Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein (RNP) electroporation.

BACKGROUND: Genome editing has been considered as powerful tool in agricultural fields. However, genome editing progress in cattle has not been fast as in other mammal species, for some disadvantages including long gestational periods, single pregnancy, and high raising cost. Furthermore, technically demanding methods such as microinjection and somatic cell nuclear transfer (SCNT) are needed for gene editing in cattle. In this point of view, electroporation in embryos has been risen as an alternative. RESULTS: First, editing efficiency of our electroporation methods were tested for embryos. Presence of mutation on embryo was confirmed by T7E1 assay. With first combination, mutation rates for MSTN and PRNP were 57.6% ± 13.7% and 54.6% ± 13.5%, respectively. In case of MSTN/BLG, mutation rates were 83.9% ± 23.6% for MSTN, 84.5% ± 18.0% for BLG. Afterwards, the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing. Thirteen recipients were transferred for MSTN/PRNP, 4 calves were delivered, and one calf underwent an induction for double KO. Ten surrogates were given double-KO embryos for MSTN/BLG, and four of the six calves that were born had mutations in both genes. CONCLUSIONS: These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied. Finally, MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

Multi-Object Tracking on SWIR Images for City Surveillance in an Edge-Computing Environment.

Although Short-Wave Infrared (SWIR) sensors have advantages in terms of robustness in bad weather and low-light conditions, the SWIR images have not been well studied for automated object detection and tracking systems. The majority of previous multi-object tracking studies have focused on pedestrian tracking in visible-spectrum images, but tracking different types of vehicles is also important in city-surveillance scenarios. In addition, the previous studies were based on high-computing-power environments such as GPU workstations or servers, but edge computing should be considered to reduce network bandwidth usage and privacy concerns in city-surveillance scenarios. In this paper, we propose a fast and effective multi-object tracking method, called Multi-Class Distance-based Tracking (MCDTrack), on SWIR images of city-surveillance scenarios in a low-power and low-computation edge-computing environment. Eight-bit integer quantized object detection models are used, and simple distance and IoU-based similarity scores are employed to realize effective multi-object tracking in an edge-computing environment. Our MCDTrack is not only superior to previous multi-object tracking methods but also shows high tracking accuracy of 77.5% MOTA and 80.2% IDF1 although the object detection and tracking are performed on the edge-computing device. Our study results indicate that a robust city-surveillance solution can be developed based on the edge-computing environment and low-frame-rate SWIR images.

Germline transmission of MSTN knockout cattle via CRISPR-Cas9.

Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.

Effect of glycerol addition time on the cryopreserved Korean native brindle cattle (Chikso) sperm quality.

Although cryopreservation is an efficient method for maintaining the biological and genetic resources of sperm, the sperm damage during the cryopreservation process cannot be ignored. It should be possible to obtain the most effective cryopreservation performance by accurately grasping the effects of various factors on the cryopreservation of sperm. The previous study demonstrated that a suitable standard protocol for cryopreservation of Korean native brindled cattle (Chikso) does not exist, based on the methods for semen cryopreservation of Chikso differ in each research center. The most obvious difference between most of protocols is the addition of glycerol before and after cooling during the Chikso cryopreserved semen process. Therefore we focused on the effects of glycerol addition time on the quality of cryopreserved Chikso sperm. In the present study, 27 individual Chikso samples were collected by transrectal massage and divided into two parts: the "cryopreservation method A" group (adding glycerol before cooling) and the "cryopreservation method B" group (adding glycerol after cooling). Meanwhile, the values of various sperm parameters were derived from each group, including sperm motility, kinematics, capacitation status, cell viability, and intracellular ATP levels, which we used to compare and evaluate sperm function. The results of this study indicated that during the semen cryopreservation process of the Chikso, the addition of glycerol after cooling yielded superior results in a variety of sperm parameters, such as sperm motility, progressive motility, rapid motility, VCL, VSL, VAP, ALH, capacitation status, viability, and intracellular ATP level after freezing and thawing. Our study is suggested that the glycerol addition time during the cryopreservation process for Chikso should be considered. In addition, our results may be provided reference to develop suitable the cryopreservation procedure of the Chikso sperm.

Production of MSTN-mutated cattle without exogenous gene integration using CRISPR-Cas9.

