• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.129-134
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• 1995

This study was carried out to investigate on the survival rate and in vitro developmental rate of bisected porcine embryos and immature oocytes by manipulator and micropipette. Bisected embryos and immature oocytes cultured for 1∼5 days in TCM 199 medium with 20% FCS. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos and oocytes were 26.1%, 22.7%, respectively. The survival rate of bisected embryos and oocytes was significantly lower than that of non-bisection embryos(62.5%). 2. The survival rate of bisected porcine embryos in cultured for 12, 24, 48, 72 hrs with 20% FCS＋TCM-199 medium were 26.9%, 19.2%, 19.2% and 11.5%, respectively. 3. The in-vitro developmental rate with and without zona pellucida of bisected porcine embryos by micromanipulator were 30.8%, 25.0%, respectively.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.108-108
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• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.117-123
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• 1992

Development in vitro of 2-cell mouse embryos was examined after appropriate exposure to oviductal milieu to demonstrate biological activity present in the oviducts. ICR and ($C57Bl/6{\times}Balb/c$) $F_1$ hybrid mice were superovulated and mated for the recovery of early embryos. Embryos were recoverd at every 2h intervals from 32h post-hCG(hph) to 56 hph. The proportions of developmental stages were determined in the recovered embryos. Development in vitro of 2-cell embryos was more rapid in $F_1$ hybrid than in ICR, showing high proportions of 4-cell embryo and blastocyst at 120 hph. 100% of blastocyst development was obtained at 38hph in $F_1$ hybrid and at 50 hph in ICR when 2-cell embryos were cultured upto 120hph in vitro. Moreover, in vitro culture of oviducts containing 2-cell embryos in ICR mice for 12h from 34hph to 46hph increased developmental capacity of ICR mouse embryo in vitro. The results indicate that oviductal environment contains substances having mitogenic activity and overcoming early cell block in vitro. The mitogenic activity is effective in vitro as well as in vivo.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.113-119
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• 1994

To improve nuclear transplantation(NT) efficiency and to produce a large scale genetically identical cloned calves, examined the in vitro development capacity after co-culture of bovine oviductal epithelial cells (BOEC) and granulosa cells in TCM-199 supplemented with 10% fetal calf serum (FCS) with early bovine embryos derived from in vitro matured fertilized(IVM-IVF) oocyte. In addition, the age dependence of IVM oocyte on electro-stimulation and the effective electric voltage on in ivtro development of bovine NT embryos were examined. The results obtained were summerized as follows; 1. The cleavage rates of IVM-IVF bovine embryos in co-culture with bovine oviductal epithelial cells and granulosa cells were not significantly different(P<0.05), but the developmental rate into morula and blastocyst stage were different showing 38.3 and 20.2%, respectively. 2. The activation (82.5%) and development in vitro(8.6%) into later embryo stages of the aging oocytes of 32 hours post-maturation (hpm) were significantly higher than those of 24 hpm at direct current (DC) voltage of 1.5kV/cm, 60$\mu$sec pulse duration and 1 pulse time. 3. The fusion rates of NT eggs of 32 hpm following to different DC voltages from range 0.75 to 1.5kV/cm were not differ, but the developmental rate into morula and blastocyst stages at DC voltages of 0.75 and 1.0kV/cm were higher(11.4 and 12.6%, respectively) than those of 1.5kV/cm(0%). From these results, it can be suggested the optimal culture system for in vitro culture of IVM-IVF bovine embryos is a co-culture system with BOEC in TCM-199 supplemented 10% FCS. The effective time and the DC voltage for activation, electrofusion and in vitro development of NT embryos derived from IVM-IVF bovine embryo are 32hpm and 0.75~1.0kV/cm. But to improve NT efficiency, the advanced research (cell cycle synchronization, micromanipulation, culture system, etc.) is needed.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.167-175
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• 2008

This study was performed to investigate the result that in-vivo or in-vitro embryos of Hanwoo cows were transferred to Holstein cows. Seventeen Hanwoo cows were used as donors for production of in-vivo embryos and fresh hanwoo in-vivo embryos were transferred to 1,150 Holsteins. And 2 embryos were transferred to 188 Holstein recipients to produce twin calves. Diagnosis on pregnancy was performed by rectal palpation at $60\sim90$ days after transfer. The pregnancy rate of Holstein recipients was 55.8% after transfer with Hanwoo in-vivo embryos and 38.2% after transfer with Hanwoo in-vitro embryos. The delivery rate of pregnant Holstein recipients was 88.4% after transfer with Hanwoo in-vivo embryos and 75.6% after transfer with Hanwoo in-vitro embryos. The rate of delivery of Holstein recipients transferred with two Hanwoo embryos was 36.2% and the rate of twin production was 25.9%. The rate of twin production by embryo transfer with in-vivo embryos was 30.4%, whereas the fate with in-vitro embryos was 15.6%. The pregnancy rate according to the grade of corpus luteum of Holstein recipients transferred with Hanwoo in-vitro embryos was 41.5 and 36.0% for A and B grade, respectively. The pregnancy rate according to the transfer in site in the uterine lumen of recipients was 40.9 and 32.7% for anterior and middle site, respectively. The pregnancy rate according to day of embryo transfer after estrus of recipients was 45.5, 38.8 and 39.7% for day 6, day 7 and day 8, respectively. There was difference of pregnancy rate according embryo transfer technician ($30.5\sim45.8%$) individual dairy farm ($21.1\sim51.0%$). These results are supposed to indicate that the rate of pregnancy after transfer with Hanwoo embryos to Holstein recipients was similar to that within the same breed, and consequently that this method would be beneficial to enhance the productivity in Hanwoo reproduction.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.261-265
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• 2007

