Bovine Cells Research Articles

Immortalized mammosphere-derived epithelial cells retain a bioactive secretome with antimicrobial, regenerative, and immunomodulatory properties.

The secretome of primary bovine mammosphere-derived epithelial cells (MDECs) has been shown to exert antimicrobial, regenerative, and immunomodulatory properties in vitro, which warrants its study as a potential biologic treatment with the potential to be translated to human medicine. Currently, the use of the MDEC secretome as a therapy is constrained by the limited life span of primary cell cultures and the decrease of secretome potency over cell passages. To address these limitations, early-passage bovine MDECs were immortalized using hTERT, a human telomerase reverse transcriptase. The primary and immortal MDECs were compared morphologically, transcriptomically, and phenotypically. The functional properties and proteomic profiles of the secretome of both cell lines were evaluated and compared. All experiments were performed with both low and high passage cell cultures. We confirmed through in vitro experiments that the secretome of immortalized MDECs, unlike that of primary cells, maintained antimicrobial and pro-migratory properties over passages, while pro-angiogenic effects of the secretome from both primary and immortalized MDECs were lost when the cells reached high passage. The secretome from primary and immortalized MDECs, at low and high passages exerted immunomodulatory effects on neutrophils in vitro. High passage immortalized MDECs retain a bioactive secretome with antimicrobial, regenerative, and immunomodulatory properties, suggesting they may serve as a consistent cell source for therapeutic use.

Analysis of Characteristics of Bovine-Derived Non-Enterotoxigenic Bacteroides fragilis and Validation of Potential Probiotic Effects

Bacteroides fragilis is a new generation of probiotics, and its probiotic effects on humans and some animals have been verified. However, research on B. fragilis in cattle is still lacking. In this study, 24 stool samples were collected from two large-scale cattle farms in Wuzhong, Ningxia, including 12 diarrheal and 12 normal stools. A non-toxigenic Bacteroides fragilis (NTBF) was isolated and identified by 16S rRNA high-throughput sequencing and named BF-1153; genome composition and genome functional analyses were carried out to reflect the biological characteristics of the BF-1153 strain. A cluster analysis of BF-1153 was performed using Mega X to explore its genetic relationship. In addition, Cell Counting Kit-8 (CCK8) was used to determine the toxic effects of the strain on human ileocecal colorectal adenocarcinoma cell line cells (HCT-8), Madin-Darby bovine kidney cells (MDBK), and intestinal porcine epithelial cells (IPECs). The results showed that BF-1153 conformed to the biological characteristics of B. fragilis. BF-1153 had no toxic effects on HCT-8, MDBK, and IPEC. Animal experiments have shown that BF-1153 has no toxic effects on healthy SPF Kunming mice. Notably, the supernatant of BF-1153 enhanced cell activity and promoted cell growth in all three cell lines. At the same time, a cluster analysis of the isolated strains showed that the BF-1153 strain belonged to the same branch as the B. fragilis strain 23212, and B. fragilis strain 22998. The results of the animal experiments showed that BF-1153 had a certain preventive effect on diarrhea symptoms in SPF Kunming mice caused by a bovine rotavirus (BRV). In summary, the strain BF-1153 isolated in this experiment is NTBF, which has no toxic effect on MDBK, HCT-8, and IPEC, and has obvious cell growth-promoting effects, especially on MDBK. BF-1153 promotes the growth and development of SPF Kunming mice when compared with the control group. At the same time, BF-1153 alleviated the diarrhea symptoms caused by BRV in SPF Kunming mice. Therefore, BF-1153 has the potential to be a probiotic for cattle.

Optimisation of cell fate determination for cultivated muscle differentiation.

Production of cultivated meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Although these formulations can drive myogenic differentiation levels comparable to serum-starvation-based protocols, the resulting cultures are often heterogeneous, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. To address this problem, we employed RNA sequencing to analyse heterogeneity in differentiating bovine satellite cells at single-nucleus resolution, identifying distinct cellular subpopulations including proliferative cells that fail to exit the cell cycle and quiescent 'reserve cells' that do not commit to myogenic differentiation. Our findings indicate that the MEK/ERK, NOTCH, and RXR pathways are active during the initial stages of myogenic cell fate determination, and by targeting these pathways, we can promote cell cycle exit while reducing reserve cell formation. This optimised medium formulation consistently yields fusion indices close to 100% in 2D culture. Furthermore, we demonstrate that these conditions enhance myotube formation and actomyosin accumulation in 3D bovine skeletal muscle constructs, providing proof of principle for the generation of highly differentiated cultivated muscle with excellent mimicry to traditional muscle.

Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells.

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis.

Transcriptome analysis identifies the NR4A subfamily involved in the alleviating effect of folic acid on mastitis induced by high concentration of Staphylococcus aureus lipoteichoic acid

BackgroundStaphylococcus aureus (S. aureus) mastitis results in economic losses during dairy production. Understanding the biological progression of bovine S. aureus mastitis is vital for its prevention. Lipoteichoic acid is a key virulence factor of S. aureus (aLTA), but the main biological pathways involved in its effect on bovine mammary epithetionallial cells (Mac-T) apoptosis and necrosis have not been fully explored. Folic acid (FA) has anti-inflammatory and anti-apoptotic effects. However, the role of FA in mediating the effects of aLTA on apoptosis and necrosis remains unknown.ResultsWe found that low concentration of aLTA inhibited apoptosis and necrosis and that high concentration promoted the apoptosis and necrosis of Mac-T. FA pretreatment alleviated high concentration of aLTA induced apoptosis. Through transcriptomic analysis, we found that nuclear receptor subfamily 4 group A (NR4A), which alters the expression of downstream genes involved in apoptosis, proliferation, and inflammation, decreased under stimulation with a low concentration of aLTA and increased under stimulation with a high concentration of aLTA. Under stimulation with a high concentration of aLTA, the expression of the NR4A subfamily could be inhibited by FA. The results showed that aLTA may affect apoptosis and necrosis through the NR4A subfamily by targeting genes involved in bacterial invasion of epithelial cells, the IL-17 signaling pathway, DNA replication, longevity regulation, the cell cycle, and tight junction pathways. We further found that the expression trends of NR4A1 and the target genes of the NR4A subfamily (PTGS2, ESPL1, MCM5, and BUB1B) in the blood of healthy cows (Healthy), subclinical mastitis cows (SCM), and SCM supplemented with FA (SCM_FA) were consistent with those observed at the cellular level in this study.ConclusionsOur study revealed that low and high concentrations of aLTA have opposite effects on apoptosis and necrosis of Mac-T and that FA can alleviate the apoptosis induced by high concentration of aLTA. Transcriptome analysis revealed that the NR4A subfamily play a role in the ability of FA to alleviate the apoptosis and necrosis induced by high concentration of aLTA.

Integrative analysis of whole genome bisulfite and transcriptome sequencing reveals the effect of sodium butyrate on DNA methylation in the differentiation of bovine skeletal muscle satellite cells

Butyric acid as a short-chain fatty acid (SCFA) is one of the key microbial metabolites of ruminants. Numerous studies indicate that butyrate is crucial in muscle growth and development, and plays an important molecular regulatory role mainly by inhibiting histone deacetylation. DNA methylation, a major epigenetic modification, is involved in cell differentiation. Butyrate, in addition to its role in acetylation modifications, can alter the DNA methylation status of cells. However, the impact of butyrate on the DNA methylation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. In this study, we developed a differentiation model of SMSCs and employed RNA sequencing (RNA-seq) alongside whole genome bisulfite sequencing (WGBS) to explore the effects of butyrate treatment on DNA methylation status and its relationship with gene expression. Treatment of SMSCs with sodium butyrate (NaB) at 1.0 mM for 2 days significantly inhibited the expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A) at the mRNA and protein levels while promoting the expression of demethylases (TET1, TET2, TET3) at mRNA levels. WGBS identified 4292 differentially methylated regions (DMRs), comprising 2294 hypermethylated and 1998 hypomethylated regions. These DMRs were significantly enriched in the MAPK, cAMP, and Wnt signaling pathways, all of which are implicated in myogenesis and development. Combining RNA-seq and WGBS data revealed a total of 130 overlapping genes, including MDFIC, CREBBP, DMD, LTBP2 and KLF4. These genes are predominantly involved in regulating the FoxO, MAPK, PI3K-Akt, and Wnt signaling pathways. This study provides new insights into the effects of butyrate-mediated DNA methylation on SMSC development and enhances our understanding of butyrate as an epigenetic modifier beyond its role in acetylation.

