Analysis of Acute Phase Response Using Acute Phase Proteins Following Simultaneous Vaccination of Lumpy Skin Disease and Foot-and-Mouth Disease.

Since 2011, South Korea has implemented biannual vaccinations against foot-and-mouth disease (FMD) and recently, lumpy skin disease (LSD), to mitigate the spread of transboundary animal diseases. However, due to past adverse reactions, potentially linked to acute phase responses from FMD vaccinations, there is hesitancy among Korean livestock farmers regarding new strategies for simultaneous vaccinations against both FMD and LSD. This study was conducted to assess possible adverse reactions to the LSD vaccination by analyzing acute phase proteins (APPs) in three groups: cows vaccinated against FMD (G1-FMDV), LSD (G2-LSDV), and both (G3-FMDV/LSDV). In G1-FMDV, APP levels peaked on day 3 post-vaccination (p < 0.001) and returned to baseline. In G2-LSDV, APP levels increased gradually, peaking on day 10 post-vaccination. In G3-FMDV/LSDV, APP levels peaked on day 3 post-vaccination and remained high until day 10 (p < 0.001). These results indicate that LSD vaccines trigger a later immune response compared to FMD vaccines, possibly due to different adjuvants. Therefore, a longer follow-up period for monitoring adverse reactions to LSD vaccinations may be required to understand and mitigate potential risks.

Application of tenotomy on Korean native cattle (Hanwoo) with spastic paresis symptoms in the field.

Bovine spastic paresis (BSP) is a neuromuscular disorder characterized by hypertension and stiffness of hindlimb. Two Korean native cattle (Hanwoo) calves developed BSP or BSP-like symptoms, and a tenotomy of superficial tendon of medial head and deep tendon of lateral head of gastrocnemius muscle was performed for treatment. A cast was applied postoperatively to prevent muscle rupture and was removed three weeks later. The prognosis was evaluated at 3 weeks, 6 and 18 months postoperatively. Neither calf showed any other postoperative sequelae. This is the first case study to report the diagnosis, treatment, and prognosis of BSP in Hanwoo.

Comparison of prepartum blood parameters in dairy cows with postpartum ketosis and new risk prediction candidates.

Introduction: Ketosis is a predominant metabolic problem and a risk factor for several postpartum diseases. This retrospective study aimed to evaluate the complete blood count (CBC), plasma biochemistry, and osteocalcin and identify significant prepartum and early postpartum values expressed in ketotic cows. Methods: In 135 Holstein Friesian cows, 210 parturitions of 114 primiparous and 96 multiparous cows were examined. According to the plasma concentrations of ß-hydroxybutyrate (BHB; ≥ 1.4 mmol/L) or non-esterified fatty acids (NEFA; ≥ 0.7 mmol/L) in the postpartum period, cows were divided into healthy cows (CON) and ketotic cows (KET). Analyses of CBC and biochemistry profiles were performed from -6 to 4 weeks of parturition every 2 weeks (prepartum; BW-5, BW-3, and BW-1, postpartum; BW1 and BW3), and osteocalcin ELISA tests were performed using blood samples from -2 to 2 weeks of parturition (BW-1 and BW1). Results: In primiparous KET (n = 114) before parturition, lower lymphocyte (Lym) in BW-5 and BW-3, lower red blood cell (RBC) in BW-5, higher mean corpuscular volume (MCV) in BW-1, and higher NEFA in BW-3 were significant compared with CON. Primiparous KET showed lower carboxylated osteocalcin (cOC) levels and a significant decrease after parturition. In multiparous KET (n = 96) before parturition, lower neutrophil (Neu) in BW-5, higher hemoglobin (HGB) in BW-5, higher MCV in BW-5 and BW-1, higher MCH in BW-5, lower total cholesterol (TC) in BW-5, higher triglyceride (TG) in BW-3, higher NEFA in BW-1, higher glucose (Glu) in BW-3, lower γ-glutamyl transferase (GGT) in BW-5, lower inorganic phosphate (iP) in BW-3, and higher body condition score (BCS) in BW-5 and BW-3 were significant compared with CON. Multiparous KET showed decreased cOC and uncarboxylated osteocalcin (ucOC) after parturition, which was lower than that in the CON group. Discussion: The blood parameters expressing different values between CON and KET in prepartum or early postpartum periods are presumed to show individual nutrition and health states, liver function, and overweight status. These parameters could be valuable indicators that can be used to prevent the occurrence of ketosis and improve management practices by recognizing these differences in ketotic cows before calving.

Evaluation of Corpus Luteum and Plasma Progesterone the Day before Embryo Transfer as an Index for Recipient Selection in Dairy Cows.