Many genome-edited animals have been produced using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique to cattle. The goal of this study was to produce trait-improved cattle using the genome-editing technology CRISPR-Cas9. Myostatin (MSTN) was selected as a target locus, and synthetic mRNA of sgRNA and Cas9 were microinjected into fertilized bovine embryos in vitro. As a result, 17 healthy calves were born, and three of them showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had a biallelic mutation (-12 bps) and a double-muscling phenotype. In conclusion, we demonstrate that the genome-editing technology CRISPR-Cas9 can produce genetically modified calves with improved traits.

Robust Channel Allocation with Heterogeneous Requirements for Wireless Mesh Backbone Networks.

When multiple mobile sensors and actuators share a common wireless mesh backbone network of defence systems, the channel allocation mechanism must guarantee the heterogeneous link requirements under conditions of uncertainty. In this paper, a robust channel allocation mechanism is proposed by exploiting partially overlapped channels for directional multi-channel wireless mesh networks. The approach relies on a chance-constrained optimization problem, in which the objective is to minimize the spectrum usage of the network, and the constraints are the signal-to-interference-plus-noise ratio requirements of links with uncertainty. We convert the proposed integer non-linear optimization problem into a mixed-integer convex problem by using efficient transition and approximation. The optimal channel allocation is obtained by solving the proposed optimization problem which adapts to the heterogeneous link and robustness requirements. The simulation results show that the proposed method ensures the heterogeneous link requirements under uncertain conditions while minimizing the spectrum usage of the network.

Effects of Kaolinite (Macsumsuk) and Herb Mixtures on the Quality and Physicochemical Properties of Pork.

The current study was conducted to identify technology for the production of high quality pork, based on the meat consumption habits of consumers. Macsumsuk, a type of kaolinite (a clay mineral), and/or a mixture of herbs (Mori Folium, Sophorae Radix, Glycyrrhizae Radix, Citri Leiocarpae Exocarpium, and Pogostemi Herba) were added to the diets of fattening pigs. Sixty barrow pigs (4 kinds of treatment × 5 pigs/treatment × 3 replicates) were randomly assigned to either the Control (no additives), T1 (3% Macsumsuk), T2 (3% Herb mixtures), or T3 (3% Macsumsuk + 3% Herb mixtures) groups, and were fed the diets for 60 d. Dressed weights were in the order of T1 (93.40±4.68kg) > T2 (91.40±6.52kg) > Control (88.80±1.57kg) > T3 (86.80±2.01kg). Back-fat thickness of the Control animals (23.2±1.03) was significantly greater than that of the various treatment groups (p<0.01). Numeric values representing the carcass yield and quality grade were higher for all the treated groups than the Control group, thought the difference was not statistically significant. Crude fat content was significantly higher in the Control group (2.23±0.34%) than in the treated groups (p<0.05). The addition of both Macsumsuk and herb mixtures into the diets of the pigs significantly reduced cooking loss of the pork compared to the Control (p<0.05). No statistically significant changes were observed in the shear force (average 5.87±0.54 kg/cm(2)), water holding capacity (average 54.59±3.16%), or CIE values of the pork, whereas cholesterol levels significantly decreased (p<0.01) in those fed Macsumsuk and/or the herb mixtures. No significant changes in fatty acid composition, total saturated fatty acid (SFA), total unsaturated fatty acid (UFA), or UFA/SFA ratios were observed by any of the treatments. In conclusion, the results obtained from this study suggest that the addition of Macsumsuk and/or herb mixtures into the diets of growing and fattening pigs improves the pork quality by reducing cooking loss, decreasing cholesterol content, and enhancing sensory characteristics.

Spinal cord herniation after multilevel anterior cervical corpectomy and fusion for ossification of the posterior longitudinal ligament of the cervical spine.

The authors report the case of a 52-year-old man who had undergone resection of an ossified posterior longitudinal ligament via the anterior approach. The patient experienced postoperative neurological deterioration that may have been caused by a massive cord herniation associated with a dural defect at the corpectomy site. Spinal cord herniation may develop as a complication of anterior cervical decompression. Surgeons should be alert to this condition when planning to treat cervical ossification of the ossified posterior longitudinal ligament via the anterior approach.