본 연구는 티모시 건초와 농후 사료 위주의 사료를 급여한 한우 씨수소 정소상체 정자 체외수정 효율 조사를 통해 정자의 활용 가능성을 조사하였다. 농후 사료는 체중의 1.8%를 급여하고 양질의 티모시 건초를 자유채식 시킨 14개월령 거세우의 정소에서 분리된 정소상체 미부의 정자를 회수하고 동결 흉해 후 체외수정을 실시한 결과는 다음과 같다. 웅성전핵과 자성전핵이 형성(2PN)된 난자는 정상수정으로, 1개의 전핵(1PN), Expanded Sperm Head (ESH), Polyspermy 형태는 비정상적인 수정의 형태로 평가하였다. 정상적으로 수정된 난자의 비율은 정소상체 정자의 경우 전체 침투율은 49.7% 그리고 정상적인 2PN을 가진 난자는 18.5%를 보였으며, 대조구 정자의 전체 침투율은 54.4%로서 정소상체 정자 보다 높은 결과를 보였으나 유의적인 차이를 보이지는 않았다. 정상적으로 2PN을 형성한 비율은 36.7%로서 정소상체 정자를 이용한 정자 보다 높았으나 유의적인 차이는 없었다. 체외수정 후 발달률 조사에서 정소 상체 정자의 분할률은 81.2%, 대조구 정자는 82.7%로 유사한 결과를 보였으나, 배반포 발달률은 정소상체 정자 24.4%와 대조구 정자 12.2%로 정소상체 정자를 사용한 난자의 발달에서는 유의적으로 높았다(p<0.05).

In this study, we examined sperm penetration and blastocyst developmental rate of oocytes to determine fertilizability of cauda epididymal spermatozoa in Hanwoo bull. One testicle with epididymides were castrated from one Hanwoo bull (14 months of age) and transported to laboratory. Spermatozoa recovered from cauda epididymis by mincing with semen extender (Optixcell, IMV, France) and cryporeserved in liquid nitrogen tank until use. As control, frozen Hanwoo semen was used. Cumulus oocyte complexes (COCs) were collected from follicles (2-8 mm) of slaughtered ovaries and 10 to15 COCs were matured in 50μl droplet with M-199 media supplemented with 10% fetal bovine serum, 10μg/ml FSH, 10μg/ml LH, 10μg/ml EGF for 22 to 24 hours in a humidified atmosphere of 5% CO2 in air. After maturation of COCs, matured COCs were co-incubated with cauda epididymal spermatozoa in 100μl droplet in modified Brackett and Oliphant media supplemented with 2.5 mM theophylline for 12 or 18 hours under 5% CO2 in air. Sperm concentration was adjusted to 5 × 106cells/ml. After IVF for 18 hours, presumptive zygotes were cultured in modified synthetic oviductal fluid with 1mM glutamine, 12 essential amino acids, 10 μg/ml insulin under 5% CO2, 5% O2 in air. In experiment 1, we examined sperm penetration rate at 12 hours of IVF of frozen-thawed epididymal sperm. Total penetration rate among cauda epididymis and control were not significantly different (mean±standard error, cauda epididymis and control vs. 49.7±11.3 and 54.4±12.8%) In experiment 2, cleavage and blastocyst development rate were evaluated at day 2 and day 8 after IVF for 18 hours. Cleavage rate among cauda epididymis and control was similar different (cauda epididymis and control vs. 81.2±3.4 and 82.7±2.5%). However, blastocyst developmental rate of cauda epididymis group was significantly higher than that of control group (cauda epididymis and control vs. 24.4±1.6 and 12.2±2.8%, p<0.05). In conclusion, cauda epididymal spermatozoa in Hanwoo bull has high fertilizability and embryo development. Cauda epididymal sperm can be used as an alternative to ejaculated frozen sperm in vitro.

