Melatonin is a neurotransmitter that modulates various physiological phenomena including regulation and maintenance of the circadian rhythm. Nicotinic acetylcholine receptors (nAChRs) play an important role in oral functions including orofacial muscle contraction, salivary secretion, and tooth development. However, knowledge regarding physiological crosstalk between melatonin and nAChRs is limited. In the present study, the melatoninmediated modulation of nAChR functions using bovine adrenal chromaffin cells, a representative model for the study of nAChRs, was investigated. Melatonin inhibited the nicotinic agonist dimethylphenylpiperazinium (DMPP) iodide-induced cytosolic free Ca2+ concentration ([Ca2+]i) increase and norepinephrine secretion in a concentrationdependent manner. The inhibitory effect of melatonin on the DMPP-induced [Ca2+]i increase was observed when the melatonin treatment was performed simultaneously with DMPP. The results indicate that melatonin inhibits nAChR functions in both peripheral and central nervous systems.

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/ c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

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 use of bovine serum albumin (BSA) as alternatives to fetal bovine serum (FBS) in in vitro maturation medium. The oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression and cryo-tolerance. Oocytes were cultured in TCM-199 supplemented with 1 μg/ml estradiol-17ß, 10 μg/ml FSH, 10 ng/ml EGF, 0.6 mM cysteine, 0.2 mM sodium pyruvate and either 8% BSA (BSA group), 10% FBS (FBS group), or neither BSA nor FBS (TCM group), and followed by in vitro fertilization and the zygotes were cultured in SOF-BE1 medium. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. We have shown that the percentages of embryos that underwent cleavage and formed a blastocyst were non significantly different among all experimental groups (37.4 ± 1.5% for FBS group vs. 31.1 ± 3.9% for BSA group and 34.5 ± 1.6% for TCM group, six replicates were performed). Furthermore, there was no significant difference between the percentage of MII oocyte between FBS (71.8 ± 1.9%) and BSA groups (69.3 ± 2.3%). However, culture of oocytes with FBS increased (P < 0.05) the cumulus cell expansion as well as expression of gape junction proteins, CX37 and CX43, at both transcriptional and translation levels. We also found that FBS significantly increased total cell number and decreased the apoptotic index in day-8 blastocyst comparing to BSA group. The beneficial effects of BSA on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocyst. Taken together, these data suggest that supplementation of maturation medium with BSA, as alternatives to FBS, can be used as defined medium that support consistently the development of IVP bovine embryos.

The expression of MMPs in the development of the fertilized egg has a very important role in cell configuration. Objective To evaluate the clinical, the effect of differentially expressed MMPs on serum and serum - free medium on the maturation of blastocysts. The expression patterns of MMPs in serum and serum-free medium were compared at 6 h, 18 h and at the blastocyst stage using real-time PCR, ELISA and immunofluorescence. The results showed that the expression of MMPs was increased in the embryos of the serum medium, as a result of analysis of MMPs and TIMPs, MMP-2 was expressed in the cytoplasm of embryos in the serum-free medium, And it was found to be higher in expression than MMP-9. The serum medium was different from the bloodless badge: overall, TIMPs showed a higher expression in the ovarian cells than cyanosis, and TIMP-3 was more pronounced. Development rate of blastocyst according to in vitro culture method was higher than that of serum - free medium (61.22% 60/98) and serum - free medium (48.28% 28/58). Analysis of the protein release locations of MMPs and TIMPs showed that MMPs and TIMPs are highly expressed in serum mediums, focusing on the inner cell mass. However, very low expression appeared in the tropoblast. On the other hand, serum - free medium showed different expression from serum medium and TIMPs expression was generally low. Therefore, in the case of serum media, the expression of MMPs is highly expressed in the cytoplasm of the fertilized egg, increasing the reconstruction of cells.