Insulin, transferrin and selenium (ITS) complex is reported to improve in vitro development of oocytes and embryos. This study was carried out to investigate the effects of ITS during in vitro culture (IVC) of porcine parthenogenetic and nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. The electrically activated oocytes were cultured in Porcine Zygote Medium (PZM-3) with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 7 days. Also, the electrically activated reconstructed embryos were cultured in PZM-3 with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 6 days. Addition of ITS to culture medium did not affect development of porcine parthenogenetic embryos in vitro. To test the effect of ITS on the in vitro development of porcine NT embryos, factorial experiments were also performed for in vitro maturation (IVM) medium (TCM-199) with or without 1% ITS and culture medium (PZM-3) with or without 0.5% ITS. Addition of 0.5% ITS to culture medium increased (p<0.05) the proportion of NT blastocysts compared with non-treated group. In contrast, addition of 1% ITS to culture medium was ineffective or had a detrimental effect. Also, addition of ITS only to maturation medium increased (p<0.05) the percentage of NT blastocysts formation compared with the control group. In conclusion, addition of ITS to IVM or IVC medium could improve subsequent blastocyst development of porcine NT embryos.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.275-281
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• 2008

The ability to preselect the sex of piglets is advantageous in the pig industry. The objective of this study was to examine the feasibility of using intracytoplasmic sperm injection (ICSI) with sorted spermatozoa to produce piglets with a preselected sex. Pig embryos were produced by ICSI of frozen X- and Y-sperm that had been separated by flow cytometry. The developmental competence of the embryos was investigated in vitro and in vivo. The populations of X- and Y-spermatozoa were 52.7% and 47.3%, respectively in our samples. The in vitro development of ICSI embryos was enhanced by longer of in vitro maturation of oocytes ($44{\sim}48\;h$ vs. $40{\sim}43\;h$). Their cleavage ($65{\sim}70%$) and blastocyst formation ($9{\sim}12%$) rates were not significantly different between male and female ICSI embryos, or between sorted and unsorted sperm-derived embryos. One pregnancy was established in a recipient that was transferred with 110 female ICSI embryos, but the pregnancy was terminated on Day 89 of gestation. Our results suggest that the separation X- and Y-spermatozoa by flow cytometric sorting can be a useful tool in combination with ICSI for the production of pig embryos and piglets of preselected sex.

• 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.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.919-927
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• 1996

In the last few years, methods for in vitro culture of early embryo stages from oocytes matured and fertilized in vitro using suitable cell culture systems have been established. But the factors affecting pregnancy rates following transfer of bovine embryos produced in vitro were not evaluated enough. So this study was performed to investigate the effects of quality and stage of embryos, parity and Corpus Luteum quality of recipients on pregnancy rates following non-surgical transfer of bovine embryos produced in vitro. Oocytes aspirated from small antral follicles of ovaries obtained at a local slaughter house were matured, fertilized with frozen-thawed semen and co-cultured for 6-7 days by utilizing co-culture system with bovine oviduct epithelial cell in vitro. After co-culture, embryos were transfered to recipients on day 7 (estrus=day 0). Recipients were monitored by ultrasonic scanning method or observation for estrus and rectal palpation after 50 days from transfer. The results of this study are follows. 1. Of the 70 recipients, 70%(49 of 70) had not showed estrus sign between day 0 and day 50, but 22.9%(16 of 70) was diagnosed not pregnant. Therefore the overall pregnancy rate of this study was 47.1%(33 of 70). 2. The pregnancy rate of recipients transfered with excellent(66.7%) and good(54.5%) embryos were higher than that of recipients transfered with fair embryos(15.8%) (p<0.05). 3. The pregnancy rate of recipients transfered with morula, compacted morula, blastocyst and expanded blastocysts were 46.2, 55.0, 62.5 and 50.0%, respectively. 4. The pregnancy rates of recipients transfered to heifer and cow were 54.5 and 55.2%, respectively. 5. The pregnancy rates of recipients with CL score I, II(66.7, 63.6%) were higher than those of recipients with CL score III (10%), (p<0.05). Success of transfer of embryos produced in vitro depends on many variables. The important factors identified in this study were the quality of embryos and the CL score of recipient animals after non-surgical transfer of embryos matured, fertilized and cultured in vitro.

• 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.