NR1D1 activation alleviates inflammatory response through inhibition of IL-6 expression in bovine endometrial epithelial cells

NR1D1 activation alleviates inflammatory response through inhibition of IL-6 expression in bovine endometrial epithelial cells

Fiber-based Biomaterial Scaffolds for Cell Support towards the Production of Cultivated Meat

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track. The fibers supported cell adhesion, proliferation, and guided muscle cell orientation, facilitating myotube formation per differentiation. A progressive increase in biomass was also documented. Interestingly, iBSC proliferation was enhanced with coatings of recombinant proteins while C2C12 cells showed minimal response. Thus, both cotton and silk yarns were suitable as fiber-based scaffolds towards cell supportive goals, suggesting an alternative path toward structured protein-rich foods via this initial stage of textile engineering for food. Biomass prediction models were generated, enabling forecasts of cell growth and maturation across various scaffold conditions and cell types. This capability enhances the precision of the cultivation process towards an engineering approach, building on the inherent benefits of hierarchical muscle tissue structure, but here via textile engineering with these initial muscle-coated edible fibers. Further, the approach offers to reduce costs by optimizing cultivation time and media needs. These approaches are part of a foundation for future scalable and sustainable cultivated meat production. Statement of SignificanceThis research investigates the use of one-dimensional fiber-based scaffolds for cultivated meat production, contributing to advancements in cellular agriculture. It introduces a method to measure changes in biomass and scaffold degradation throughout the cultivation process. Additionally, our development of biomass prediction models improves the precision and predictability of cultivated meat production. This research not only aids in scaling up cultivated meats but also enhances the use of textile engineering techniques in tissue engineering, paving the way for producing complex, three-dimensional meat structures more sustainably.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1%, fibroblasts: 33.1%, control: 27.3%). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(-). In Expt. 3, addition of 10% fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2%; 10% FCS, 43.4%; 1% ADSC lysate, 34.2%; 10% ADSC lysate, 48.1%). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10% addition, corresponding to 1 x 105 cells/mL, is as effective as 10% FCS without a decrease in cryotolerance due to the increased lipid content.

Hybrid Wavelet‐Deep Learning Framework for Fluorescence Microscopy Images Enhancement

ABSTRACTFluorescence microscopy is a powerful tool for visualizing cellular structures, but it faces challenges such as noise, low contrast, and autofluorescence that can hinder accurate image analysis. To address these limitations, we propose a novel hybrid image enhancement method that combines wavelet‐based denoising, linear contrast enhancement, and convolutional neural network‐based autofluorescence correction. Our automated method employs Haar wavelet transform for noise reduction and a series of adaptive linear transformations for pixel value adjustment, effectively enhancing image quality while preserving crucial details. Furthermore, we introduce a semantic segmentation approach using CNNs to identify and correct autofluorescence in cellular aggregates, enabling targeted mitigation of unwanted background signals. We validate our method using quantitative metrics, such as signal‐to‐noise ratio (SNR) and peak signal‐to‐noise ratio (PSNR), demonstrating superior performance compared to both mathematical and deep learning‐based techniques. Our method achieves an average SNR improvement of 8.5 dB and a PSNR increase of 4.2 dB compared with the original images, outperforming state‐of‐the‐art methods such as BM3D and CLAHE. Extensive testing on diverse datasets, including publicly available human‐derived cardiosphere and fluorescence microscopy images of bovine endothelial cells stained for mitochondria and actin filaments, showcases the flexibility and robustness of our approach across various acquisition conditions and artifacts. The proposed method significantly improves fluorescence microscopy image quality, facilitating more accurate and reliable analysis of cellular structures and processes, with potential applications in biomedical research and clinical diagnostics.

ALKBH5 Reduces BMP15 mRNA Stability and Regulates Bovine Puberty Initiation Through an m6A-Dependent Pathway.

The timing of puberty significantly influences subsequent reproductive performance in cattle. N6-methyladenosine (m6A) is a key epigenetic modification involved in the regulation of pubertal onset. However, limited research has investigated alterations in m6A methylation within the hypothalamic-pituitary-ovarian (HPO) axis during the onset of puberty. In this study, combined analysis of methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-seq) is used to describe the overall modification pattern of m6A in the HPO axis, while GSEA, KEGG, and GO analyses are used to describe the enrichment pathways of differentially expressed genes and differentially methylated genes. The m6A modifications of the differential genes KL, IGSF10, PAPPA2, and BMP15 and the pathways of cell adhesion molecules (CAMs), TGF-β, cell cycle, and steroid hormone synthesis may play roles in regulating the function of the HPO axis tissue during pubertal transition. Notably, BMP15's m6A modification depends on the action of the demethylase ALKBH5, which is recognized by the reader protein YTHDF2, promoting bovine granulosa cell proliferation, steroid production, and estrogen secretion. This study reveals for the first time the modification mechanism of BMP15 m6A during the initiation of bovine puberty, which will provide useful information for improving the reproductive efficiency of Chinese beef cattle.