The aim of this study was to investigate the role of Src homology 2-containing phosphotyrosine phosphatase SHP2 in intricate signaling network invoked by oocyte to achieve cytoplasmic maturation and also blastocyst development. Activation of SHP2 regulates multicellular differentiation, proliferation and survival through numerous signal pathways. The most prominent pathway is RAS/PI3K and p-AKT signaling cascade, as a result mitogenic effect become enhanced. Oocytes were cultured in cisplatin an anticancer drug, but selective activator of SHP2 and our grouping were SOF medium alone, SOF + EGF, SOF + CISPLATIN 0.3 μM, and SOF + EGF + CISPLATIN 0.3 μM. We evaluated that EGF neutralizes the apoptotic effect of cisplatin as well as maintain the high expression of SHP2, as a result blastocyst development become boosted up. We also found that inhibition of SHP2 with its specific inhibitor PHPS1 5 μM decreases the blastocyst development and neutralizes growth factors effect. The developmental ability and quality of bovine embryos were determined by assessing their cell number, gene expression, immunofluorescence, and immunoblot. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. Our results show that SHP2 have significant effect on MAP kinase pathways which expand the cumulus cells during oocyte maturation and blastocyst development as compare to inhibition of SHP2 with PHPS1. SHP2 not only transduce the signaling of epidermal growth factor but it also has a role in signal transduction of FGF and IGF. The expression of ERK, PI3K/p-AKT and mTOR was increased with EGF, but with the treatment of SHP2 inhibitor the expression of these genes become drop done. So we can conclude from these results that SHP2 is important for oocyte maturation as well as for blastocyst development.

Variance of conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants, essential for the maintenance of early pregnancy. Similar to other IFN genes such as IFNA and IFNB, multiple IFNT genes are present. However, some kinds of IFNT genes actively transcribed and regulated in bovine conceptuses have not been well characterized. In this study, during the course of bovine IFNT gene transcription through the use of next generation sequencer SOLiD3, revealed that among 38 IFN genes registered, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses during early pregnancy. Also, to identify a transcription factor(s) involved in the regulation of IFNT genes, mRNAs for various known transcription factors were investigated by real-time PCR in conceptus tissues, respectively. Furthermore, compared to the IFNT genes, IFNT1 and IFNTc1 had same active levels, which were previously shown to correlate with the appearance of effective antiviral activity. However, the expression levels of these Luc activities differed. Bovine ear fibroblast (EF) cells were cotransfected with luciferase reporter constructs carrying upstream (–631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1(JUN), ETS2 and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP). CDX2, either alone with the other 2 transcription factors, was found to increase luciferase activity approximately 14- and 11-folds, respectively. The degree of transcriptional activation of the IFNTc1 gene was not similar to that IFNT1 gene by AP1, ETS2 or/and CREBBP, expression plasmid. These results suggest that two isoforms of bovine conceptus IFNT genes are regulated differently in conceptuses during early pregnancy.

Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide) assays showed that 100 nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.01). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl, two membrane surface molecule genes (P<0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.

Until recently, there have been many researches about the freezing methods and several methods of cryopreservation. Hypothermic preservation has been used to complement the embryo freezing technology. There is a study to show the successful results for long-term hypothermic preservation. For that reason, FBS and BSA are commonly added to the culture medium to support embryo development. We investigated the effectiveness of hypothermic preservation method at 4℃ according to embryonic developmental stages for Hanwoo embryos and evaluated the effect of FBS and BSA on Hanwoo embryos as a supplemental reagent in hypothermic preservation medium after recovering preserved embryos from hypothermic preservation. The present study reported that survival and hatching rates of embryos at morula stage following storage at 4℃ is Day 7 group was significantly higher (p < 0.05) compared than those of other groups (p < 0.05). As a result, the survival and hatching rates of embryos at the blastocyst stage following storage at 4℃ result is showed that significantly higher (p < 0.05) survival rates than those of other groups an Day 6. The result showed that hatching rate at Day 6 and 7 were significantly lower (p < 0.05) compared with other groups. The result regarding the survival and hatching rates of bovine embryos following storage at 4℃ for 72 h in various concentrations of BSA are shown The results showed that survival rate of 1% BSA group was not significantly different (p < 0.05) compare with control (FBS) group. Also, the results showed that hatching rate of control (FBS) and 1% BSA were significantly different (p < 0.05) compared with other groups. In conclusion, our result demonstrated that the hypothermic preservation did not effect on the survival and hatching rates of embryos after recovering. In addition, the supplementation of BSA in preservation medium showed no difference in the embryo developmental competence after hypothermic preservation compared to FBS treatment. With that, BSA can be an alternative reagent for the hypothermic preservation medium as an energy source and pH buffer.

