Environment-friendly, efficient process for mechanical recovery of waste lithium iron phosphate batteries.

Technology for recycling retired lithium batteries has become increasingly environment-friendly and efficient. In traditional recovery methods, pyrometallurgy or hydrometallurgy is often used as an auxiliary treatment method, which results in secondary pollution and increases the cost of harmless treatment. In this article, a new method for combined mechanical recycling of waste lithium iron phosphate (LFP) batteries is proposed to realize the classification and recycling of materials. Appearance inspections and performance tests were conducted on 1000 retired LFP batteries. After discharging and disassembling the defective batteries, the physical structure of the cathode binder was destroyed under ball-milling cycle stress, and the electrode material and metal foil were separated using ultrasonic cleaning technology. After treating the anode sheet with 100 W of ultrasonic power for 2 minutes, the anode material was completely stripped from the copper foil, and no cross-contamination between the copper foil and graphite was observed. After the cathode plate was ball-milled for 60 seconds with an abrasive particle size of 20 mm and then ultrasonically treated for 20 minutes with a power of 300 W, the stripping rate of the cathode material reached 99.0%, and the purities of the aluminium foil and LFP reached 100% and 98.1%, respectively.

High-precision and rapid binocular camera calibration method using a single image per camera.

This study proposes a precise and rapid binocular camera calibration (BCC) method based on a stereo target composed of 12 coded planar targets on which each calibration corner has a unique coded number. Unlike Zhang's method which requires numerous pairs of images in a binocular calibration process and fails to realize the matching of homonymous corners in the case of the incomplete target projection, the proposed method can implement an accurate BCC using a single calibration image per camera even in the case of target incompete projection. The proposed method greatly decreases the complexity of the calibration process. An optimization method based on multi-constraint is also presented to improve the accuracy of the BCC. The reprojection error and the 3D measurement errors are combined to evaluate the precision of the BCC more comprehensively. A binocular camera is calibrated by utilizing the proposed method and Zhang's method for comparison. The reprojection error and 3D measurement errors are remarkably reduced by applying the proposed method. The supplementary experiment further verifies the advantage of the proposed optimization method.

Robust and accuracy calibration method for a binocular camera using a coding planar target.

High-accuracy binocular camera calibration is a vital basis of precise binocular vision 3D measurement. In this work, a high-precision and robust binocular camera calibration method based on a coding target is proposed. First, a coding target with the simple patterns is designed. Every corner on the coding target has a unique code number, which can make the identification of homonymous corners easier and more valuable, even if the target is partially occluded. The decoding of the coding target is rapid, robust, and accurate at a complex background. Subsequently, the zenith and azimuth angles are introduced in the proposed calibration method to study the effect of the orientation of the coding target on the stability of calibration results and improve the robustness of the calibration results. Finally, to fully utilize the 3D information of the calibration corners on the coding target, we combine the reprojection and 3D geometric constraints to propose a multi-constraint optimization method for refining the parameters of binocular camera and improving the accuracy of binocular camera calibration. The comparison experiments have been done to verify the performance of the proposed calibration method. The standard deviations of the intrinsic and extrinsic parameters are greatly decreased, compared with Zhang's method. The mean reprojection and 3D geometric errors calculated by the proposed method have a large reduction. And the application experiment furtherly validates the effectiveness of the proposed method.

Environmentally friendly automated line for recovering aluminium and lithium iron phosphate components of spent lithium-iron phosphate batteries.

Lithium iron phosphate (LFP) batteries contain metals, toxic electrolytes, organic chemicals and plastics that can lead to serious safety and environmental problems when they are improperly disposed of. The published literature on recovering spent LFP batteries mainly focuses on policy-making and conceptual design. The production line of recovering spent LFP batteries and its detailed operation are rarely reported. A set of automatic line without negative impact to the environment for recycling spent LFP batteries at industrial scale was investigated in this study. It includes crushing, pneumatic separation, sieving, and poison gas treatment processes. The optimum retaining time of materials in the crusher is 3 minutes. The release rate is the highest when the load of the impact crusher is 800 g. An air current separator (ACS) was designed to separate LFP from aluminium (Al) foil and LFP powder mixture. Movement behaviour of LFP powder and Al foil in the ACS were analysed, and the optimized operation parameter (35.46 m/s) of air current speed was obtained through theoretical analysis and experiments. The weight contents of an Al foil powder collector from vibrating screen-3 and LFP powder collector from bag-type dust collector are approximately 38.7% and 52.4%, respectively. The economic cost of full manual dismantling is higher than the recovery production line. This recycling system provides a feasible method for recycling spent LFP batteries.

