• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.127-134
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• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.253-257
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• 2009

To enhance the embryo preservation technology and better application of embryo transfer technique to the field (dairy science or animal reproduction. etc.), we examined the viabilities of bovine embryos produced in vitro and in vivo after cryopreservation according to their developmental stage and thawing temperature. Bovine embryos from in vivo/vitro fertilization (Hanwoo) were examined at day 7, 8, and 9. Survival rates and total cell numbers of in vivo fertilized embryos were as follows: morulae 68.8% and $67\;{\pm}\;6.0$; blastocysts 80.5% and $120\;{\pm}\;10$; expanded blastocysts 77.4% and $138\;{\pm}\;9.7$, respectively. Rates of embryo development for blastocysts and expanded blastocysts after thawing were significantly higher than that of morula stage embryos (p<0.05). While survival rates of in vitro fertilized embryos according to developmental stage showed no significant difference among groups (morula 67.9%; blastocyst 74.3%; and expanded blastocyst 79.4%), total cell numbers were significantly lower than those of other groups (morula $64\;{\pm}\;5.9$; blastocyst $116\;{\pm}\;8.7$; and expanded blastocyst $135\;{\pm}\;9.1$) For the viability according to thawing temperature, survival rate was higher in $37^{\circ}C$.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.171-178
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• 1993

The present study was carried out to investigate the developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulse at output voltage of 2.0 kV/cm for $100{\mu}$ sec to induce cell fusion. The recovery rate and developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos was investigated. 1. The recovery rate of nuclear transplanted 2-cell embryos in normal morphology after freezing and thawing was significantly higher in rapid freezing(DMSO 4.5M) than in slow cooling(p<0.01). 2. When the recovered embryos in normal morphology were cultured in vitro, there were no significant differences in the developmental potency to blastocyst between the freezing methods and the concentrations of cryoprotectant. In summary, these experiments have proved that rapid freezing method(DMSO 4.5M) is effective in nuclear transplanted 2-cell mouse embryos. If improved micromanipulation techniques and freezing are combined, nuclear transplantation technique will contribute to the improvement of productivity in livestock animals.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.141-149
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• 1997

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.75-85
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• 2019

Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E₂, 17β-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E₂ synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17β-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17β-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.221-227
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• 1998

This study was carried out to determine the effects of ionomycin and 6-dimethylaminopurine (6-DMAP) treatments on parthenogenesis of rabbit oocytes. The oocytes were randomly assigned to the activation treatments with either ionomycin plus 6-DMAP or electric stimulation. The oocytes were colected from the oviducts of superovulated rabbits at 13~14 hours and 19~20 hours post hCG injection and were activated with 5$\mu$M ionomycin for 5 min and 2 hours incubation in 2mM 6-DMAP. The other oocytes were stimulated by three pulses of 3.6kV/cm for 60 $\mu$sec each 30 min apart, starting 19 hours post in hCG in 0.28M mannitol solution with 100$\mu$M Ca2+ and Mg2+. The results obtained were summarized as follows: 1. Following treatment of the oocytes with ionomycin plus 6-DMAP, the cleavage rate and in vitro developmental rate to blastocyst were significantly(P<0.01) higher in the oocytes collected bet ween 19~20 hours than between 13~14 hours after hCG injection. 2. When the oocytes were treated with ionomycin plus 6-DMAP, 85(98.8%) of 86 treated oocytes extruded the second polar bodies, with the entire chromatin complements outside ooplasm. However when the oocytes were restored during subsequent incubation in the drug-free medium, the cytoplasts regain their full capacity for parthenogenetic activation and nuclear remodelling. 3. The cleavage rate and the in vitro developmental rate to blastocyst were not significantly different in the oocytes activated by ionomycin plus 6-DMAP treatment(91.2 and 45.6%) or electrical stimulation(89.6 and 34.3%).