Cryopreservation of bovine embryos is used to efficiently implant surrogate mothers. It has been widely accepted that high lipid content in the oocyte interrupts its survival during freeze-thaw cycles. Serum component in the culture medium is thought to increase the embryo`s lipid contents. Conversely, L-carnitine stimulates lipid metabolism by transporting long chain fatty acids into the mitochondria. Objective of this study was to analyze the effect of L-carnitine supplementation in IVM medium and defined IVC medium on the development, lipid contents and the cryosurvival of bovine IVF embryos. 0.0, 1.5, 3.0 and 6.0 mM L-carnitine was supplemented in IVM medium, respectively (IVM-LC 0.0, LC 1.5, LC 3.0 and LC 6.0). Development rate from the 2cell to the morula stages was higher in IVM-LC 3.0 groups than those of IVM-LC 6.0 (p<0.05). But there were no significant differences among the other groups in the blastocyst rates and lipid content results. When 0.0, 1.5, 3.0 and 6.0 mM L-carnitine were supplemented in IVC medium (IVC-LC 0.0, LC 1.5, LC 3.0 and LC 6.0), development competence was not significantly different between those embryos. Lipid contents of embryos treated L-carnitine (IVC-LC 1.5, 3.0 and 6.0) were significantly lower than embryos of non-treated group. L-carnitine was supplemented 0.0, 1.5, 3.0, 6.0 mM during IVM and 3.0 mM during IVC (LC 0.0 - 3.0, LC 1.5 – 3.0, LC 3.0 – 3.0, LC 6.0 – 3.0) and cryosurvival of blastocysts confirmed after freezing-thawing. There were no significant differences on development, but LC 3.0 – 3.0 was significantly lower lipid contents than other groups. And LC 3.0 – 3.0 had better survival rates and hatched rates of blastocysts than LC 0.0 – 0.0. In conclusion, supplementation of L-carnitine in defined IVC medium decreases lipid contents. And L-carnitine supplementation improves cryosurvival and developmental ability of bovine IVF embryos.

Successful cryopreservation of bovine oocytes is a very important technology for research and commercial applications. However, the survival and development rate of vitrified-thawed oocytes is lower than non-vitrified oocytes. Hydroxypropyl Cellulose supplementation (HPCs) has extremely high viscosity, which permits transitions to a glassy state at low temperatures. This characteristics of HPCs have been reported to help the survival of human oocytes. In this study, we investigated the survival rate, fertilization rate and ROS levels to confirm the effect of cryoprotectant solutions with HPC for oocyte vitrification in bovine. For vitrification, bovine MII oocytes were pretreated with EG10 (added 0, 10, 50 and 100 ㎍/㎖ HPC) for 5 min, exposed to EG30 (added 0, 10, 50 and 100 ㎍/㎖ HPC) for 30 sec, and then directly plunged into LN2. Thawing was taken by 4-step procedures [1 M sucrose and 10% FBS added D-PBS (SFD) -> 0.5 M SFD -> 0.25 M SFD -> 0.125 M SFD] for 1 min, respectively. After thawing, oocytes were washed with TL-HEPES, incubated in a droplet of previous cultured IVM medium for 1 h to recover. IVF drop (44 ㎕) contained 10 vitrified-thawed oocytes with sperm concentration of 1 × 106 cells ㎖, and then 2 ㎕ heparin and 2 ㎕ PHE were added. At 2 days after IVF, cleaved embryos were cultured in CR1aa + 3 mg/mL FAF-BSA for 48 h and cultured in CR1aa + 10% FBS for 4 days. In the results, in vitro survival rate of bovine vitrified-thawed MII oocyte was significantly higher in 50 (85.5%) and 100 ㎍/㎖ (80.2%) HPC groups than 0 (71.2%) and 10 ㎍/㎖ (71.3%) groups (p<0.05). The ROS level was lower in 50 ㎍/㎖ HPC group than in control group. After in vitro fertilization, cleavage rate and blastocyst development rate were not significantly different among treatment groups. Therefore, these results indicated that HPC treatment has a positive effect on the survival of vitrified-thawed bovine oocytes.