Recycling of electrode materials from spent lithium-ion power batteries via thermal and mechanical treatments.

This study developed a physical separation process that recovers active cathode materials from current collectors in spent lithium-ion power batteries (LIBs). The physical separation process, implemented via thermal and mechanical treatments, was examined based on cohesive zone models (CZMs) and verified by physical separation experiments. The most efficient condition was determined by optimising the key parameters (temperature and time) of selective heating. Among several mechanical separation methods, high-speed shearing best separates positive electrode materials into active cathode materials (LiFePO4) and current collectors (Al fragments). The separation effect was verified by computing the dissociation rate and microscopic observation of the separated materials. The feasibility and efficiency of the above process were assessed in a work-of-force analysis, flow field simulation, high-speed crushing experiment and material property analysis. The above analyses realised a feasible, efficient and environmentally friendly separation route without changing the chemical structure and properties of the electrode materials. Under non-high (energy-conserving) temperature conditions, the LiFePO4 dissociation rate stabilises at 80-85%. Under high-speed crushing, the LiFePO4 dissociation rate reaches 85% at 32,000-r/min crushing and a maximum shearing velocity of the blade edge v ≈ 500 m/s. This approach can effectively recycle electrode materials, gain valuable resources and can be used to recycle and utilise spent LIBs, thus addressing two grave issues - environmental pollution and resource wastage to achieve the sustainable development of LIBs and electric vehicle industry.

Low-temperature thermal pretreatment process for recycling inner core of spent lithium iron phosphate batteries.

Spent lithium iron phosphate (LFP) batteries contain abundant strategic lithium resources and are thus considered attractive secondary lithium sources. However, these batteries may contaminate the environment because they contain hazardous materials. In this work, a novel process involving low-temperature heat treatment is used as an alternative pretreatment method for recycling spent LFP batteries. When the temperature reaches 300°C, the dissociation effect of the anode material gradually improves with heat treatment time. At the heat treatment time of 120 minutes, an electrode material can be dissociated. The extension of heat treatment time has a minimal effect on quality loss. The physicochemical changes in thermally treated solid cathode and anode materials are examined through scanning electron microscopy with energy-dispersive X-ray spectroscopy. The heat treatment results in the complete separation of the materials from aluminium foil without contamination. The change in heat treatment temperature has a small effect on the quality of LFP material shedding. When the heat treatment temperature reaches 300°C and the time reaches 120 minutes, heat treatment time increases, and the yield of each particle size is stable and basically unchanged. The method can be scaled up and may reduce environmental pollution due to waste LFP batteries.

Environment-friendly technology for recovering cathode materials from spent lithium iron phosphate batteries.

The consumption of lithium iron phosphate (LFP)-type lithium-ion batteries (LIBs) is rising sharply with the increasing use of electric vehicles (EVs) worldwide. Hence, a large number of retired LFP batteries from EVs are generated annually. A recovery technology for spent LFP batteries is urgently required. Compared with pyrometallurgical, hydrometallurgical and biometallurgical recycling technologies, physical separating technology has not yet formed a systematic theory and efficient sorting technology. Strengthening the research and development of physical separating technology is an important issue for the efficient use of retired LFP batteries. In this study, spent LFP batteries were discharged in 5 wt% sodium chloride solution for approximately three hours. A specially designed machine was developed to dismantle spent LFP batteries. Extending heat treatment time exerted minimal effect on quality loss. Within the temperature range of 240°C-300°C, temperature change during heat treatment slightly affected mass loss. The change in heat treatment temperature also had negligible effect on the shedding quality of LFP materials. The cathode material and the aluminium foil current collector accounted for a certain proportion in a sieve with a particle size of -1.25 + 0.40 mm. Corona electrostatic separation was performed to separate the metallic particles (with a size range of -1.5 + 0.2 mm) from the nonmetallic particles of crushed spent LFP batteries. No additional reagent was used in the process, and no toxic gases, hazardous solid waste or wastewater were produced. This study provides a complete material recovery process for spent LFP batteries.

Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves.

