Sero-Prevalence and Risk factors of Contagious Bovine Pleuropneumonia (CBPP) in Afgoye District Lower Shabelle Region, Somalia

Contagious bovine Pleuropneumonia (CBPP) is an infectious and highly contagious respiratory disease of cattle and water buffalo, caused by the Mycoplasma mycoides subspecies mycoides. It induces significant economic losses and leads to a severe livestock production problem, negatively influencing people’s livelihoods of affected countries. In Somalia, there is no updated data on the prevalence and distribution of the disease. Hence, a descriptive cross-sectional study was conducted from September 2022 to June 2023 in different villages under the Afgoye District of lower Shabelle region, Somalia. The main purpose of this study is to assess the sero-prevalence and identify the associated risk factors for the occurrence of the disease. In this study, villages, age, sex, breed, and body condition were considered as risk factors. A total of 90 blood samples were collected and tested in the laboratory using the Anti-CBPP Elisa kit test. Out of 90 serum samples from herd cattle, 32 were positive, resulting in an overall prevalence of 35.5%. In addition, we found a statistically significant variation between the prevalence of the disease and factors such as sex, age, body condition and breeds. In summary, the overall prevalence of Contagious bovine pleuropneumonia in this study area is worth to be considered because there is a low quality of health care and less awareness of the Contagious bovine Pleuropneumonia effects on herds, which warrants the official authorities to act and follow appropriate preventive and control measures to reduce the incidence of the disease and generate appropriate controlling and prevention measures in all regions of Somalia.

Molecular Identification of Mycobacterium Strains Responsible of Bovine Tuberculosis Cases in Bobo-Dioulasso Slaughterhouse, Burkina Faso

Bovine tuberculosis (bTB) is an endemic zoonosis significantly affects animal health in Burkina Faso. The primary causative agent is Mycobacterium tuberculosis (M. tuberculosis) complex, mainly M. bovis. Cattle are considered as natural reservoir of M. bovis. However, in Burkina Faso, the circulation of these strains remains poorly understood and documented. This study aimed to identify and characterize Mycobacterium strains from suspected carcasses during routine meat inspection at Bobo-Dioulasso refrigerated slaughterhouse. A prospective cross-sectional study was conducted from January 2021 to December 2022 on cases of seizures linked to suspected bovine tuberculosis. Microbiological and molecular analyzes were used for mycobacterial strain isolation and characterization. Out of 50 samples, 24% tested positive by microscopy and 12% by culture. Molecular analysis identified 6 strains of Mycobacteria, exclusively Mycobacterium bovis specifically the subspecies bovis (Mycobacterium bovis subsp bovis). In conclusion, M. bovis subsp bovis is the primary agent responsible for bovine tuberculosis in Bobo-Dioulasso. Continuous monitoring of mycobacterial strains is therefore necessary for the effective control of this pathology in the local cattle population.

Classical bovine spongiform encephalopathy and chronic wasting disease:two sides of the prion coin

Transmissible spongiform encephalopathies(TSEs)are a group of progressive and ultimately fatal neurologic diseases of man and animals,all resulting from the propagated misfolding of the host's normal cellular prion protein.These diseases can be spontaneous,heritable,anthropogenic/iatrogenic,or in some cases horizontally transmissible,and include such notable TSEs as bovine spongiform encephalopathy(BSE)of cattle and chronic wasting disease(CWD)of cervids.Although they are both unequivocally protein misfolding disorders,they differ markedly in their pathogenesis,transmissibility,and zoonotic potential.While the BSE epidemic has largely abated over the past three decades following global feed bans on ruminant meat and bone meal,CWD,which is readily transmitted through various forms of excreta,has rapidly expanded from its original endemic zone to encompass much of North America,along with recently identified foci in Scandinavia.Most importantly,although the classical form of BSE has proven transmissible to humans consuming contaminated beef or beef products,so far there have been no conclusive reports on the zoonotic transmission of cWD to humans.The underlying basis for these differences-whether host or agent directed-are not well understood,though may be due to inherent differences in the three-dimensional structure of the misfolded BSE or CWD prion proteins or the expression levels and tissue distribution of respective cellular prion proteins.With the uncontrolled geographic spread of CWD,it is imperative that we improve our understanding of the factors governing prion disease pathogenesis,transmission,and zoonotic potential.

Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Investigating Prevalence of Bovine Tuberculosis in Cattle in an Agro-Pastoral Community in Awdal Region, Somaliland

Awdal region is the most northwesterly province of Somaliland. The region is one of the agro-pastoral livelihood zones in Somaliland, where farming and agricultural production are the predominant livelihood sources. IGAD (Intergovernmental Authority on Development) Sheikh Technical University of Science (ISTUS) worked to study the prevalence of Bovine Tuberculosis (TB) among cattle in Awdal region of Somaliland. The aim was to inform public health and Veterinary experts on having one-health approach to infectious diseases among humans and animals. The serum that was stored at -20?C was transported to IGAD Sheikh Technical University of Science (ISTUS) for further analysis using BOVIGAM serological test (Sandwich ELISA). The results indicate that Bovine Tuberculosis is highly prevalent in the study area (10.1%). The high prevalence recorded in the current study could be due to the consumption of raw milk and lack of proper control strategies in place to control the transmission of the disease between animals, between animals and wildlife and between animals and humans. Hence, an awareness creation campaign should be created on bTB transmission and its public health significance to cattle owners, milk and meat consumers and people who are in close proximity to cattle. In addition, testing and eradication programme should be implemented where applicable.

Expression of miR‑138 in cryopreserved bovine sperm is related to their fertility potential

Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene expression.In the last decade,miRNAs have been described in sperm of several mammals,including cattle.It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans;however,little is known about the content,expression,and function of sperm-borne miRNAs in early bovine embryo.In this study,total sperm RNA was isolated from 29 cryopreserved sperm samples(each coming from a separate bull)using a RNeasy kit and treatment with DNase I.RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer.The expression of 10 candidate miRNAs in bovine sperm(bta-miR-10a,bta-miR-10b,bta-miR-138,bta-miR-146b,bta-miR-19b,bta-miR-26a,bta-miR-34a,bta-miR-449a,bta-miR-495 and btamiR-7),previously identified in testis and/or epididymis,was evaluated with RT-qPCR.The cel-miR-39-3p was used as a spike-in exogenous control.Nonparametric Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility[HF;non-return rates(NRR)ranging from 39.5 to 43.5]and those with subfertility(SF;NRR ranging from 33.3 to 39.3).Several sperm functionality parameters(e.g.,viability,membrane stability or oxygen consumption,among others)were measured by multiplexing flow cytometry and oxygen sensing technologies.Results RNA concentration and purity(260/280 nm ratio)(mean±SD)from the 29 samples were 99.3±84.6 ng/μL and 1.97±0.72,respectively.Bioanalyzer results confirmed the lack of RNA from somatic cells.In terms of the presence or absence of miRNAs,and after applying the Livak method,8 out of 10 miRNAs(bta-miR-10b,-138,-146b,-19b,-26a,-449a,-495,-7)were consistently detected in bovine sperm,whereas the other two(bta-miR-10a,and-34a)were absent.Interestingly,the relative expression of one miRNA(bta-miR-138)in sperm was significantly lower in the SF than in the HF group(P=0.038).In addition to being associated to fertility potential,the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption.The expression of three other miRNAs(bta-miR-19b,bta-miR-26a and bta-miR-7)was also correlated with sperm function variables.Conclusions In conclusion,although functional validation studies are required to confirm these results,this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond,and supports its use as a fertility biomarker in cattle.

Financial Losses Associated with Bovine Brucellosis (Brucella abortus) in Carchi-Ecuador

Brucellosis is an infectious disease of worldwide distribution, which has a great economic impact due to the productive and reproductive losses that it causes, in addition to the serious public health problem. The aim of this study is to estimate the economic losses, through financial analysis, caused by bovine brucellosis in the province of Carchi, over a one-year period. A random sampling was used to determine the prevalence in the study area, where 2976 animals were considered, and the Rose Bengal (RB) test was used as a screening test and the Fluorescence Polarized Assay (FPA) as a confirmatory test, obtaining a prevalence of 8.2% (244/2976). In addition, parameters associated to the losses caused by brucellosis in cattle were determined by literature review. To estimate costs, field information was collected through a survey of a total of 100 randomly selected farmers. The loss estimated due to calves lost as a result of abortions and neonatal death was USD. 79170.00. The loss due to death of 4 cows as a result of metritis was estimated at USD. 5000.00. The cost of examination and treatment of aborted cows was USD. 20100.00. The losses due to reduction in milk production from aborted and non-aborted seropositive cows were estimated at USD. 158114.21. The financial losses due to brucellosis in province of Carchi were estimated at USD. 262384.21.