Interferon tau (IFNT), has known as a key signal molecule for a period of pregnancy in ruminants owing to the need on maternal recognition of pregnancy. It is generated in trophectoderm cells of the elongation bovine conceptus at day 13-21 and a peak output is at day 15-17 of pregnancy period. Moreover, other studies indicated that it can be effective in the embryonic development and quality. In previous study, there were 8 bovine IFNT, but only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the peri-implantation. In this study, we target the one between the two, IFN-tau-c1 and then the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. In order to proceed this study, the immature oocytes from the ovaries at local slaughterhouse have been matured in vitro for 22 hours. For preparing the donor cell that have a mutation on IFNT gene, somatic cells were transiently transfected with Cas9 protein and single guide RNA targeting IFNT, and various single derived colonies with high proliferation were isolated and confirm the mutation by PCR. Finally, one colony had mono-allelic mutation (4bps deletion) was picked out and applied as the donor cell to SCNT. A donor cell was injected into an oocyte that nucleus was removed. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation and cultured for 7 days in chemically defined medium. In this study, control (n=199) and IFNT knockout-group (n=219) were compared with four replications. As results, there was no significant difference between control-and IFNT-knockout group not only in cleavage rate, but also blastocyst formation rate (Control: 12.3% ± 9.2, IFNT knockout-group: 20.1 ± 11%). In addition, the number of blastocyst cell was not different between control (91.7 ± 26.2) and IFNT knockout group (83.5 ± 21.3). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT and all samples were positive for mutation. In conclusion, these data indicated that the interruption of IFNT did not influence the embryonic development. In future study, we will transfer these mutated embryos toto test the effect of IFNT for pregnancy period. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972) and the Technology Development Program (S2566872) by MSS.