The national natural monument of Korea, Jeju Black Cattle (JBC), it is a native species with unique blood line. This cattle breed needs mass production and industrialization to further improve and preserve their characteristics. This study was to examine whether there were differences in in vitro developmental rates according to body weight (<300, 300 ~ 350, 350 ~ 400 and >400 kg) and grade (1++, 1+, 1, 2 and 3), and oocyte donors or non-donors. As a method of IVM, groups of ten cumulus oocyte complexes (COCs) were cultured in 50 μl droplets of maturation medium (TCM199 supplemented with 10% FBS, 0.2 mM sodium pyruvate, 1 μg/ml follicle-stimulating hormone, 1 μg/ml estradiol-17β) under mineral oil at 38.8℃ in an incubator with a 5% CO2 atmosphere for 22 to 24 h. For IVF, 44 ul IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 μl heparin and 2 μl PHE (20 μM peicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. For IVC, after 44±2 h of incubation, cleaved embryos were incubated in CR1aa medium containing 3 mg/ml FAF-BSA until day 4 at 38.8℃ in a 5% CO2 incubator. Embryos were then cultured in CR1aa medium containing 10% FBS until day 8. As a result, in vitro development rates were the highest in 350 ~ 400 kg body weight group and in 1++ grade group than other groups (p<0.05). However, there was no difference in in vitro developmental capacity of classified donor and non-donor oocyte groups. This result demonstrated that the better in vitro developmental capacity was obtained in high level originated oocyte groups (350 ~ 400kg, 1++ grade) than in others, while there was no different in donor types.

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein 50 μM for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and 50 μM and genestein 1 and 50 μM for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and 50 μM, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin 1 μM for 3 hr incubation. In mitochondrial activities, addition of quercetin 50 μM for 6 hr incubation increased mitochondrial activity but decreased at 100 μM concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin (1∼10 μM), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin (25∼50 μM). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin 10 μM (p<0.05). The developmental rates to morula or blastocysts at low (0.01∼1 μM) and high (5∼10 μM) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and 10 μM) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations (0.01∼10 μM).

The purpose of this experiment was to compare the pregnancy rate (PR) according to the state of the ovaries and uterus, according to the number of embryos transferred from cows and heifers and to investigate the method of artificial twin induction with Hanwoo in vitro fertilized (IVF) embryos by embryo transfer (ET). Looking at the PR according to the condition of the ovaries and uterus, the result was not influenced by the condition of the ovaries, but was significantly influenced by the state of the uterus. The PR according to the number of embryos transferred from cows was 36.8%, 53.0%, 50.5% for 1, 2, and 3 embryos, respectively and although there was a higher frequency of twin calves with 3 embryos than 2, the calving rate was the highest with 2 embryos. In case of heifers, the transfer of 1 embryo showed the best pregnancy and calving rate, and although the PR was similar with 2 embryos (67.7 versus 66.4), in case of 2 embryos transferred there was high frequency of embryonic loss (6.1%) occurred when a cow was diagnosed at 28 and 53 d after ET, total loss (21.3%); sum of fetal death, abortion and stillbirth after pregnant diagnosis at 60 day.

Cathepsin B, a lysosomal cystein protease that plays an important role in the degradation of intracellular proteins in lysosomes, is detected in a wide variety of cells including bovine oocytes and embryos. Although the mode of action of cathepsin B is not fully understood, a strong relationship was observed between cathepsin B and apoptosis in many types of cells. Cathepsin B was found to induce the apoptotic pathway through activating initiator caspases rather than executioner caspases. Thus, the aim of this study was evaluated the effect of capthesin B inhibitor, E-64, on blastocyst developmental competence and subsequent preimplantation quality of the IVF and SCNT bovine embryos. After IVF and SCNT procedures, presumptive bovine embryos were cultured in CR1aa medium supplemented with E-64 for 24 h. Then, samples were additionally cultured in CR1aa medium without E-64 for 5 days. In our results, the frequency of blastocyst formation was higher when treated with E-64 compared with the control group (p<0.05). Furthermore, the blastocyst cell number was enhanced and apoptosis reduced (TUNELpositive nuclei number) by E-64 treatment in both IVF and SCNT bovine embryos (p<0.05). In the real-time quantitative RT-PCR, the expression of anti-apoptotic Bcl-xL gene was shown to be increased in the blastocyst stage, whereas expression of proapoptotic Bax was decreased. In conclusion, our results indicate that E-64 improves the developmental competence and embryonic qualities of bovine IVF and SCNT embryos by modulating cathepsin B induced apoptosis during the preimplantation stage.

The purpose of this experiment is to compare the pregnancy rate (PR) according to the state of the ovaries and uterus, according to the number of embryos transferred from cows and heifers and to investigate the method of artificial twin induction with Hanwoo in vitro fertilized (IVF) embryos by embryo transfer (ET). Looking at the PR according to the condition of the ovaries and uterus, the result was not influenced by the condition of the ovaries, but was significantly influenced by the state of the uterus. The PR according to the number of embryos transferred from cows was 36.8%, 53.0%, 50.5% for 1, 2, and 3 embryos respectively, and although there was a higher frequency of twin calves with 3 embryos than with 2, the calving rate was the highest with 2 embryos. In case of heifers, the transfer of 1 embryo showed the best pregnancy and calving rate, and although the PR was similar with 2 embryos (67.7 versus 66.4), in case of 2 embryos transferred there was high frequency of embryonic loss( 6.1%) occurred when a cow was diagnosed at 28 and 53 d after ET, total loss (21.3%; sum of fetal death, abortion and stillbirth after pregnant diagnosis at 60 day).

