, 39.8$\pm$1.9${\mu}{\textrm}{m}$, respectively.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.171-176
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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 conception of embryos transferred, and pregnancy and twin birth rates after transfer of fresh or frozen-thawed bovine blastocysts produced in vitro were also evaluated. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol. The results obtained were as the following. The pregnancy rate after transfer was higher in co-culture group than in mTALP group, but was not signficantly different, and there is no difference between fresh embryo group and frozen-thawed embryo group in conception rate. The conception rate was not different whether 3∼4 blastocysts or 2 blatocysts transferred into a recipient, but the production rate of twin calves was significantly higher (p<0.05) when 3∼4 embryos transferred. The average birth weight of twin calves(24.38kg) was numerically, but not significantly lighter than that of single calves(26.68kg).

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.9-12
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• 2016

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors that regulate many critical processes involved in early folliculogenesis and oocyte maturation. In this study, effects of GDF9 and BMP15 treatment during in vitro maturation of porcine oocytes upon development after parthenogenetic activation were investigated. Neither GDF, BMP15 alone nor in combination affects the number and viability of cumulus cells or the rates of oocyte maturation and blastocyst development. However, the treatment of GDF9 on porcine oocytes increased the number of trophectodermal (TE) cells of blastocysts derived from activated oocytes (P<0.05). The treatment of BMP15 increased the cell numbers of both inner cell mass (ICM) and TE cells (P<0.05). The treatment with the combination of GDF9 and BMP15 further increased the numbers of ICM and TE cells, compared with GDF9 or BMP15 treatment alone (P<0.05). In conclusion, the treatment of GDF9 or BMP15 (or both) enhanced the quality of blastocysts via the increased number of ICM and/or TE cells.

• Journal of Embryo Transfer
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• v.29 no.1
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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).

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1245-1252
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• 2017

Objective: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense ($0.01{\mu}g/mL$) and H. japonicus ($0.01{\mu}g/mL$). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense ($28.9%{\pm}2.9%$), H. japonicus ($30.9%{\pm}1.5%$), and a mixture of P. amurense and H. japonicus ($34.8%{\pm}2.1%$) treated groups compared with the control group ($25.4%{\pm}1.6%$). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. Conclusion: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

• Development and Reproduction
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• v.20 no.3
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• pp.219-225
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• 2016

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ${\geq}38$ years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen ($LN_2$) and then directly immersed into the first WS for 1 min at $37^{\circ}C$ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrified-warmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.9-14
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• 2010

This study was conducted to find out the effects of artificial shrinkage (AS) on post-thaw development of bovine embryos. The blastocoelic cavity of blastocyst was punctured to remove its fluid contents and then incubated in the holding medium (HM) for 10 min. The punctured and non-punctured (control) blastocysts were equilibrated in vitrification solution 1 (VS1; TCM-199+20% FBS+10% EG) for 5 min and vitrification solution 2 (VS2; TCM199+20% FBS+35% EG+5% PVP+0.5 M Sucrose) for 1 min and vitrified by direct dropping into the liquid nitrogen. Vitrified blastocysts (punctured and control) were thawed and cultured in vitro (12 hr) for studying survival and hatching rates. The levels of shrinkage were measured by the volume of the blastocyst during equilibration in VS1 (at 1, 3 and 5 min of equilibration) and VS2 (at 30 and 60 sec of equilibration) that was considering the volume of non-punctured blastocyst in HM as 100%. The levels of shrinkage were higher in punctured group (62.4, 64.6, 64.3% at 1, 3 and 5 min in VS1; 50.6 and 52.7% at 30 and 60 sec in VS2) than control group (84.8, 86.6, 86.4% at 1, 3 and 5 min in VS1; 72.1 and 68.8% at 30 and 60 sec in VS2), but within each group the levels of shrinkage were similar. The survival (90.9%) and hatching (50.0%) rates of vitrified blastocysts at 12 hr post-thaw were higher in punctured group than that in control group (76.9% and 0.0% respectively). We confirmed that vitrification solutions (VS1 and VS2) have no toxic effect on the survival of blastocysts because the survival rates of blastocysts exposed to VS1 and VS2 for 24 hr were similar between punctured and control groups (94.3 vs. 96.0%; p>0.05). In conclusion, the preliminary data show that AS of blastocyst may improve survival and hatching rate after thawing.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.307-311
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG ($33.1{\pm}9.6$ vs $21.7{\pm}8.3%$). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP-treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The mRNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.