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.127-132
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• 2014

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 ($0.5{\mu}g/ml$), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at $39^{\circ}C$ in a humidified atmosphere of 5% (v/v) $CO_2$, 5% (v/v) $O_2$ and 90% (v/v) $N_2$. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group ($174.3{\pm}2.5$) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst ($182.3{\pm}2.1$) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; $165.7{\pm}4.2$, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.283-291
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• 1997

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.7
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• pp.956-965
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• 2019

Objective: The main goal of this study was to provide a morphological indicator that could be used to select high-quality oocytes of appropriate meiotic and developmental capabilities in pig. The higher quality of immature oocytes, the higher success rates of in vitro maturation (IVM) and in vitro fertilization (IVF). Thus, prior to the IVM culture, it is important to characterize oocytes morphologically and biochemically in order to assess their quality. Two of the largest indicators of oocyte quality are the presence of cumulus cells and status of chromatin. To investigate the effects of porcine oocyte chromatin configurations on the developmental capacity of blastocysts, we assessed oocyte chromatin status according to follicle size and measured the developmental potency of blastocysts. Methods: To sort by follicle size, we divided the oocytes into three groups (less than 1 mm, 1 to 3 mm, and more than 3 mm in diameter). To assess chromatin configuration, the oocytes were assessed for their stages (surrounded nucleolus [SN] germinal vesicle [GV], non-surrounded nucleolus [NSN] GV, GV breakdown, metaphase I [MI], pro-metaphase II [proMII], and metaphase II [MII]) at different maturation times (22, 44, and 66 h). To assess the development rate, oocytes of each follicle size were subjected to parthenogenetic activation for further development. Finally, GV oocytes were grouped by their chromatin configuration (SN, SN/NSN, and NSN) and their global transcriptional levels were measured. Results: SN GV oocytes were more suitable for IVF than NSN GV oocytes. Moreover, oocytes collected from the larger follicles had a greater distribution of SN GV oocytes and a higher developmental capacity during IVM, reaching MII more quickly and developing more often to blastocysts. Conclusion: Porcine oocytes with high-level meiotic and developmental capacity were identified by analyzing the relationship between follicle size and chromatin configuration. The porcine oocytes from large follicles had a significantly higher SN status in which the transcription level was low and could be better in the degree of meiotic progression and developmental capacity.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.51-57
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• 2008

The present study investigated the efficient methods to produce in vitro Hanwoo embryos, and to improve the pregnancy rate. The developmental rate, total cell number and ICM ratio of in vitro embryos were compared amongst different culture media. Comparisons were also made on the status of recipients, pregnancy rate along with day of transfer after the estrus. Development of embryos into blastocyst stage in IVMD101 supplemented with 5% fetal bovine serum (FBS) group was significantly higher (34.2%) than that of TCM-199 supplemented with 5% FBS (26.8%) and IVMD101 without FBS (25.9%) (p<0.05). The development rate to blastocyst stage was significantly faster in IVMD101(5% FBS) than that of other groups ($0.2{\sim}2.3%$) (p<0.05). The average number of inner cell mass and trophectoderm were similar among treatment groups, which were $36.0{\sim}44.7$ and $83.3{\sim}106.7$. However, total cell number in IVMD101(5% FBS and 0% FBS) was significantly higher than that of TCM199(5% FBS). There were no differences in the pregnancy rate among treatment groups (32.0%, 33.9% and 28.6%, respectively). However, the pregnancy rate of Day 6 embryos cultured in IVMD101(5% FBS) was significantly (p<0.01) higher than IVMD101 without FBS and TCM-199 + 5% FBS (38.0% vs. 17.2% and 32.4%, respectively). No significant difference was observed for the pregnancy rate between heifer and cow transferred with Day 6 embryos cultured in IVMD 101(5% FBS) (42.7% and 39.3%, respectively). However, there was a significant difference of pregnancy rate (p<0.05) in heifer between one and two embryos transferred (31.4% and 41.9%). There was no difference of pregnancy rate among transfer days after estrus between heifer and cow, but the pregnancy rate of transfer to heifer with day 6 after estrus was significantly higher (p<0.05) than that of day 7 and 8 (22.2% vs. 49.0% and 38.7% respectively). Based on the above findings, there is a possibility to produce in vitro produced embryos cultured in IVMD101(5% FBS) showed higher blastocyst rate and the increased cell number. In terms of the pregnancy rate of in vitro produced embryos, the highest pregnancy rate was observed when two embryos were cultured in IVMD101(5% FBS) and transferred.