Freezing of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification of bovine expanded, hatched and SCNT embryos on the survival rate, apoptosis index and further development after thawing. Expanded, hatched and SCNT embryos were vitrified after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. Artificial shrinkage of the blastocyst was achieved after pushing a pulled pasteur pipet into the blastocoele cavity until it contracted. The shrunken and not shrunken embryos were exposed to cryoprotectant solution in 7.5% ethylene glycol-7.5% DMSOPBS with 20% FBS for 5 min. They were placed in a small volume of vitrification solution (15% ethylene glycol+15% DMSO+PBS+20% FBS+0.5 M sucrose) and plunged into liquid nitrogen on a cryotop. Then, after thawing, cryoprotectant was diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min. Under the optimal conditions, overall efficiency of the survival rate of bovine expanded, hatched, SCNT embryos in artificial shrinkage groups was higher compared with non-artificial shrinkage groups (p< 0.05). Especially, the numbers of TUNEL-positive nuclei in artificial shrinkage groups were significantly reduced than those of non-artificial shrinkage groups among frozen-thawed expanded, hatched, and SCNT blastocysts (p< 0.05). Our results showed that survival rates in cryopreserved expanded, hatched, SCNT embryos could be improved by reducing the fluid content. Therefore, we suggest that artificial shrinkage method is a effective pretreatment technique for the cryotop vitrification of expanded, hatched, SCNT bovine blastocysts.

This study was carried out to confirm the effects of luteotrophin, human chorionic gonadotrophin (hCG), and an anti-luteolytic agent, flunixin meglumin (FM), on pregnancy rates in Hanwoo with in vitro produced (IVP) embryo transfers (ET), and to research the effects on the estrus cycle. Treatments included hCG and FM administration 3~10 minutes prior to ET. Also, pregnancy rates were compared with lidocane treatment and FM treatment prior to ET. The results are shown below. 30-day pregnancy rate was 76.7% in the hCG-treated group and 75.7% in the FM-treated group. Both rates were higher than the 70% rate for the control group. 42-day pregnancy rate was 76.7% in the FM-treated group. This was higher than 66.7% recorded for both the hCG-treated and control groups. The pregnancy rate of the hCG-treated group was high at Day 30 (76.7%) but low at Day 40 (66.7%), and there were no differences from the FM-treated and control groups. The recurrent estrus rate of infertile individuals at 2 weeks after ET was 36.4% in the hCG-treated group, under 71.4% in the FM-treated group and 80.0% in the control group. The non-pregnancy rate of individuals without recurrent estrus was 18.2% in the hCG-treated group, which was higher than the 0% rate in both the FM-treated and control groups. The pregnancy rates were higher in the FM-treated group than the Lidocane-treated group with 72.3% versus 67.5% in the heifers and 48.9% versus 43.6% in the cows. From the above results, the FM treatment proved more effective than the hCG treatment and no treatment whatsoever in increasing pregnancy rates after ET. In addition, hCG treatment was shown to be undesirable due to the deviations it caused in the reproductive physiology of the hCG-treated recipients. Therefore, in our study, the FM treatment resulted in a higher pregnancy rate than either lidocaine treatment or no-treatment in the trials of ET.

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a ɤ-tubulin spot. However, ɤ-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

This study was carried out to collect the basic data about gestation lengths and offspring's birth weights and sex ratio of the dairy recipients transferred with Hanwoo IVF embryos. Blastocysts cultured for days were transferred to 96 and 167 heads for the basic data about gestation length and offspring's birth weight and sex ratio of the dairy recipients, respectively. The gestation lengths of the dairy recipients transferred with Hanwoo IVF embryos were () and () days in male and female of the offspring, respectively. The gestation lengths of the recipients were , , and days in spring, summer, autumn and winter of the calving season, respectively, and were significantly different among the calving season (p<0.05). The birth weights of male and female calves were () and ( kg in offsprings of the dairy recipients transferred with Hanwoo IVF embryos, respectively. The sex ratio was 90.7 in the offsprings of the dairy recipients transferred with Hanwoo IVF embryos.

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-p , EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-p gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.