• 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.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.199-205
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• 2013

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.111-115
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• 2011

In general, zona pellucida (ZP) of the blastocyst has to be removed first, then either isolated the inner cell mass (ICM) or ZP-removed whole blastocyst, which is then cultured on the feeder layer to induce ICM outgrowth for the generation of embryonic stem cells (ESC). However, it is unclear whether ICM isolation before seeding on feeder layer is beneficial or not because the interaction between ICM and trophoblasts may affect cellular growth and/or pluripotency during the culture on the feeder. In the present study, two ZP removal methods (mechanically by splitting with a 28-gauge needle versus chemically by the treatment of acid-Tyrode's solution) and two ICM isolation methods (ZP-free whole blastocyst seeding versus mechanical isolation of ICM) were evaluated for the efficient isolation and culture of putative parthenogenetic bovine ESC. The number of maintained outgrown colonies was counted in each experimental group. As the result, mechanical removal of ZP with a needle and followed by whole ZP-free blastocyst seeding on feeder cells tended to attach more on the feeder layer and resulted in more outgrown colonies with its simple and less time-costing benefits. Currently we are generating ESC lines in HanWoo cattle by using this method for initial outgrowth of the parthenogenetic bovine blastocysts.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.307-313
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• 1997

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

• 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

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.133-137
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• 2004

Sexing from bovine embryos which were fertilized in vitro implicate a possibility of the sex-controlled cattle production. This study was carried out to investigate the possibility of determining of embryo sex by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe which constructed from the btDYZ-1 sequences. To evaluate Y-chromosome specificity of the FISH probe, metaphase spreads of whole embryos and lymphocytes were prepared and tested. A male-specific signal was detected on 100% of Y chromosome bearing metaphase specimens. Using the FISH technique with a bovine Y-specific probe, 232 whole embryos of 8 cell- to blastocyst-stage were analyzed. Observing the presence of the Y-probe signal on blastomeres, 102 embryos were predicted as male, and 130 embryos as female. The determining rate of embryo sex by FISH technique was about 93% regardless of embryonic stages. In conclusion, the FISH using a bovine Y-specific DNA probe is an accurate, reliable and quick method for determining the sex of bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.6
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• pp.599-605
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• 1995

The ultrastructures of in vitro-derived bovine blastocysts have been compared with those of blastocysts obtained from a superovulated cow. In vivo blastocysts obtained from the uterus showed well-differentiated features, while in vitro-derived embryos, which were developed from in vitro fertilized ovum, showed insufficient cellular organizations. In vitro-derived embryos contained many undefined cellular organizations in the perivitelline spaces compared with in vivo-derived blastocysts. Other features of in vivo and in vitro blastocysts were characterized by differential development of microvilli projection into blastocoele from the surface of the trophoblast cells. The conceivable reason for the difference between in vivo and in vitro developments of bovine embryos is that it is likely that in vitro culture system adopted in the present experiment may not be sufficient for better embryonic development.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.805-812
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• 2006

The purpose of this study was to investigate the effect of Matrigel on the development of bovine embryos after in vitro fertilization. Bovine embryos were cultured in Ⅰ) SOF+ 0.8% BSA(SOF-B), Ⅱ) SOF+ 0.8% BSA plus 0.8% Matrigel(SOF-M) and III) SOF+0.8% BSA and 10% FBS(SOF-BF). The addition of Matrigel appeared not to increase the proportion of blastocysts (SOF-B, 26.6%; SOF-M, 28.2%; SOF-BF, 26.2%). However, the proportion of hatched blastocysts were significantly increased(P<0.05) by Matrigel(SOF-B, 23.7%; SOF-M, 48.7%; SOF-BF, 18.5%). The means of cell number blastocyst was not significantly different among the treatment groups(SOF-B, 172.7±35.5; SOF-M, 175.1±37.4; SOF-BF, 172.8±38.1). The proportion of apoptotic cells in blastocyst was also found to be not significant among the treatment groups(SOF-B, 3.6±3.2%; SOF-M, 4.3±2.6%; SOF-BF, 4.9±4.3%). In this experiment, Matrigel appeared to support embryonic hatching of bovine embryos. Results suggest that Matrigel, as extracellular matrix components, may be another avenue for formulating more physiological culture system in serum-free culture.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.253-256
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• 2004

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p<0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-cell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro.