Because multiple ovulation embryo transfer (MOET) in cattle includes several benefits such as wide spreading of genetically superior offspring for long distance, this biotechnological method has been widely applied to Hanwoo. When the recipients are not stayed close after embryo recovery from donor, the embryos are moved to other farms via several vehicles (car, train, and airplane). However, air travel induces lesser oxygen level, increased vibration, lower air pressure, higher noise, and increased exposure of cosmic radiation to living things than ground level. It was still unknown that fresh embryos obtained from multiple ovulation of Hanwoo could maintain their fertility after being transported via air plane, the present case report introduced a clinical case of MOET in Hanwoo after shipping fresh embryos via air transportation. The donor was multi-ovulated via follicle-stimulating hormone series of injection, which was followed by a gonadotrophin-releasing hormone injection and artificial insemination twice. The embryos were recovered by the uterine flushing, packed in ministraws, transported to recipients for 6 h including 1 h air flight, and then transferred to the synchronized recipients. During pregnancy diagnosis of early gestation period, 5 of 7 recipients (71.4%) presented no heat signs and showed fetal sacs with fluid under transrectal ultrasonography. After normal gestation period, all recipients naturally delivered healthy calves (male n = 2 and female n = 3) without abortion, stillbirth, and premature birth. The present case report indicated that transportation of fresh embryos for MOET via domestic flight in Korea did not affect to their fertility.

The ability to determine the sex of bovine embryos before the transfer is advantageous in livestock management, especially in dairy production, where female calves are preferred in milk industry. The milk production of female and male cattle benefits both the dairy and beef industries. Pre-implantation sexing of embryos also helps with embryo transfer success. There are two approaches for sexing bovine embryos in farm animals: invasive and non-invasive. A non-invasive method of embryo sexing retains the embryo’s autonomy and, as a result, is less likely to impair the embryo’s ability to move and implant successfully. There are lists of non-invasive embryo sexing such as; Detection of H-Y antigens, X-linked enzymes, and sexing based on embryo cleavage and development. Since it protects the embryo’s autonomy, the non-invasive procedure is considered to be the safest. Invasive methods affect an embryo’s integrity and are likely to damage the embryo’s chances of successful transformation. There are different types of invasive methods such as polymerase chain reaction, detection of male chromatin Y chromosomespecific DNA probes, Loop-mediated isothermal amplification (LAMP), cytological karyotyping, and immunofluorescence (FISH). The PCR approach is highly sensitive, precise, and effective as compared to invasive methods of farm animal embryonic sexing. Invasive procedures, such as cytological karyotyping, have high accuracy but are impractical in the field due to embryonic effectiveness concerns. This technology can be applicable especially in the dairy and beef industry by producing female and male animals respectively. Enhancing selection accuracy and decreasing the multiple ovulation embryo transfer costs.

체외 환경에서 생산되는 배아 (Embryo)는 활성산소종 (Reaction oxygen species, ROS) 수준이 일정 수준을 초과함에 따라 산화적인 손상을 받게 된다. 선행연구에 따르면, 항산화제는 ROS를 감소시켜주는 효과를 가지기 때문에 ROS로부터 오는 배아의 단백질, DNA의 손상, 세포 자멸사를 방지하여 배아의 발달률을 향상시킨다. 이전연구에 따르면 항산화제로써 엘라그산 (Ellagic acid, EA)은 ROS를 효과적으로 제거하고, 난자의 산화스트레스를 방지하는 효과를 가지고 있다고 보고되었다. 그리하여, 본 연구를 통해 우리는 소의 수정란 배양체계 중 in vitro culture (IVC) 단계에서 EA의 농도 (0, 5, 10 μM) 별 첨가가 소의 수정란 발달률과, 질적 수준에 미치는 영향을 조사하고자 실험을 진행하였다. 결과적으로, 배반포의 단계별 발달 수준에서 cleavage 형성률은 EA첨가군과 대조군 간의 차이를 발견할 수 없었으나 배반포 형성률에서는 모든 EA 첨가군들이 대조군보다 높았고 EA 첨가군 중에 5 μM 첨가군이 가장 높았다 (p < 0.05). 생산된 배반포의 총 세포 수는 5 μM EA 첨가군이 대조군과 10 μM EA 첨가군 보다 유의적으로 높았으며, 대조군과 10 μM EA 첨가군 사이의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). 세포 자멸사 세포 수는 모든 EA 첨가군들이 대조군보다 유의적으로 낮았다 (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). ROS 수준에서 모든 EA 첨가군들과 대조군 간의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18, p < 0.05). qRT-PCR 실험 결과에서 Nrf2 gene expression은 대조군과, 5 μM 첨가군에서 유의적 차이가 없었으나, 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다. Keap1 gene expression은 5 μM 첨가군에서 유의적으로 하향 조절된 것을 관찰하였다. 하지만 EA의 농도가 10 μM으로 높아짐에 따라 발현 수준이 증가한 것을 관찰할 수 있었다. CAT gene expression은 5 μM 첨가군에서 유의적으로 상향조절 되었으나 10 μM 첨가군에서는 유의적인 차이를 보이지 않았다. SOD1 gene expression은 대조군과 5 μM 첨가군은 유의적인 차이를 보이지 않았으나 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다.

Embryos produced with serum show the alterations in their ultrastructure, impaired compaction, abnormal blastulation, aberrant mRNA expression profiles and large calf syndrome with greater incidences of stillbirths and deaths after birth. The aim of the present study was to describe in vitro embryo production by analyzing embryo production, fetal production and pregnancy rate in free-serum medium. The OPU-IVP data used in this study from 2016. Approximately, sixteen cows (Hanwoo), which belonged to the Institute of Gyeongsang National University, were used. Two experimental group is used in this study. Serum groups were conducted in March to July and free-serum group was conducted in September to December. The recovered cumulus-oocyte complexes were morphologically classified to four grades based on the compaction of cumulus cells layers and homogeneity of the cytoplasm. The number of oocyte was significantly greater in serum groups than that in free-serum groups (29.61 ± 0.63 vs. 15.6 ± 0.62; p < 0.05). Between serum and free-serum groups indicate that average of 1st and 2nd grade oocytes were no difference (2.38 ± 1.67 vs. 2.38 ± 1.48; p > 0.05), but number of 3rd and 4th grade oocytes were greater in serum groups than that in free-serum groups (7.31 ± 7.64 vs. 5.60 ± 6.29; p < 0.05). Embryo cleaved competence was higher in rate in free-serum groups than that in serum groups (62.1% vs. 58.3; p < 0.05). However, blastocyst developmental rate was no difference between serum and free-serum groups (33.1% vs. 43.5%; p < 0.05). 986 recipients were used for embryo transfer. Pregnancy rate was indicated that between serum and free-serum group was no difference (54.6% vs. 56.3%; p < 0.05). In conclusion, we developed the free-serum system for production of in vitro bovine embryos in order to meet the developmental and qualitative requirements for large scale commercial use.

Thiamethoxam (TMX) is a neonicotinoid insecticide. Residues of TMX have been detected in various crops. Although it has specific high toxicity to insects and is designed to exterminate them, the toxicity has also found in mammals recently. Differ from acetylcholine toxicity, TMX has peroxide toxicity in mammals. Matured oocytes have the capacity of fertilization, but oocytes own abundant mitochondria and its maturation is vulnerable to reactive oxygen species (ROS). Excessive production of reactive oxygen species (ROS) can override antioxidant defenses, produce oxidative stress and DNA damage that triggers apoptosis and necrosis in organisms. However little is known about the harm of ROS induced by TMX during oocytes maturation. Here, bovine germ-vesicle (GV) oocytes were cultured to metaphase of the second meiosis (MII) stage in vitro with or without TMX. During this process, oocytes were evaluated by various methods. Microscopic examination showed that 1.6 mM TMX significantly inhibited the maturation process in which oocytes were arrested before MI stage or between MI and MII stage. Correspond to this two periods, immunofluorescence staining and enzyme activity analysis showed that active CDC25 and CDC2 reduced in TMX group compared to control; time lapse and immunofluorescence staining gave results that Cyclin B could not be degraded, actin cap could not form, and Bub3 could not be removed from kinetochores. In addition, MII oocytes exposed to TMX showed disordered chromosomes and spindle. To study further, oocytes cultured for 24 h were analyzed. On the one hand, these oocytes in TMX group accumulated more ROS and produced significantly decreased mitochondrial membrane potential and increased apoptotic signal compared to control by methods of quantities for dichlorodihydrofluorescein diacetate (DCHFDA), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide and Annexin-V, but the level of γH2AX protein in TMX group did not decline significantly compared with control. On the another hand, these oocytes were activated to be parthenogenetic embryos and cultured. Assessment for embryo development showed decreased rates of cleavage, morula and blastocyst in TMX group compared to control in vitro. In conclusion, these results suggest that ROS induced by TMX results in dysfunction of mitochondria and apoptosis, which block bovine oocytes to MI stage, trap them at AI/TI stage and trigger disordered chromosomes and spindle at MII stage. Additionally, MII oocytes with poor qualities result from TMX lose abilities to cleavage and develop to be morulae and blastocysts.

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

To have a better understanding of pluripotency, whole gene expression of embryo-derived stem cells (EdSCs) in bovine species was investigated. EdSCs were established from the embryos produced by in vitro fertilization, parthenogenesis and somatic cell nuclear transfer. Then, the microarray was performed and analyzed. Differently expressed genes (DEGs) were also confirmed by Real-time PCR. Among 10,203 DEGs, little difference was found in gene expression among three kinds of EdSCs. Conversely, all EdSCs have an immensely different gene expression when compared with somatic cells, consistent with scatter plat results. To investigate shared pathways for pluripotency in all EdSCs, 2,415 co-DEGs were identified which compared with somatic cells. By KEGG database, there were 54 signaling pathways in co-DEGs and some of them were related with pluripotency maintenance such as TGFβ, WNT and JAK-STAT signaling. In TGFβ signaling, BMP family and SMAD family were involved in co-up-regulated DEGs. In WNT signaling, WNT family and receptors were included in co-up-regulated DEGs, while inhibitors of WNT signaling were associated with co-down-regulated DEGs. In JAK-STAT signaling, STAT3 belonged to co-down-regulated DEGs. These DEGs were also confirmed by Real-time PCR. Taken together, BMP and WNT pathways may be activated and paly central roles to retain pluripotency in bovine EdSCs, whereas the LIF/STAT3 pathway may not be operated well. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and No. 2015048003 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea.

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

In mammal, oocytes are arrested at the metaphase Ⅱ until fertilization. However, unfertilized oocytes that remain in the oviduct or under in vitro culture, which is called "oocyte aging". Asynchrony negatively affects fertilization, pre- and post-implantation embryo development. Caffeine is known to phosphodiesterase inhibitor that rescues oocyte aging in several species. Nevertheless, the effect of caffeine was not clear in bovine aging oocytes. In this study investigated the cytoskeleton distribution in aged oocytes and the embryo development ability of aged oocytes from treated with or without caffeine during maturation. The cumulus and oocyte complexes (COCs) were cultured in 10% FBSTCM199 for up to 22h at 38.5℃ in 5% CO₂. For oocyte aging study, the COCs were cultured in 10% FBS-TCM199 supplemented with or without 10 mM caffeine for 40hs. And then oocytes underwent in vitro fertilization using highly motile sperm recovered from frozen and than thawed bull semen. As a result normal cytoskeleton percentage of caffeine treatment group more than the aging group (67.57%±4.11 VS 44.61%±6.40) and no significantly different compared to control group. Aged oocytes derived from addition of caffeine to the in vitro maturation medium significantly increased the percentage of 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%±10.18 VS 67.88%±7.72). Apoptotic fragmenting of genomic DNA was measured in individual embryos using the TUNEL assay. Blastocyst derived from caffeine treatment group significantly decereased the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocytes aging periode can improved the develpmental rate and quaility in bovine embryos developing in vitro.

The aim of this study was to evaluate the effect of α-tocopherol on blastocysts development and subsequent cryosurvival of the vitrification. The α-tocopherol(0, 100, 200, 400 μM) was added in to culture medium for the bovine embryos. The blasocysts from the α-tocopherol and untreated control groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. There were no differences in the overall cleavage rate(56.14±4.66, 58.18±4.70, 62.97±6.86 and 51.17±7.28) among four treatment groups. However, in blastocyst development and total cell number were significantly higher in α-tocopherol 200 μM(38.60±7.12; 106.33±3.50) to culture medium than other treatment groups(29.30±5.24, 31.60±7.12 and 26.37±4.18; 101.36±5.12, 97.27±2.87, and 91.23±7.52 respectively). Before and after vitrification, the total cell number and blastocyst development of embryo were significantly higher in July to August than September to October. In conclusion, addition of α-tocopherol 200 μM to in vitro bovine embryo culture medium was beneficial for improving embryo quality by decreasing the embryo damage blsstocysts cell number and improving the tolerance of the embryos to cryopreservation.