Importance of the in vitro model of tissues or organs is now evident in tissue engineering and cell biology research. Till now, two-dimensional culture systems have been using for in vitro cell culture, and have contributed to cell function studies despite their limitations. Three-dimensional (3D) culture has been utilized in cell biology research because it appears to mimic morphology and physiology of cells in living tissues and organs, unlike conventional monolayer cell culture. In our laboratory, we are developing 3D culture systems of bovine endometrial cells as a tool for the analysis of uterine endometrial functions. Among them, this lecture introduces spheroid culture and Matrigel culture. 1. Spheroid culture; Spheroids are a spherical mass composed of cells and extracellular matrices (ECMs). We have regenerated multicellular spheroids composed of bovine endometrial stromal and epithelial cells using ascorbate (1). Expression of MMPs, which are key enzymes for the tissue remodeling of the endometrium, were analyzed using the spheroid. E2, P4 and type-I IFN did not affect the gene expression of MMPs in the spheroid. However, treatment of type-I IFN increased the clearance of MMPs in the supernatant. These results suggest that IFN indirectly regulates endometrial tissue remodeling through clearance of MMPs. 2. Matrigel culture; It is reported that cells form lumens automatically by culturing cells in Matrigel (2). Matrigel is a solubilized basement membrane extracted derived from EHS mouse sarcoma cells. The bovine endometrial epithelial cells cultured in 15% Matrigel formed a circular or elliptical gland-like structure. Gene expressions of glandular epithelial specific factors (FOXA2, SERPINA14 and GRP) were significantly high in the Matrigel, compared to the monolayer cultured cells, except FOXA2. Further, SERPINA14 expression was affected by neither P4 nor IFN. However, when epithelial cells in Matrigel were co-culture with stromal cells, SERPINA14 expression increased significantly in the treatment of both P4 and IFN. These results suggest that bovine endometrial epithelial cells cultured in Matrigel show properties similar to the glandular epithelial cells in vivo, and regulated by the factors produced by the stromal cells. Finally, by using these 3D culture systems, it becomes possible to clarify not only factors regulating embryo elongation and implantation but also regulation of their expression. It will be able to reveal the mechanism of the embryo elongation and implantation to contribute to the improvement of the embryo transplantation technique. (1) Yamauchi N, Yamada O, Takahashi T, Imai K, Sato T, Ito A, Hashizume K. A three-dimensional cell culture model for bovine endometrium: regeneration of a multicellular spheroid using ascorbate. Placenta. 2003; 24(2-3):258-69. (2) Eritja N, Llobet D, Domingo M, Santacana M, Yeramian A, Matias-Guiu X, Dolcet X. A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis. Am J Pathol 2010; 176:2722-2731.

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.

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

Bovine colostrum is necessary for newborn calves to survive, grow and receive immunity from their mother. Cows in Korea produce about 35kg of colostrum, 4Kg of which is fed to the calf, and the rest is discarded. The bovine colostrum causes the harmful side effects to human, such as allergies and digestive problems; so, it is prohibited by law to consume colostrum itself as a food. However, many scientific research data have suggested that components in the colostrum can improve human health and has the ability to help treat diseases. In line with the trend of food and pharmacy industries using natural product materials, which attract positive attention, recently, some ingredients in colostrum have been used in the production of food supplements, and it has been used in its raw form in some cosmetics. This review introduces the active ingredients and physiologically active substances contained in bovine colostrum, summarizes the efficacy of physiological enhancement of the colostrum, which has been proven by scientific methods to date, and also suggests the possibility of industrial applications of colostrum as an animal-derived natural material.

ompared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte’s maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

The major focus of this study is to analyze the expression of bovine MMPs and to monitor their activity during the estrus cycle and pregnancy. During pregnancy, MMP-2 expression was detectable around 30 days but became insignificant by 60 days, then started to increase again around 90 days and reached the maximum at 250 days. The activity of MMP-2 protein changed in accordance with its expression level. As expected, the level of TIMP-2 exhibited a reverse pattern. About MMP-9, high level expression was observed as early as 30 days and gradually increase until 90 days. Then started to decrease after 250 days. Again, the sites of MMP-9 expression were similar to those of MMP-2. On the other hand, expression of TIMP-3 remained low until 90 days but showed a small and temporal increase around 250 days. In summary, expression of different MMPs were differentially regulated during estrus cycle and pregnancy. While the expression of MMP-2 was high in estrus cycle, MMP-9 slowly takes over with the progression of pregnancy. These results indicated that the luteal tissue perform distinct functions during pregnancy and estrus. Perhaps the activity of MMP-2 is required for the structural remodeling of luteum, resulting the suppression of P4 inflow from blood. On the other hand, steady maintenance of MMP-9 throughout luteal development is important for the activation of cell proliferation, maturation and angiogenesis.

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 μg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 μM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

We observed the external genitalia and uterus of a 24-month-old freemartin Hanwoo. The vulva was smaller than observed in a normal female Hanwoo, while the clitoris was larger in the freemartin. The angle between the external genitalia and the perineum also varied. Upon internal genital examination, the uterus of the freemartin was a thin tube approximately 18 cm in size and had not differentiated into a normal uterus and uterine horns.