Wild and cultivated rice show a significant difference in anthocyanin biosynthesis in the leaf. The regulation system of anthocyanin biosynthesis in rice leaf and the causal mechanism of the difference in this biosynthesis between wild and cultivated rice remain largely unknown. In this study, a genome-wide association study and transcriptome analysis were performed to identify the determinant factors and dissect the regulatory system for anthocyanin biosynthesis in rice leaves. OsC1, OsRb and OsDFR were identified as the determinants of anthocyanin biosynthesis in rice leaves. Artificial selection of certain null mutations of OsC1 and OsRb was the main causal mechanism underlying the loss of anthocyanin pigmentation in most cultivated rice. OsP1 and the MYB-bHLH-WD40 complexes regulate anthocyanin biosynthetic genes in rice leaves with partial functional overlap. OsP1 specifically activates upstream biosynthetic genes (OsCHS, OsCHI and OsF3'H) for anthocyanin biosynthesis, whereas the ternary MYB-bHLH-WD40 complex activates all anthocyanin biosynthetic genes including OsCHS, OsCHI, OsF3'H, OsF3H, OsDFR and OsANS. OsC1 and OsRb are tissue-specific regulators that do not influence anthocyanin biosynthesis in the pericarp. Our results reveal the determinant factors, regulatory system and domestication of anthocyanin biosynthesis in rice leaves, and show the potential of engineering anthocyanin biosynthesis in rice.

Pneumatic separation and recycling of anode and cathode materials from spent lithium iron phosphate batteries.

A novel approach to recycling of copper and aluminum fragments in the crushed products of spent lithium iron phosphate batteries was proposed to achieve their eco-friendly processing. The model of pneumatic separation that determines the optimal airflow velocity was established using aerodynamics. The influence of the airflow velocity, and the density and thickness, and their ratios, of the aluminum and copper fragments on pneumatic separation were evaluated. The results show that the optimal airflow velocities of copper and aluminum fragments with and without the electrode materials are 3.27m/s and 1.67m/s, respectively. The accuracy and reliability of the present model was verified using a pneumatic separation experiment. It is concluded that graded pneumatic separation is unnecessary for the crushed particle size more than 9 mm. The experimentally determined optimal airflow velocity of the copper and aluminum fragments with and without the electrode materials is 3.3m/s and 1.7m/s, respectively. The mass fractions of the copper and aluminum fragments upon removal of the electrode materials after pneumatic separation are 97% and 96%, respectively, and both with the electrode material achieve 97.0%. The theoretically obtained optimal airflow velocities have good agreements with the experimentally obtained ones.

Combined mechanical process recycling technology for recovering copper and aluminium components of spent lithium-iron phosphate batteries.

The recycling processes of spent lithium iron phosphate batteries comprise thermal, wet, and biological and mechanical treatments. Limited research has been conducted on the combined mechanical process recycling technology and such works are limited to the separation of metal and non-metal materials, which belongs to mechanical recovery. In this article the combined mechanical process recycling technology of spent lithium iron phosphate batteries and the separation of metals has been investigated. The spent lithium iron phosphate batteries monomer with the completely discharged electrolyte was subjected to perforation discharge. The shell was directly recycled and the inner core was directly separated into a positive electrode piece, dissepiment, and negative electrode piece. The dissociation rate of the positive and negative materials reached 100.0% after crushing when the temperature and time reached 300 °C and 120 min. The crushed products were collected and sequentially sieved after the low-temperature thermal treatment. Then, nonferrous metals (copper and aluminium) were separated from the crushed spent lithium iron phosphate batteries by eddy current separation with particle size -4 + 0.4. The optimised operation parameters of eddy current separation were fed at speeds of 40 r min-1, and the rotation speed of the magnetic field was 800 r min-1. The nonferrous metals of copper and aluminium were separated by the method of pneumatic separation. The optimal air speed was 0.34 m s-1 for the particle-size -1.6 + 0.4 mm and 12.85-14.23 m s-1 for the particle-size -4 + 1.6 mm. The present recycling process is eco-friendly and highly efficient and produces little waste.

Eddy current separation for recovering aluminium and lithium-iron phosphate components of spent lithium-iron phosphate batteries.

With the rapid development of the electric vehicle market since 2012, lithium-iron phosphate (LFP) batteries face retirement intensively. Numerous LFP batteries have been generated given their short service life. Thus, recycling spent LFP batteries is crucial. However, published information on the recovery technology of spent LFP batteries is minimal. Traditional separators and separation theories of recovering technologies were unsuitable for guiding the separation process of recovering metals from spent LFP batteries. The separation rate of the current method for recovering spent LFP batteries was rather low. Furthermore, some wastewater was produced. In this study, spent LFP batteries were dismantled into individual parts of aluminium shells, cathode slices, polymer diaphragms and anode slices. The anode pieces were scraped to separate copper foil and anode powder. The cathode pieces were thermally treated to reduce adhesion between the cathode powder and the aluminium foil. The dissociation rate of the cathode slices reached 100% after crushing when the temperature and time reached 300℃ and 120 min, respectively. Eddy current separation was performed to separate nonferrous metals (aluminium) from aluminium and LFP mixture. The optimized operation parameters for the eddy current separation were feeding speed of 1 m/s and magnetic field rotation speed of 4 m/s. The separation rate of the eddy current separation reached 100%. Mass balance of the recovered materials was conducted. Results showed that the recovery rate of spent LFP can reach 92.52%. This study established a green and full material recovery process for spent LFP batteries.