The aim of the present study was to evaluate the effects of synchronization method, season, parity, corpus luteum (CL) size, and progesterone (P4) levels on the pregnancy rate after bovine embryo transfer (ET). Among 165 recipient candidates who received 1 of 2s estrus synchronization treatments, 96 heifers and 43 cows were selected through rectal examination and used as recipients. The day before ET, the CL size and plasma P4 concentration were evaluated. The CL sizes and plasma P4 levels were not different between the selected and unselected candidates, and the pregnancy rates with the two synchronization methods were not different. However, the pregnancy rates were higher in heifers than in lactating cows, and also higher after ET performed from September to February than from March to August (p < 0.05). The recipients with a CL larger than 1.5 cm showed statistically higher pregnancy rates, and although there was no statistical significance, the pregnancy rate was higher when the plasma P4 levels were between 2.0 and 4.0 ng/mL. Exposure to a stressful environment and repeated manipulations can reduce the success rate of ET, and recipient selection with an optimal CL size and P4 level can increase the success rate of ET.

Identification AAVS1-like locus from the porcine genome and site-specific integration of recombinase-mediated cassette exchange using CRISPR/Cas9.

Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Comparison of the bovine blood gas parameters produced with three types of portable blood gas analyzers.

BACKGROUND: A definite diagnosis should be made in the bovine practice field, however, it was difficult to perform laboratory analysis immediately. Currently, three types of portable blood gas analyzers are available in Korea. OBJECTIVES: This study aimed to evaluate the correlations among these three analyzers. METHODS: Seventy-two plasma samples from Holstein-Friesian cows were used for blood gas analysis, and three instruments (EDAN i15 Vet, VETSCAN i-STAT, and EPOC) were operated simultaneously. Moreover, plasma calcium levels were compared between these portable analyzers and blood chemistry device, which is usually used in a laboratory environment. Pearson analysis was performed to confirm the correlation of each parameter produced with the three instruments and blood chemistry analyzer. RESULTS: As results, high correlation was observed in parameters of pH, pO2, potassium ion, ionized calcium, and glucose (p < 0.001, r > 0.7). In addition, pCO2 showed a moderate correlation among the three analyzers (p < 0.001, r > 0.5), and there was no correlation among all instruments for sodium ions. There was also a high correlation between ionized calcium from the three portable devices and total calcium from the biochemistry analyzer (p < 0.001, r > 0.9). CONCLUSIONS: In conclusion, there was a high correlation between results from the three different blood gas analyzers used in the bovine clinical field in Korea. Thus, a consistent diagnosis can be made even with different equipment if the operator is aware of the strengths and weaknesses of each piece of equipment and operates it properly.

Spontaneous Alteration of Blood pH By a Bicarbonate Buffer System During Experimental Hypercalcaemia in Cows.

INTRODUCTION: Maintaining mineral homeostasis as well as the secretion and metabolism of mineralotropic hormones is important for healthy of periparturient dairy cows. To increase the activity of mineralotropic hormones, blood pH can be adjusted. The purpose of this study was to investigate changes in blood pH and the mechanism of action of this change in induced hypercalcaemic cows. MATERIAL AND METHODS: Six non-lactating Holstein cows were used in a 2 × 2 crossover design. To induce hypercalcaemia, calcium borogluconate was administered subcutaneously to experimental cows and normal saline was administered subcutaneously to control cows. Blood and urine samples were collected serially after administration. Whole blood without any anticoagulant was processed with a portable blood gas analyser. Plasma concentration and urinary excretion of calcium were measured. RESULTS: In hypercalcaemic cows, both blood and urine calcium levels were significantly increased at 8 h compared to those at 0 h (P < 0.05), and a spontaneous increase in blood pH was also observed. The calcium concentration in plasma was highest at 2 h after administration (3.02 ± 0.27 mmol/L). The change in pH correlated with that in bicarbonate (r = 0.781, P < 0.001) rather than that in partial pressure of CO2 (r = 0.085, P = 0.424). CONCLUSION: Hypercalcaemia induced a spontaneous change in blood pH through the bicarbonate buffer system and this system may be a maintainer of calcium homeostasis.

Evaluation of ruminal motility using an indwelling 3-axis accelerometer in the reticulum in cattle.