Punch‑excised explants of bovine mammary gland to model early immune response to infection

Background Mammary gland(MG)infections(mastitis)are frequent diseases of dairy cows that affect milk quality,animal welfare and farming profitability.These infections are commonly associated with the bacteria Escherichia coli and Staphylococcus aureus.Different in vitro models have been used to investigate the early response of the MG to bacteria,but the role of the teat in mastitis pathogenesis has received less attention.In this study,we used punch-excised teat tissue as an ex vivo model to study the immune mechanisms that arise early during infection when bacteria have entered the MG.Results Cytotoxicity and microscopic analyses showed that bovine teat sinus explants have their morphology and viability preserved after 24 h of culture and respond to ex vivo stimulation with TLR-agonists and bacteria.LPS and E.coli trigger stronger inflammatory response in teat when compared to LTA and S.aureus,leading to a higher produc-tion of IL-6 and IL-8,as well as to an up-regulation of proinflammatory genes.We also demonstrated that our ex vivo model can be applied to frozen-stored explants.Conclusions In compliance with the 3Rs principle(replacement,reduction and refinement)in animal experimenta-tion,ex vivo explant analyses proved to be a simple and affordable approach to study MG immune response to infec-tion.This model,which better reproduces organ complexity than epithelial cell cultures or tissue slices,lends itself particularly well to studying the early phases of the MG immune response to infection.

Calcitriol induces post-thawed bovine sperm capacitation

Background:Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity.In vivo,this process is directly associated with high calcium levels in sperm cytoplasm.Calcitriol,the vitamin D hypercalcemic metabolite,is related to human sperm motility,capacitation,and acrosome reaction.This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation.Methods:One million freezethawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min.Untreated cells(negative control)and treated ones with calcitriol or heparin(100μg/mL,positive capacitation control)were evaluated for motility,viability,and functional parameters.Menadione(70μM,30 min)treatment was included as a reactive oxygen species(ROS)positive sperm agent.Results:The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability,vigor,and capacitation than their positive and negative controls.The percentage of sperm with intact plasma and acrosome membranes,mitochondrial membrane potential(ΔΨm),and phosphatidylserine externalization was similar in all the conditions evaluated,while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control.Conclusion:Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility,viability,and vigor without altering key biological parameters such as redox status,ΔΨm,and cell death.

Evaluation and application of a milk antibody ELISA for assessing the prevalence and incidence of bovine tuberculosis in dairy herds in Hubei Province,China

Bovine tuberculosis(bTB)is a chronic zoonotic disease that is endemic in China.Current in-vitro tests for bTB are mainly based on blood assays.Collection of samples results in some stress to the sampled cattle and associated economic losses for the herd owner.This study was designed to investigate the relationship between milk and serum antibody tests for bTB in dairy cows using 85 cows with milk and corresponding blood samples.Totally 4,395 milk samples were used to assesse the apparent(test)prevalence and incidence of bTB using the milk antibody ELISA.The association between levels of bTB milk antibody and milk quality was also evaluated.Milk and serum antibody tests showed a good correlation with a 87.5%(95%CI:61.7%,98.4)positive agreement and 98.7%(95%CI:95.4,99.8)negative agreement.The animal level lactoprevalence ranged from 0.3%(95%CI:0,1.2)to 33.3%(95%CI:26.6,40.6)in different farms and the incidence rate ranged from 0 head/cow-month(95%CI:0,0.02)to 0.04 head/cow-month(95%CI:0.02,0.07).Twenty percent of sampled farms met the criteria for bTB control in China.The prevalence on large-scale farms was lower(p<0.001)than on small farms.The bTB milk antibody levels had a negative correlation with milk yield and a positive correlation with somatic cell count(SCC),milk protein percentage(MPP)and percentage of total solids(TS).According to this research,milk ELISA could be used as a supplement of blood samples to assist in the surveillance for bTB and for alerting control and eradication of bTB.

