• 한국가축번식학회지
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• 제20권1호
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• pp.71-76
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• 1996

This experiment was carried out to investigate the developmental ability of bovine embryos matured and fertilized in vitro to the gastrulation stage. The bovine oocytes were collected from 2∼5mm follicles, matured for 20∼24hrs in 5% CO2 incubator and then fertilized with frozen-thawed semen. On day 9 after IVF and after freezing and thawing the hatching abilities of expanding blastocysts were examined. Cleavage rate and production rate to expanding blastocysts were 59.7%(955/1604) and 20.7%(333/1604), respectively. Hatching rate of day-9 expanding blastocysts was 54%(40/74), that after freezing and thawing was 56%(79/141). Also, developmental ability of hatched blastocysts to the primitive streak stage was 26%(6/23).

• 한국수정란이식학회지
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• 제15권1호
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.281-287
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• 2013

In mammal, unfertilized oocytes remain in the oviduct or under in vitro culture, which is called "oocyte aging". This asynchrony negatively affects fertilization in pre- and post-implantation embryo development. Caffeine a phosphodiesterase inhibitor is known to rescue oocyte aging in several species. The objective of this study is to determine the cytoskeleton distribution in aged oocytes and the embryo developmental ability of aged oocytes in the present or absence of caffeine during maturation. Caffeine treatment increased the incidence of normal spindle assembly of aged oocytes (treatment, $67.57{\pm}4.11%$ aging, $44.61{\pm}6.4%$) and no significant differences compared to control group. Fluorescence values were compared using ROS (Reactive oxidation species) stain. Fluorescence values appear of control group intensity rate ($51.53.{\pm}3.80$), aging group ($68.10{\pm}5.54$) and treatment of caffeine ($45.04{\pm}2.98$). Aged oocytes that were derived from addition of caffeine to the IVM (in vitro maturation) medium had significantly increased 2-cell that developed to the blastocyst stage compared to the aging group. Blastocysts, derived from caffeine treatment group, significantly increased the total cell number compare aging ($90.44{\pm}10.18$ VS $67.88{\pm}7.72$). Apoptotic fragments of genomic DNA were measured in individual embryo using TUNEL assay. Blastocyst derived from caffeine treatment group decreased significantly the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocyte aging can improve the developmental rate and quality in bovine embryos developing in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.31-36
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• 1993

The effects of freezing and 1,2-propanediol on early and late pronucleate stage mouse ova were investigated in terms of survival after thawing and development in vitro. The samples were divided into two groups according to different age in pronucleate ova: ova in(1) early pronuclear stage with two distant pronuclei at 18h after hCG injection, and (2) late pronuclear stage with adjacent pronuclei at 30h. Zygotes in the late pronuclear stage have been proven to be more resistant to 1,2-propanediol, showing a significantly higher developmental rate than zygotes in early stage (80.3 versus 66.3%, <0.05), but survival rate was similar in the two groups (91.0 versus 93.5%). After freezing and thawing, survival and developmental rates were decreased in both groups when compared to the control group (54.3 versus 92.3%, 47.7 versus 73.3%. respectively). And developmental rate in the late pronuclear stage zygotes showed significantly higher than in early (55.4 versus 40.0%) after thawing. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than late ova. These findings suggest that the timing of freezing could be important for survival and further development in vitro in cryopreservation of human pronucleate ova.

• 대한수의학회지
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• 제38권2호
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• 농업과학연구
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• 제43권1호
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• pp.52-60
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• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.257-257
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• 2004

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes on in vitro maturation of cat oocytes and development of IVM/IVF embryos. 1. When recovered from ovaries collected at different stages of the reproductive cycle (inactive, follicular and luteal stage), the developmental rates of oocytes to GV and MI stage were 72.5% and 27.5%, 57.5% and 7.5%, 62.5% and 17.5%, respectively. (omitted)

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.127-133
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• 2016

Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. Inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage cause of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.25-32
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• 2010

The present study aimed to examine the effects of ${\gamma}$-linolenic acid (GLA) supplementation to in vitro culture (IVC) medium on in vitro developmental competence, freezability and morphology of in vitro matured and fertilized bovine embryos. In vitro produced (IVP) bovine zygotes were cultured in IVC medium supplemented with 0 (negative control), 15, 31, 62, 125, 250, 500 or 1,000 ppm GLA, 250 ppm linoleic acid albumin (LAA) and without any supplement as a control. Day 6 blastocysts derived from culture control were cultured in IVC medium containing either 62, 250 GLA or 250 LAA for 24 h, and at Day 7 were subjected to freezing or morphological examination by electron microscope. GLA 15 showed a tendency to have a higher cleavage rate at Day 2 (70.3%) than other groups. The hatching rate at Day 9 in LAA (38.2%) was significantly higher than the control and all treatment groups (p<0.05), while the blastocyst rate in LAA (32.4%) did not differ from those of 15 (30.5%), 31 (27.1%), and 62 GLA (33.1%) or the control (35.1%). GLA in concentrations of 125, 250, 500, and 1,000 ppm had significantly detrimental effect on the blastocyst rate compared to 15, 31 and 62 ppm GLA, LAA, and control groups (p<0.05). In contrast, the highest post-thaw survival rate (100%) was observed in the control group (p<0.01). Large lipid droplets were observed in the cytoplasm of trophoblastic cells, even in the control, but were abundant in GLA groups. Taking the results of the study into consideration, the addition of GLA to the culture medium for IVP bovine embryos at the dose of 15 ppm increased the developmental competence of zygotes and enhanced the cleavage rate up to Day 2. However, blastulation rate and post-thaw survival were not increased when GLA was added to the culture media.

• 한국수정란이식학회지
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• 제13권2호
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• pp.147-157
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• 1998

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.