Attempts to increase production and improve farm environments have been made for several years. Rumen motility (RM) is one of the biological parameters that provides essential information of individuals in ruminants, and it is usually evaluated by auscultation. The study was aimed to examine RM using the 3-axis accelerometer (3XA) in cattle. The manufactured 3XA were placed in the reticulum (3XA-R) and implanted in the subcutaneous layer of the brisket (3XA-SC), respectively, and the accelerations were compared following intramuscular injection of xylazine (0.05 mg/kg) or saline in experiment 1 and of xylazine (0.05 mg/kg) or atropine (0.04 mg/kg) in experiment 2. In experiment 3, the dose-dependent decrease of RM was evaluated following xylazine administration (0, 0.05, 0.1 mg/kg) in the 3XA-R equipped cows via a 3 × 3 Latin square method. In experiment 1, saline-treated animals showed a continuous fluctuation while the frequency and amplitude of 3XA-R in xylazine-injected cows were reduced after administration. The acceleration of 3XA-SC was changed after administration, but not abruptly. Among the motion parameters, V2 was calculated only using X- and Z-axis acceleration in consideration of the cylindrical shape, and it showed the apparent difference between pre- and post-xylazine administration. In experiment 2, the V2 of 3XA-R was decreased after atropine administration while that of 3XA-SC was maintained. In experiment 3, a dose-dependent V2 decrement of 3XA-R after xylazine administration was observed and lasted for 40 and 80 min in doses of 0.05 mg/kg and 0.1 mg/kg, respectively. In conclusion, The 3XA detected the decrease in RM efficiently and processed the data wirelessly without interference from body movement. This technology will help detect problems early and prevent a decline in cattle productivity.

Gigapixel confocal imaging using a massively parallel optical probe array with single directional infinite scanning.

Here we demonstrate high-throughput gigapixel confocal imaging using a massively parallel optical probe array with single directional infinite scanning. For implementation of the single directional infinite scan with high lateral resolution, a parallelogram array micro-objective lens module, composed of two wafer-level microlens arrays, is proposed to generate a massively parallel optical probe array for integration into the confocal imaging system, including an objective-side telecentric relay lens with a low-magnification. To test the feasibility of the proposed system with single directional infinite scanning, we designed and constructed a confocal imaging system using a parallelogram array of multi-optical probes with a massively parallel array size of 200 × 140. The constructed system provides a full width-half maximum lateral resolution of 1.55 µm, as measured by the knife-edge detection method, and a field-of-view width of 28.0 mm with a sampling interval of 1 µm/pixel.

Changes in plasma pH and blood and urinary macromineral concentrations in experimentally induced hypocalcemic cows with Na2EDTA.

This study was performed to confirm the alterations of blood and urine parameters in artificially induced hypocalcemic cows. For a 2 × 2 cross-over design, four non-pregnant, non-lactating Holstein Friesian cows (623 ± 63 kg) were utilized. Cows in the treatment and control group were infused with ethylenediaminetetraacetic acid (Na2EDTA) solution and normal saline through an intravenous catheter for 3 hr, respectively. Laboratory analyses included complete blood cell count, plasma chemistry, blood gas analysis and urine chemistry. During the hypocalcemic period, abnormal signs were not observed clinically, hematologically nor biochemically either in groups. But, plasma calcium and magnesium concentrations continued to decrease throughout Na2EDTA infusion, and significant group differences (P<0.05 or P<0.001) were detected until 5 hr after the initiation of infusion. Urinary excretions of these minerals were significantly reduced compared to the control group by 6 hr (Ca, P<0.05; Mg, P<0.001). Moreover, there is a significant group difference in the change in plasma pH at 1 hr after Na2EDTA infusion (P<0.05) and maintained a decreased level until 6 hr. Consequently, the blood pH was diminished simultaneously with hypocalcemia and hypomagnesemia induction in cows infused with Na2EDTA. This phenomenon may be one of the mechanisms to recover normocalcemia including maximizing the effect of parathyroid hormone, however, further studies are needed to elucidate the mechanism to alter the blood pH in hypocalcemia.

Replication of high refractive index glass microlens array by imprinting in conjunction with laser assisted rapid surface heating for high resolution confocal microscopy imaging.

In a multi optical probe confocal imaging system utilizing a microlens arrays as an objective lens, a high numerical aperture is required to improve resolving power. Glass microlens arrays are suitable for high-resolution imaging since they provide outstanding optical properties with a high refractive index. We demonstrated the rapid fabrication of microlens arrays on a high refractive index optical glass substrate via laser assisted thermal imprinting. The optical performance of the fabricated glass microlens arrays were evaluated and compared to that of a polymer microlens. In contrast to the polymer, the real image afforded by, and the calculated resolution of, the imprinted glass microlens arrays were significantly better, at about 0.73 µm compared to the polymer (∼1.56 µm). Our results reveal the considerable potential of direct thermal imprinting as a rapid, single-step, low cost fabrication method for replication of glass microlens array of high dimensional accuracy affording excellent optical performance.

Temperature dependence of the superconducting energy gaps in Ca9.35La0.65(Pt3As8)(Fe2As2)5 single crystal.

We measured the optical reflectivity R(ω) for an underdoped (Ca0.935La0.065)10(Pt3As8)(Fe2As2)5 single crystal and obtained the optical conductivity [Formula: see text] using the K-K transformation. The normal state [Formula: see text] at 30 K is well fitted by a Drude-Lorentz model with two Drude components (ωp1 = 1446 cm-1 and ωp2 = 6322 cm-1) and seven Lorentz components. Relative reflectometry was used to accurately determine the temperature dependence of the superconducting gap at various temperatures below Tc. The results clearly show the opening of a superconducting gap with a weaker second gap structure; the magnitudes for the gaps are estimated from the generalized Mattis-Bardeen model to be Δ1 = 30 and Δ2 = 50 cm-1, respectively, at T = 8 K, which both decrease with increasing temperature. The temperature dependence of the gaps was not consistent with one-band BCS theory but was well described by a two-band (hence, two gap) BCS model with interband interactions. The temperature dependence of the superfluid density is flat at low temperatures, indicating an s-wave full-gap superconducting state.

Long-term health and germline transmission in transgenic cattle following transposon-mediated gene transfer.

BACKGROUND: Transposon-mediated, non-viral gene delivery is a powerful tool for generating stable cell lines and transgenic animals. However, as multi-copy insertion is the preferred integration pattern, there is the potential for uncontrolled changes in endogenous gene expression and detrimental effects in cells or animals. Our group has previously reported on the generation of several transgenic cattle by using microinjection of the Sleeping Beauty (SB) and PiggyBac (PB) transposons and seeks to explore the long-term effects of this technology on cattle. RESULTS: Transgenic cattle, one female (SNU-SB-1) and one male (SNU-PB-1), reached over 36 months of age with no significant health issues and normal blood parameters. The detection of transgene integration and fluorescent signal in oocytes and sperm suggested the capacity for germline transmission in both of the founder animals. After natural breeding, the founder transgenic cow delivered a male calf and secreted milk containing fluorescent transgenic proteins. The calf expressed green fluorescent protein in primary cells from ear skin, with no significant change in overall genomic stability and blood parameters. Three sites of transgene integration were identified by next-generation sequencing of the calf's genome. CONCLUSIONS: Overall, these data demonstrate that transposon-mediated transgenesis can be applied to cattle without being detrimental to their long-term genomic stability or general health. We further suggest that this technology may be usefully applied in other fields, such as the generation of transgenic animal models.

Development of genome engineering technologies in cattle: from random to specific.

The production of transgenic farm animals (e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos (zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure. However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer (SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies (e.g., ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies will accelerate our understanding of genetic traits in bovine and will be readily adapted for bio-medical applications in cattle.

Prepubertal growth and single nucleotide polymorphism analysis of the growth hormone gene of low birth weight Holstein calves.

Holstein calves weighing less than 20 kg at birth have been noted in Korea. Due to insufficient information, we raised small calves with age-matched normal birth weight Holstein calves and determined body weights before puberty. In addition, 3 single nucleotide polymorphisms (SNPs) of the growth hormone (GH) gene were analyzed. Up to 10 months of age, low birth weight calves were smaller than normal weight calves. In exon 5 of the GH gene, SNP genotype variation was detected in some small calves; however, this did not appear to be the only factor inducing low birth weight and slow growth.

Design methodology for a confocal imaging system using an objective microlens array with an increased working distance.

In this study, a design methodology for a multi-optical probe confocal imaging system was developed. To develop an imaging system that has the required resolving power and imaging area, this study focused on a design methodology to create a scalable and easy-to-implement confocal imaging system. This system overcomes the limitations of the optical complexities of conventional multi-optical probe confocal imaging systems and the short working distance using a micro-objective lens module composed of two microlens arrays and a telecentric relay optical system. The micro-objective lens module was fabricated on a glass substrate using backside alignment photolithography and thermal reflow processes. To test the feasibility of the developed methodology, an optical system with a resolution of 1 µm/pixel using multi-optical probes with an array size of 10 × 10 was designed and constructed. The developed system provides a 1 mm × 1 mm field of view and a sample scanning range of 100 µm. The optical resolution was evaluated by conducting sample tests using a knife-edge detecting method. The measured lateral resolution of the system was 0.98 µm.

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing.

Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science.

Developmental competence and cryotolerance of caprine parthenogenetic embryos cultured in chemically defined media.

In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 µM, Ca(2+) ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for goat embryo production.

Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells.

Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle.

Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells.

Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6-203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science.