Differentially expressed whey proteins of donkey and bovine colostrum revealed with a label-free proteomics approach

This study aimed to analyze and compare the differentially expressed whey proteins(DEWPs)of donkey and bovine colostrum using high-performance liquid chromatography with tandem mass spectrometry-based proteomics.A total of 620 and 696 whey proteins were characterized in the donkey and bovine colostrum,respectively,including 383 common whey proteins.Among these common proteins,80 were identified as DEWPs,including 21 upregulated and 59 downregulated DEWPs in donkey colostrum compared to bovine colostrum.Gene Ontology analysis revealed that these DEWPs were mainly related to cellular components,such as extracellular exosome,plasma membrane,and mitochondrion;biological processes,such as oxidation-reduction process,cell-cell adhesion,and small guanosine triphosphate(GTP)ase-mediated signal transduction;and molecular functions,such as GTP binding,GTPase activity,and soluble N-ethylmaleimide-sensitive factor(NSF)attachment protein receptor activity.Metabolic pathway analysis suggested that the majority of the DEWPs were associated with soluble NSF factor attachment protein receptor interactions in vesicular transport,fatty acid biosynthesis,and estrogen signaling pathways.Our results provide a vital insight into the differences between donkey and bovine colostrum,along with important information on the significant components as nutritional and functional factors to be included in infant formula based on multiple milk sources.

Bovine Placental Bioactive Proteins Preparation and Efficacy Evaluation

Taking the qualified bovine placenta as the research object,a series of pure physical separation and extraction processes such as tissue fragmentation,ultrasound,freeze-thawed,centrifugation and filtration were used to prepare the bovine placental bioactive proteins.The results of protein concentration showed that bovine placental bioactive proteins prepared in this study could reach(11.33±2.09)mg/mL under optimal experimental condition.The determination of specific components showed that there were abundant exosome like vesicles in the product with particle size ranging from 30 to 150 nm,pie structure under transmission electron microscope and expressing CD9,CD63 and TSG101 by western blot detection.The results of cell efficacy evaluation showed that bovine placental bioactive proteins could significantly promote the proliferation of human skin fibroblasts and had anti-inflammatory effect via down-regulating IL-6 and TNF-αexpressionin RAW264.7 cells.

Bovine <i>β</i>-casein: Detection of two single nucleotide polymorphisms by bidirectional allele specific polymerase chain reaction (BAS-PCR) and monitoring of their variation

Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platforms are available for semiautomated or fully automated SNP geno-typing. However, in some cases the use of simple and rather cheap methods is an effective tool to be implemented within one’s own laboratory for the routine analysis of a specific SNP. The present paper describes two simple tests based on the bidirectional allele-specific polymerase chain reaction (BAS-PCR) developed for the identification of β-casein (CSN2) B and I genetic variants. The practical application of the two methods on a panel of 84 Italian Brown bulls and 100 Italian Friesian cows is also discussed, including the biological significance of the two genetic variants and the importance of taking their occurrence into account when linkage analyses are performed on milk functional properties. A combined system for analysing milk protein variants by isoelectrofocusing (IEF) and the BAS-PCR assay developed for CSN2*I is described.

Antitumor Effect of Cationic INKKI Peptide from Bovine <i>β</i>-Casein on Melanoma B16F10

