The purpose of this study is to develop transgenic cell line expressing targeted human granulocyte colony stimulating factor (hGCSF) and green fluorescence protein (GFP) genes as well as production of Somatic Cell Nuclear Transfer (SCNT) embryos derived from co-expressed transgenic donor cells. Constructed pPiggy-mWAP-hGCSF-EF1-GFP vector was chemically transfected into bovine fetus cells and then, only GFP expressed cells were selected as donor cells for SCNT. Cleavage and blastocyst rates of parthenogenetic, SCNT embryos using non-TG cell and hGCSF-GFP dual expressed SCNT embryos were examined (cleavage rate: 78.0±2.8 vs. 73.1±3.2 vs. 70.4±4.3%, developmental rate: 27.2 ±3.2 vs. 21.9±3.1 vs. 17.0±2.9%). Result indicated that cleavage and blastocyst rates of TG embryos were significantly lower (P<0.05) than those of parthenogenetic and non-TG embryos, respectively. In this study, we successfully produced hGCSF-GFP dual expressed SCNT embryos and cryopreserved to produce transgenic cattle for bioreactor system purpose. Further process of our research will transfer of transgenic embryos to recipients and production of hGCSF secreting cattle.

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG (81.1±15.1), 1,2-PROH (88.0±21.1) or the control (99.9±21.3) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO (14.7±1.3), EG (12.1±1.1), Glycerol (15.2±1.8), and 1,2-PROH (11.5±1.3) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.5±0.7, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

본 연구에서 배아의 생식세포 동결에 가장 흔히 쓰이고 있는 두 가지 동결 보호제, 즉 DMSO와 EG의 독성을 비교하고자 생쥐 수정란 모델을 이용한 실험을 하였다. 생후 6주령의 암컷 생쥐 F1 hybrid mice에 10 IU의 PMSG를 복강 주사하여 과배란을 유도하고, 2-세포기 배아를 획득하고 DMSO와 EG 각각 노출시킨 후, 배양을 하였다. 배반포의 전체 세포수는 2-세포기 단계에서 DMSO(68.1±24.1)로 EG(81.2±27.0) 혹은 control(99.0±18.3)(p<0.001) 처리구에 비해서 유의적으로 낮았다. DMSO 처리구가 EG 처리구에 비해 세포수가 적었다. DMSO(15.4±1.5)와 EG(10.2±1.4) 두 처리구는 대조구(6.1±0.9, p<0.0001)와 비교해서 배반포에서 세포사 비율이 더 높음을 확인했다. 또한, DMSO 처리구는 EG 처리구(p<0.001)보다 더 많은 세포사멸된 세포가 확인되었다. DMSO 또는 EG 처리군과 대조군 사이에는 배아 부화율에 있어서 차이가 있었으며, 이는 배아에 대한 동결 보호제의 잠재적인 독성을 확인한 결과였다. 이번 연구에서 장기간 처리했을 때 EG 처리군보다 DMSO 처리군에서 배아발달과 세포수가 저하된 것은 DMSO의 독성이 더 높을 것으로 사료된다.

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

Pigs are considered an ideal source of human disease model due to their physiological similarities to humans. However, the low efficiency of in vitro embryo production (IVP) is still a major barrier in the production of pig offspring with gene manipulation. Despite ongoing advances in the associated technologies, the developmental capacity of IVP pig embryos is still lower than that of their in vivo counterparts, as well as IVP embryos of other species (e.g., cattle and mice). The efficiency of IVP can be influenced by many factors that affect various critical steps in the process. The previous relevant reviews have focused on the in vitro maturation system, in vitro culture conditions, in vitro fertilization medium, issues with polyspermy, the utilized technologies, etc. In this review, we concentrate on factors that have not been fully detailed in prior reviews, such as the oocyte morphology, oocyte recovery methods, denuding procedures, first polar body morphology and embryo quality.

Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of Hanwoo following embryo transfer has not yet been reported. In this study, we investigated the correlation between oxygen consumption rate and pregnancy rates of Hanwoo embryo using a SECM. In addition to, the expression of apoptosis-related genes was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos (blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). The oxygen consumption or GI blastocysts were significantly higher than those of GII blastocysts (10.2x1014/mol s-1 versus 6.4x1014/mol s-1, p＜0.05). Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0 and over 12.0x1014/mol s-1, respectively. Apoptosis regulatory genes, Hsp-70.1 were significantly increased in over-10.0 group than below 10.0 group but in Caspase-3, Bax and P53 gene, there was no significant difference. In conclusion, These results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of Hanwoo embryos.

Sexed semen is commonly used for the production of calves of the desired gender. Gender selection is important in animal production industries. For example, female cattle are required for the dairy industry while males are preferred in the beef cattle industry. The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each 12.58±8.31 and 13.25±7.86. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen (3.75±1.98 vs. 8.23±6.07, P＜0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P＜0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p＜0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p＜0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p＜0.01, p＜0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.

생쥐 2세포기, 4세포기, 8세포기를 각 발생 단계에서 채취하여 동결 보호제를 첨가한 서로 다른 배양액에서 배양하고, 배아의 동결 보존 및 융해 시 급속 처리와 저속 처리 단계로 비교하여 이들 조건이 배아의 생존과 발현에 미치는 영향을 조사하여 다음의 결과를 얻었다. 동결 보호제로 처리하여 배양액을 달리한 경우, 급속 단계에서는 모든 배양액에서 비슷한 발생율을 보였고, 저속단계의 4세포기와 8세포기는 D-PBS에서 높은 발생율을 보였다(P＜0.05, P＜0.01). 배아의 발생 시기에 따른 동결 보존 후, 발생율은 2, 4, 8세포기로 넘어갈수록 발생율의 증가를 보여 8세포기에서 발생율이 가장 높았다(P＜0.01). 동결 보호제의 처리단계에 따른 발생율은 2세포기의 급속 단계에서는 유사하였으나, 4세포기와 8세포기는 저속단계에서 높은 발생율을 보였으며(P＜0.05), 특히 8세포기에서 가장 높았다(P＜0.01).

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

본 연구에서는 1톤 용량의 배아미를 생산할 수 있는 중형 배아미 생산시스템의 설계, 개발 그리고 평가를 목표로 하였다. 개발된 배아정미기는 연삭과 마찰의 혼합방식으로 제조되었다. 배아미 생산시스템의 형태는 2대의 직렬 입형 배아정미기로 구성하였으며, 배아부착율, 백도, 싸래기율을 조사하였다. 또한, 연삭식, 마찰식, 연삭과 마찰의 혼합방식에 따라서 각각 배아미의 배아부착율, 백도, 싸래기율도 조사하였다. 본 시스템은 1단계 배아정미기에서는 연삭과 마찰의 혼합방식으로 미강을 깍은 후, 2번째 배아 정미기에서는 쌀의 배아가 떨어지지 않도록 미세 미강을 제거하면서 쌀의 백도를 높이도록 개발되었다. 배아정미기 시작기에서는 축 롤러 부분의 금강석 연삭돌을 3개, 스크린부에는 6개의 연삭돌을 설치하였고, 각각의 정미기 롤러축의 회전속도는 960 rpm과 780 rpm으로 하여 배아 부착율과 백도를 높였다. 그 결과, 약 20%의 배아 부착율을 증가시킬 수 있었다. 본 연구에서는 다음과 같이 요약할 수 있다. 첫째, 배아부착율은 현미의 함수율과 밀접한 관계가 있었다. 함수율 15.2±0.1%인 시료로 실험한 결과, 투입량 약 600 kg일 때 배아부착률은 약 70%를 나타내었다. 둘째, 배아미의 백도는 정미기 롤러축의 회전속도 960 rpm 과 780 rpm 조건으로 운전하였을 때 각각 35, 37 백도로 향상시킬 수 있었다. 셋째, 싸래기율은 본 시스템에서 1% 미만으로 나타냈다. 본 연구에서 개발된 연식마찰식 배아정미기를 평가해본 결과 배아부착율, 백도, 싸래기율을 효과적으로 개선할 수 있었고 30%의 에너지 이용을 절감할 수 있었다.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

Embryos formed in vivo were collected from 171 donors housed in Chung Cheong Buk-Do Institute of Livestock and Veterinary Research of the Chungbuk community during the years 2009∼2012. We evaluated annual embryo collection, effect of follicle stimulating hormone (FSH), controlled internal drug release (CIDR) and prostaglandin (PG) administration to the donor for superovulation and controlling the estrus cycle, seasonal effects of embryo collection and compared the number of embryos recovered as per the collection days and pregnancy rate. In all, 1,243 embryos were collected from 118 donors with an average of 7.31 ± 5.35 embryos per donor, out of which 69.4% were transferable. Dosages of FSH required for inducing superovulation in various donors were compared. Average number of embryos collected from donors administered with 30 AU of FSH (7.13 ± 5.74 per donor) was not significantly different from that of donors who were given an injection of 24 AU of FSH (7.53 ± 4.91 per donor). However, the percentage of transferable embryos in the 30AU FSH-administered group (63.2 %, 449 of 711) was higher than that in the 24AU FSH-administered group (77.8%, 414 of 532). In the group of donors under a natural estrus cycle, the FSH dose administered did not influence the number of transferable embryos produced (7.49 ± 6.25 per donor for 30 AU of FSH vs 7.49 ± 4.92 per donor for 24 AU of FSH). However, in donors administered with CIDR and PG for controlling the estrus cycle, the FSH dose affected the average number of transferable embryos collected (4.25 ± 2.87 per donor for 30 AU of FSH vs 8.50 ± 6.36 per donor for 24 AU of FSH). We collected embryos from donors 6, 7 or 8 days after artificial insemination (AI). Results showed that the percentage of transferable embryos among those collected 8 days after AI was significantly higher than that among embryos collected 6 or 7 days after AI. Seasonal variations did not affect number of recovered embryos and pregnancy rates in natural estrus cycle and CIDR treatment groups (48.28% and 42.55%) but higher than pregnancy rate of frozen embryos (19.63%). These results indicated that administration of FSH beyond a threshold dose (at least 24 AU) has no beneficial effect on the production embryos and that collection of embryos 7∼8 days after AI is optimal for embryo recovery. CIDR treatment induced superovulation in short term and had no influence on the natural estrus cycle. Finally, although good-quality embryos were transferred, freezing significantly reduced the pregnancy rates after transfer.

Pyracantha is a genus of thorny evergreen large shrubs in the family of Rosaceae, with common names Firethorn or Pyracantha. It's extract has also been used in cosmetics as a skin-whitening agent and functioning through tyrosinase inhibition. Recent studies have shown that pyracantha extract possesses antioxidant activities and may significantly improve lipoprotein metabolism in rats. Although the mode of action of Pyracantha extract is not fully understood, a strong relationship was observed between antioxidant and apoptosis in some types of cells. Thus, the aim of this study was to evaluated the effect of pyracantha extract on blastocysts formation and their quality of the porcine parthenogenetic embryos. After parthenogenetic activation by chemicals, presumptive porcine parthenogenetic embryos were cultured in PZM-3 medium supplemented with extracts of pyracantha leaf, stalk and root for 6 day (1, 5 and 10 μg/ml, respectively). In our results, the frequency of blastocyst formation in pyracantha root extract (5 μg/ml) treated group had increased that of other groups. Furthermore, blastocysts derived from pyracantha root extract (5 μg/ml) treated group had increased the total cell numbers and reduced apoptotic index. Blastocyst development was significantly improved in the pyracantha root extract (5 μg/ml) treated group when compared with the H2O2 treated group (p<0.05). Subsequent evaluation of the intracellular levels of ROS in pyracantha root extract (5 μg/ml) treated groups under H2O2 induced oxidative stress were decreased (p<0.05). In conclusion, our results indicate that treatment of pyracantha root extract may improve in vitro development of porcine parthenogenetic embryos through its antioxidative and antiapoptotic effects.