• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.487-495
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• 2007

The objectives of the present study were to initiate cloning of Korean native goat by somatic cell nuclear transfer (NT) and to examine whether unovulated (follicular) oocytes can support the same developmental ability of NT embryos as ovulated (oviductal) oocytes after hCG injection in stimulated cycles of the goat. The in vivo-matured and immature oocytes were collected from the oviducts and follicles of superovulated does, respectively, and the immature oocytes were maturated in vitro. Ear skin fibroblasts derived from a 3-yr-old female Korean native goat were used as the donors of nuclei or karyoplasts. Following fusion, activation and in vitro culture to a 2- to 4-cell stage, 49 in vitro-derived and 105 in vivo-derived embryos were transferred to 6 and 17 recipient does, respectively. One doe and three does of the respective groups were identified as pregnant by ultrasonography on day 30 after embryo transfer. However, only one doe, which had received in vivo-derived embryos, delivered a normal female kid of 1.9 kg on d 149. The cloned kid gained more weight than her age-matched females as much as 87% during the first 4 mo after birth (17.7 vs. $9.4{\pm}0.8$ kg) and reached puberty at 6-mo age a few months earlier than normal female does. The telomere length of the kid, which was similar to that of the donor fibroblast at 2-mo age, decreased 8% between 2- and 7-mo ages. Moreover, at 7-mo age, she had 21% shorter telomere than her age-matched goats. To our knowledge, this is the first case in which a cloned animal born with a normal weight exhibited accelerated growth and development. The unusually rapid growth and development of the cloned goat may have resulted from SCNT-associated epigenetic reprogramming involving telomere shortening.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.9
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• pp.1239-1246
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• 2016

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.237-241
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• 2012

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

• Journal of Embryo Transfer
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• v.26 no.2
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• pp.117-122
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• 2011

In this study, we examined the effectiveness of in vitro fertilization of porcine immature oocytes on the embryo development of blastocysts or hatched blastocysts and the number of cells according to the in vitro fertilization conditions. In the in vitro fertilization of in vitro matured porcine oocytes, there were no significant differences between treatment groups regarding fertilization rate, blastocyst rate, and embryo development of hatched blastocysts according to the storage periods of liquid sperm of 24, 48, and 72 hours. The embryo development rate of hatched blastocysts after the fertilization according to different spermatozoa concentrations ($0.4{\times}10^5$, $1.2{\times}10^5$, and $3.6{\times}10^5$ cells/ml) showed the highest rate in the group with a spermatozoa concentration of $1.2{\times}10^5$ cells/ml; in particular, this rate was significantly higher than that in the $0.4{\times}10^5$ cells/ml group (p<0.05). The total number of blastocysts cells as well as trophectoderms (TE) that developed in each treatment group were also significantly higher in the $1.2{\times}10^5$ cells/ml group than in any other groups (p<0.05). In contrast, the embryo development rate of blastocysts according to different co-incubation periods of sperm and oocyte (1, 3, and 6 hr) was high in the 6-hour group; in particular, the rate was significantly higher than that of the I-hour group (p<0.05). Furthermore, the total number of oocytes cells and TEs that developed was significantly higher in the 6-hour group than any other group (p<0.05). In this study, the most effective treatment conditions for porcine embryo development and high cell number were found to be as follows: a sperm storage period of less than 72 hours, a spermatozoa concentration of $1.2{\times}10^5$ cells/ml, and a 6-hour co-incubation period for sperm and ooocyte.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1117-1123
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• 2009

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro, there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.71-77
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• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.89-95
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• 2009

Successful in vitro embryo production heavily relies on the normal maturation and fertilisation of oocytes. We examined the normal and abnormal fertilisation of zebu cattle oocytes matured in vitro. Immature cumulus oocyte complexes (COCs) from zebu cattle ovaries at slaughter were matured in vitro (IVM) for 24 h. The oocytes were either fixed, stained and examined for nuclear changes or fertilised in vitro (IVF) with Percoll-separated, heparintreated spermatozoa (1.0 ${\times}$ $10^6$/mL) of zebu (n = 7) and crossbred bulls (n = 7). After 18 h of sperm-COCs co-incubation at $39^{\circ}$C with 5% $CO_2$ in humidified air, the presumptive zygotes were fixed, stained and examined for pronuclei. The number of oocytes retrieved per ovary was 5.4 ${\pm}$ 0.7. The percentage of matured oocytes was 73.0. The difference in motility of spermatozoa before and after Percoll seperation was significant (p<0.001). The percentages of normal and abnormal fertilisation (polyspermia and oocytes with one pronucleus) varied significantly depending on individual bulls (p<0.05). A protocol for IVF of IVM oocytes in Bangladeshi zebu cattle is developed. A future study may elucidate the capacity of such IVM-IVF oocytes to develop to the blastocyst stage for transfer to surrogate mother.

• Korean Journal of Veterinary Research
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• v.59 no.2
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• pp.81-88
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• 2019

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF ($113.0-114.8{\mu}m$) were significantly larger than that of no IVG-SAF ($111.8{\mu}m$). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.165-172
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• 2004

This study was carried out to examine the effects of maturation media on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-37 (mNCSU-37), modified NCSU-23 (mNCSU-23), or TCM-199 supplemented with 10% porcine follicular fluid (pFF). Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium(mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU-23. The results are as follows. 1. In the result of IVM, the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different among the media, though numeric value of them were slightly lower in TCM-199 than in mNCSU-37 or in mNCSU-23. 2. In the result of IVF, though the rate of sperm penetration was not significantly different among the maturation media, the percentage of oocytes with male pronucleus (MPN) of ones matured in mNCSU-37 (88.0%) was significantly higher than in TCM-199 (71.1%) (p<0.05). 3. In the result of IVD, the percentage of cleaved oocytes of ones matured in mNCSU-37 (52.3%) or in mNCSU-23 (53.7%) was significantly higher than in TCM-199 (43.1%) (p<0.05), but the rate of blastocysts at day 6 was not significantly different among the maturation media, though putative embryos from oocytes matured in mNCSU-37 or in mNCSU-23 were developed more than in TCM-199. These results suggested that mNCSU-37 or mNCSU-23 was more appropriate than TCM-199 as IVM medium for porcine immature oocytes.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.183-191
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• 2001

This study was designed to establish the superior method for IVF embryos from DNA marker-proved Hanwoo cattle. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from unstimulated. immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local abesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analysed using the GLM procedure of SAS. Mean number of follicles identified on ultrasound was 5.5 $\pm$2.9 in right and 4.3 $\pm$2.8 in left ovaries, respectively. The highest follicles(16.6$\pm$2.6) were found in 5101 cow compared to others. Recovery rate of follicular oocytes in individual cow was highest in 5101 cow with 89.3% in > 2mm and 94.0% in $\leq$ 2mm follicles. Total recovery rate was significantly(P<0.01) higher in $\leq$ 2mm(85.7%, 130/154) than > 2mm follicles(74.2%, 201/271). Significantly more oocytcs of Grade IV were recovered from > 2mm follicles. Mean number follicles recovered was 4.8$\pm$3.7. 3.0$\pm$3.4 and 0.3$\pm$0.6 in $\leq$2mm, 2~6mm and $\geq$6mm follicles. respectively. Our results imply that the more fertilizablc oocytes can be recovered from invisible-immature follicles by the combination of simultaneous rectal ovarian-palpation and ultrasound-guided approach in Hanwoo cattle.