Cationic peptide with the sequence INKKI 41-45 was isolated from bovine β-casein after tryptic hydrolysis and synthetized. The aim of this work was to evaluate the antiproliferative activity in vitro and antitumor effect in animal model. The in vitro cytotoxicity was evaluated on B16F10 melanoma cells by MTT assay. Detection of apoptosis was measured using the annexin V/PI double staining and cell cycle analysis performed flow cytometry. Caspase-3 activity was analyzed with substrate specific fluorogenic DEVD-MCA. In vivo, antitumor activity was evaluated in B16F10 melanoma tumor-bearing C57BL/6J mice. The animals were treated with 55 mg/kg INKKI administered into peritumoral region, while control group received saline solution. The following antitumor parameters were examined: tumor volume, number of metastases, tumor delayed time, tumor doubling time. Histological analyses were performed with H & E staining. The results showed that INKKI induced dose-response cytotoxicity selective for B16F10 melanoma cells (IC50 1.7 μM) and did not present cytotoxic effects for FN1 fibroblast cells. INKKI-induced apoptosis detected trough of annexin V/PI assay and it was accompanied with an increase of sub-G1 apoptotic fractions and significant increase of caspase-3 cleavage. The tumor-bearing mice treated with INKKI showed a significant reduction in tumor volume of 72.62% and decreased of metastasis number loci. In addition, INKKI caused a significant delay in tumor growth and prolonged the tumor doubling time. Histological analysis revealed an increased of necrosis areas and reduction of tumor cells in tumor treated with INKKI, it was a many hallmark of its antitumor effects observed from in vivo experiments. In conclusion, we show that INKKI is a peptide that could be considered a new putative candidate development to anticancer therapy drug.

牛血超氧化物歧化酶(bovine superoxide dismutase)生产工艺研究

将红细胞连续分离、热变性以及超滤技术应用在牛血 SOD生产制备中 ,使 SOD生产成本大幅降低 ,实现牛血 SOD生产工业化 .实验表明 ,冷冻血球和新鲜血球在酶的收率、纯度以及比活等方面无明显差别 ;热变性、超滤、丙酮沉淀等各工艺步骤酶的活性以及收率基本稳定 ;通过重组 SOD技术使失去铜锌离子失活的酶蛋白恢复酶的催化活性 .

Genetic characterization of antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China

Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicrobial resistance in S. aureus cultured from bovine mastitis in Northwest China in 2014. A total of 44 S. aureus were isolated for antimicrobial resistance and resistance-related genes. Antimicrobial resistance was determined by disc diffusion and the corresponding resistance genes were detected by PCR. Phenotype indicated that S. aureus isolates were resistant to penicillin （84.09%）, erythromycin （20.45%）, tetracycline （15.91%）, gentamicin （9.09%）, tobramycin （6.82%）, kanamycin （6.82%） and methicillin （2.27%）. 9.09% of the S. aureus isolates were classified as multidrug resistant. In addition, genotypes showed that the isolates were resistant to rifampicin （100%, rpoB）, penicillin （95.45%, blaZ）, tetracycline （22.73%, tetK, tetM, alone or in combination）, erythromycin （22.73%, ermB or ermC）, gentamicin/tobramycin/kanamycin （2.27%, aacA-aphD）, methicillin （2.27%, mecA） and vancomycin （2.27%, vanA）. Resistance to tetracycline was attributed to the genes tetK and tetM （r=0.558, P〈0.001）. This study noted high-level geno- and phenotypic antimicrobial resistance in S. aureus isolates from bovine mastitis cases in Northwest China.

Multi-spectroscopic characterization of bovine serum albumin upon interaction with atomoxetine

The quenching interaction of atomoxetine(ATX) with bovine serum albumin(BSA) was studied in vitro under optimal physiological condition(pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ΔH^0 and ΔS^0 indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by F?rsters theory. UV-absorption, Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD), synchronous spectra and three-dimensional(3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.

Effects of donor cells on in vitro development of cloned bovine embryos

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer （SCNT）. The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The oocytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer. The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastocyst as the parameters of SCNT efficiency. The donor cells from different individuals （04036, 06081, 06088, and 06129） had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate （P〈0.05）, and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes （P〉0.05）. It was concluded that the genetic background of the donor cells could affect the efficiency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.

Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture

Background： Bovine milk contains not only a variety of nutritional ingredients but also microRNAs （miRNAs） that are thought to be secreted by the bovine mammary epithelial cells （BMECs）. The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones. Results： According to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin （DIP） to induce a lactogenic differentiation. Quantitative polymerase chain reaction （qPCR） results showed that the expressions of miR-21-Sp （P = 0.005）, miR-26a （P = 0.016）, and miR-320a （P = 0.011） were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a （P-- 0.017） in the cell culture medium were lower in the DiP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture （P = 0.018）. The medium-to-cell expression ratios of miR- 103 （P = 0.025）, miR-148a （P 〈 0.001）, and miR-223 （P = 0.013） were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development. Conclusion： The results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.