Copper hexacyanoferrate/carbon sheet combination with high selectivity and capacity for copper removal by pseudocapacitance.

The efficient capture of copper ions (Cu2+) in wastewater has dual significance in pollution control and resource recovery. Prussian blue analog (PBA)-based pseudocapacitive materials with open frameworks and abundant metal sites have attracted considerable attention as capacitive deionization (CDI) electrodes for copper removal. In this study, the efficiency of copper hexacyanoferrate (CuHCF) as CDI electrode for Cu2+ treating was evaluated for the first time upon the successful synthesis of copper hexacyanoferrate/carbon sheet combination (CuHCF/C) by introducing carbon sheet as conductive substrate. CuHCF/C exhibited significant pseudocapacitance and high specific capacitance (52.92 F g-1) through the intercalation, deintercalation, and coupling of Cu+/Cu2+ and Fe2+/Fe3+ redox pairs. At 0.8 an applied voltage and CuSO4 feed liquid concentration of 100 mg L-1, the salt adsorption capacity was 134.47 mg g-1 higher than those of most reported electrodes. Moreover, CuHCF/C demonstrated excellent Cu2+ selectivity in multi-ion coexisting solutions and in actual wastewater experiments. Density functional theory (DFT) calculations were employed to elucidate the mechanism. This study not only reveals the essence of Cu2+ deionization by PBAs pseudocapacitance with promising potential applications but also provides a new strategy for selecting efficient CDI electrodes for Cu2+ removal.

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.

[Analysis of risk factors for re-operation due to postoperative haemorrhage following coblation-assisted tonsillectomy].

Objective:To investigate the risk factors for re-operation due to postoperative haemorrhage following coblation-assisted tonsillectomy. Methods:The clinical data of 135 patients with haemorrhage after coblation-assisted tonsillectomy in our hospital from January 2015 to May 2022 were collected, According to the patients received reoperation after tonsillectomy or not, all patients were divided into the reoperation group （n=43） and non-reoperation group （n=92）,clinical data were compared between the two groups. Results:Univariate analysis showed that there was no significant difference in gender, age, postoperative intravenous glucocorticoid use, diabetes and hypertension between the two groups（P>0.05）, Univariate and multivariate factor analyses showed that early haemorrhage, obvious hemorrhage points were independent risk factors for re-operation due to postoperative haemorrhage（P<0.05）. Conclusion:Early haemorrhage and obvious hemorrhage points are independent risk factors for re-operation due to postoperative haemorrhage following coblation-assisted tonsillectomy.

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.

BACKGROUND: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. METHODS: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. FINDINGS: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. INTERPRETATION: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. FUNDING: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

A new model of trajectory in eddy current separation for recovering spent lithium iron phosphate batteries.

Eddy current separation (ECS) is an environment-friendly technology for separating nonferrous metallic particles whose size was from 2 mm to 10 mm. No wastes are generated in ECS. ECS quality of nonferrous metals from solid wastes is rather low in the production practice of spent lithium iron phosphate (LFP) batteries recovering. Repeating separation even manual sorting is required in the production. The traditional method of falling point prediction based on eddy current mechanics uses equivalent acceleration to replace separation motion curves. These curves have low precision and are unsuitable for predicting the motion trajectory of small particle size of sorted materials. In this work, eddy current separation of positive and negative plates in a crushed product of spent lithium iron phosphate battery is used as an example to establish the force and kinematics models of different materials in the eddy current separation. An iterative method, rather than average speed method, is used to improve the accuracy of the model. Displacement interval replaces disengagement angle as a separating index to improve the model's intuitiveness and practical guidance. In the range of 2-20 mm, test results are consistent with simulation results. The copper and aluminium foils at a magnetic roller speed of 800r/min can be separated to a maximum particle size ratio of 1.72, and the maximum particle size ratio of copper and positive electrode sheets can be large. This paper provided an environmental-friendly and effective technology for separating nonferrous metals from crushed spent LFP batteries.

Generation of cloned calves from different types of somatic cells.

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respectively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from different individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P < 0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9% and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%c, respectively (pABC < 0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respectively.

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle.

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 microg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.

Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer.

The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8%, respectively) or transfected FOECs (TFOECs, 73.8 and 47.7%, respectively); nor from serum-fed (73.6 and 47.2%, respectively) or serum-starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFP-embryos (eight fresh blastocysts and nine vitrified/thawed blastocysts ) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully.