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 doublemuscling phenotype. In conclusion, we demonstrate that the genome-editing technology CRISPR-Cas9 can produce genetically modified calves with improved traits.

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How do body temperature and activity change before and after parturition in pregnant cows? Changes in body temperature such as ruminal, rectal, and vaginal temperature during the parturition have been reported, but there are no results of the simultaneous observation of body temperature and activity. The aim of this study was to simultaneously confirm changes in the ruminoreticular temperature and body activity before and after parturition using the ruminoreticular bio-capsule sensor every 1 h. The 55 pregnant cows were used for the experiment, the ruminoreticular bio-capsule sensor was inserted and stabilized, and the ruminoreticular temperature and body activity were measured. The ruminoreticular temperature was lower by 0.5° from −24 h to −3 h in parturition compared to 48 h before parturition and then recovered again after parturition. Body activity increased temporarily at the time of parturition and 12 h after parturition. Therefore, the ruminoreticular temperature and body activity before and after parturition was simultaneously confirmed in pregnant cows.

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Background Several DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS. Results In bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%). Conclusions These results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type.

In recent years, CRISPR/Cas9 has become known as a powerful tool for generating genetically modified cells or animal models with its site-specific gene editing capability. Because myostatin (MSTN) is associated with muscle differentiation and growth, ablation of MSTN would be ideal for studying phenotypes associated with muscle growth in beef cattle. The aim of this study is to produce Korean beef cattle with MSTN mutation through microinjection of CRISPR/Cas9. For this study, ovaries of Korean beef cattle were obtained from slaughterhouses; 122 immature oocytes were aspirated from 8 Korean beef cattle. The oocytes were matured in tissue culture medium 199-based medium for 22h and inseminated with frozen-thawed semen to produce zygotes. All 122 zygotes were directly injected with 200ng of Cas9 mRNA and 100ng of sgRNA for MSTN after IVF and were cultured for 7 days in two-step chemically defined medium. Each embryo from the individual cattle was cultured separately to distinguish its lineage. After 7 d, 29 (23.8±8.9) blastocysts were recovered, and all blastocysts were assessed with T7 endonuclease (T7E1) mutation assay to check for mutations at the MSTN locus. Based on T7E1, 26 out of 29 blastocysts (89.7±10.5%) had heterozygous mutation in the MSTN gene. After analysis, additional blastocysts were produced and transferred into 14 surrogates. All surrogates were diagnosed for pregnancy via rectal palpation and ultrasonography at Day 55, at which time normal fetuses and embryonic sacs were confirmed in 10 surrogates. In conclusion, these data demonstrate that CRISPR/Cas9 could be employed as an efficient method to genetically modify the MSTN gene in Korean beef cattle.

Background: Several DNA transposons, PiggyBac (PB), Sleeping beauty (SB) and Tol2 have been applied as effective means for transgenesis in many species. Cattle are not typical experimental animals, and relatively little verification has been studied in this species. Thus, the goal of this study was the applicability of three transposon systems in somatic and embryo cells in cattle, while also determining which of the three systems is appropriate for each type of cell. To conduct the experiment, green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, GFP-positive cells or blastocysts were observed through a fluorescent microscope and transfection efficiency was calculated by FACS.Results: In the bovine somatic cells experiment, the PB (63.97 ± 11.56) showed higher efficiency as compared to the other two systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Unlike the results of the somatic cells, Tol2 (75.00%) and SB (70.00%) in the embryo were more efficient as compared to PB (42.86 %).Conclusions: These results demonstrate that all three transposon systems can be used in bovine somatic cells and embryos as a gene engineering experimental method and which type of transposon system is appropriate to apply depending on the cell type.

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