Landscape transcriptomic analysis of bovine follicular cells during key phases of ovarian follicular development

BackgroundThere are many gaps in our understanding of the mechanisms involved in ovarian follicular development in cattle, particularly regarding follicular deviation, acquisition of ovulatory capacity, and preovulatory changes. Molecular evaluations of ovarian follicular cells during follicular development in cattle, especially serial transcriptomic analyses across key growth phases, have not been reported. This study aims to address this gap by analyzing gene expression using RNA-seq in granulosa and antral cells recovered from ovarian follicular fluid during critical phases of ovarian follicular development in Holstein cows.ResultsIntegrated analysis of gene ontology (GO), gene set enrichment (GSEA), protein–protein interaction (PPI), and gene topology identified that differentially expressed genes (DEGs) in the largest ovarian follicles at deviation (Dev) were primarily involved in FSH-negative feedback, steroidogenesis, cell proliferation, apoptosis, and the prevention of early follicle rupture. In contrast, DEGs in the second largest follicles (DevF2) were mainly related to loss of cell viability, apoptosis, and immune cell invasion. In the dominant (PostDev) and preovulatory (PreOv) follicles, DEGs were associated with vascular changes and inflammatory responses.ConclusionsThe transcriptome of ovarian follicular fluid cells had a predominance of granulosa cells in the dominant follicle at deviation, with upregulation of genes involved in cell viability, steroidogenesis, and apoptosis prevention, whereas in the non-selected follicle there was upregulation of cell death-related transcripts. Immune cell transcripts increased significantly after deviation, particularly in preovulatory follicles, indicating strong intrafollicular chemotactic activity. We inferred that immune cell invasion occurred despite an intact basal lamina, contributing to follicular maturation.Graphical

Discovery of Mycobacterium avium subsp. paratuberculosis Lytic Phages with Extensive Host Range Across Rapid- and Slow-Growing Pathogenic Mycobacterial Species

Background/Objectives: Developing interventions for Johne’s disease, which focuses on controlling Mycobacterium avium subsp. paratuberculosis (MAP) in contaminated environments by treating infected cows and preventing transmission from diseased animals, is a critical priority. Bacteriophage (phage) therapy, an emerging biological intervention, offers a promising alternative for the treatment and management of MAP infections. Methods: In this study, we generated an MAP-specific lytic phage library aimed at characterizing the therapeutic potential of phages under environmental and biological conditions that mimic those encountered in infected cattle such as ruminal fluid, milk, colostrum, and the bovine intestinal epithelium, a key site of MAP colonization and, later, transmission. Results: Our library contains a diverse collection of phages that have demonstrated robust lytic activity against MAP. The host range of these phages was thoroughly assessed, revealing that several isolates produce clear plaques on a range of MAP strains, as well as other pathogenic non-tuberculous mycobacterial (NTM) species and M. tuberculosis strains. This broad host range expands the therapeutic potential of the phage collection, positioning it as a potential cross-species antimicrobial tool. In vitro tests under conditions replicating the rumen, milk, and colostrum environments show that selected phages maintain stability and lytic efficacy, even in the presence of complex biological fluids. Furthermore, a subset of these phages was capable of preventing MAP colonization and invasion in cultured bovine epithelial cells, suggesting their potential for direct prophylactic application in cattle. Conclusions. Our collection of MAP phages represents a valuable source that can be developed into probiotic-like preparations, offering a cost-effective solution for prophylaxis and control of Johne’s disease.

Dynamically cultured, differentiated bovine adipose-derived stem cell spheroids as building blocks for biofabricating cultured fat

Cultured or cultivated meat, animal muscle, and fat tissue grown in vitro, could transform the global meat market, reducing animal suffering while using fewer resources than traditional meat production and no antimicrobials at all. To ensure the appeal of cultured meat to future customers, cultured fat is essential for achieving desired mouthfeel, taste, and texture, especially in beef. In this work we show the establishment of primary bovine adipose-derived stem cell spheroids in static and dynamic suspension culture. Spheroids are successfully differentiated using a single-step protocol. Differentiated spheroids from dynamic cultures maintain stability and viability during 3D bioprinting in edible gellan gum. Also, the fatty acid composition of differentiated spheroids is significantly different from control spheroids. The cells are cultured antibiotic-free to minimize the use of harmful substances. This work presents a stable and bioprintable building block for cultured fat with a high cell density in a 3D dynamic cell culture system.

Progesterone increases metabolism via the pentose phosphate pathway in bovine uterine epithelial cells.

During early pregnancy, glucose is essential for the uterine epithelium and the developing embryo. In cows, progesterone increases the secretion of glucose into the uterine lumen. The uterine epithelium can convert glucose to fructose, but other fates of glucose in the uterine epithelium have been sparsely investigated. Therefore, our objective was to investigate how progesterone influences glucose metabolism in immortalized bovine uterine epithelial (BUTE) cells. BUTE cells were grown to 80% confluence and treated with vehicle (DMSO) or 10 µM progesterone for 24h. Cells were collected and analyzed. Immunohistochemistry was performed on endometrial samples collected from the bovine endometrium on days 1 and 11 of the reproductive cycle. Progesterone treatment increased glucose consumption of BUTE cells. RNAseq identified 3,072 genes regulated by progesterone. KEGG analysis indicated that progesterone altered genes associated with metabolic pathways and glutathione metabolism. Manually examining genes unique to specific glucose metabolic pathways identified an increase in the rate-limiting enzyme in the pentose phosphate pathway-glucose-6-phosphate dehydrogenase. Functionally, a major product of the pentose phosphate pathway is NADPH, and progesterone treatment increased NADPH levels in BUTE cells. In cows, immunohistochemistry confirmed that glucose-6-phosphate dehydrogenase levels were higher in the uterine epithelium in the luteal phase when progesterone concentrations are high. Progesterone increased glucose-6-phosphate dehydrogenase expression and metabolism via the pentose phosphate pathway in the bovine uterine epithelium. This metabolism could provide substrates for cell proliferation, molecules to be secreted into the uterine lumen, or maintain reduction/oxidation balance in the uterine epithelium.

Vitamin D3 mediates autophagy to alleviate inflammatory responses in bovine endometrial epithelial cells and organoids via the PI3K/AKT/mTOR pathway

As a natural anti-inflammatory agent, it remains unclear whether the anti-inflammatory effects of VD3 (1,25 dihydroxyvitamin D3) are related to autophagy. This study investigates the impact of VD3 on inflammatory injury, autophagy, oxidative stress, and apoptosis in bovine endometrial epithelial cells (BEECs) and bovine endometrial organoids (BEOs). BEECs and BEOs were treated with LPS (1 μg/ml) for 24 hours, followed by treatment with LPS+VD3 (50 ng/ml) for 6 hours. Cell viability was assessed using the CCK8 assay. The expression levels of inflammatory factors (IL-1β, IL-6, TLR4, NF-κB), autophagy markers (Beclin-1, ATG5, ATG7, p62), and components of the PI3K/AKT/mTOR pathway (PI3K, AKT, and mTOR) were quantified using qRT-PCR and Western blot analyses. LC3B expression was detected by immunofluorescence, and the apoptosis rate was assessed using Annexin V. The results demonstrated a significant decrease in the expression levels of IL-1β, IL-6, TLR4, and NF-κB, along with a notable increase in the activity of CAT and SOD2 in the LPS+VD3 group (P < 0.05). The expression of autophagy-related factors was significantly increased, whereas the expression of signaling pathway factors was decreased in the LPS+VD3 group (P < 0.05). Additionally, apoptosis was significantly alleviated in the LPS+VD3 group (P < 0.05). Collectively, these findings indicate that VD3 modulates autophagy, attenuates oxidative stress and inflammatory damage in BEECs and BEOs, and inhibits LPS-induced apoptosis via the PI3K/AKT/mTOR pathway.

Study on the feasibility of using livestock blood as a fetal bovine serum substitute for cultured meat.

Fetal bovine serum (FBS) accounts for the largest portion of the cost of cultured meat production or cell culture experiments and is highly controversial in terms of animal welfare because it is taken from the fetus of a pregnant cow during slaughtering. Nevertheless, FBS is the most important supplement in the cell culture manufacturing process. This study aimed to develop an FBS substitute from slaughterhouse waste blood to reduce the cost of FBS in cultured meat production through various experiments. Our study successfully demonstrated that adult livestock blood obtained from slaughterhouses can effectively replace FBS. Our substitute, when cultured with bovine myosatellite cells, demonstrated cell growth that was either equivalent to or superior to that of commercial FBS. In the process of muscle generation through differentiation, the substitutes from bovine and chicken formed 70%-75% more bovine muscle compared to the control group using FBS. Furthermore, using the FBS substitute can reduce cell culture costs by approximately 61% compared to using commercial FBS. Therefore, the groundbreaking FBS substitute will not only contribute to the development of technology to mass-produce cultured meat using livestock byproducts but will also lower the production cost of media for experimental cell culture or vaccine production.

Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cellnuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

A20 Alleviates the Inflammatory Response in Bovine Endometrial Epithelial Cells by Promoting Autophagy.

Endometritis represents a prevalent condition in perinatal dairy cows. Bovine endometrial epithelial cells (BEECs), as the primary interface between cavity and the external environment, are particularly vulnerable to infection by pathogenic bacteria following parturition. A20 is essential for regulating inflammation and modulating immune responses. Nevertheless, the exact role of A20 in the BEECs in response to inflammatory response is not fully understood. An endometritis model infected by Escherichia coli (E. coli) in vivo and a BEECs inflammation model induced with lipopolysaccharide (LPS) in vitro were built to investigate the function and governing mechanisms of A20 in endometritis. The results showed that infection with E. coli resulted in endometrial damage, inflammatory cell infiltration, and upregulation of inflammatory factors in dairy cows. Furthermore, A20 expression was upregulated in the endometrium of cows with endometritis and in BEECs following LPS stimulation. A20 overexpression attenuated the level of proinflammatory cytokines in LPS-stimulated BEECs; conversely, A20 knockdown lead to an exacerbated response to LPS stimulation. The overexpression of A20 was shown to activate autophagy and suppress the NF-κB signaling pathway in LPS-stimulated BEECs. However, blocking autophagy with chloroquine notably attenuated the anti-inflammatory effect of A20, leading to the activation of the NF-κB signaling pathway. In summary, the study demonstrated that A20's suppression of inflammation in LPS-stimulated BEECs is associated with the activation of autophagy. Therefore, the A20 protein showed potential as a novel treatment focus for managing endometritis in dairy cows.

Mechanism of collagen type IV regulation by focal adhesion kinase during retained fetal membranes in dairy cows

Retained fetal membranes (RFM) is an important reproductive disease in dairy cows, caused by maternal and fetal placental tissue adhesion. The main collagen in maternal and fetal placenta tissues is collagen type IV (COL-IV) and its breakdown is the key to placental expulsion. Focal adhesion kinase (FAK) has been shown to regulate the hydrolysis of Col-IV by affecting the activity of MMP-2 and MMP-9 activity, but the regulation of the mechanisms involved in placenta expulsion in dairy cows after postpartum are still unclear. The aim of this study was to investigate the pathogenic mechanism of RFM by studying the relationship between the FAK signaling pathway and COL-IV regulation. Maternal placental tissues were collected from six healthy and six cows with RFM of similar age, parity, body condition and milk yield at 12 h postpartum. In vitro experiments were performed on bovine endometrial epithelial cells from three groups including a FAK inhibitor group, a FAK activator group and a control group without FAK inhibitor and activator. The abundance of molecules involved in the FAK signaling pathway and COL-IV was detected by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The immunohistochemical results showed that the key molecules of FAK signaling pathway FAK, Src, MMP-2 and MMP-9 and Col-IV were expressed in placental tissues. The expression level of FAK, Src, MMP-2, and MMP-9 were significantly down-regulated (P < 0.05) and the abundances of COL-IV were significantly up-regulated (P < 0.05) in maternal placental tissues of RFM cows compared with healthy cows. In the FAK inhibitor treatment group, the relative expression levels of FAK and other related proteins were significantly down-regulated (P < 0.05) and the relative expression levels of COL-IV were significantly up-regulated (P < 0.05) with the results of the FAK activation group the opposite. These results indicated that FAK in maternal endometrial epithelial cells could regulate the hydrolysis process of Col-IV through the expression of key factors of signaling pathways and promote collagen hydrolysis, which in turn facilitated the process of postpartum placenta expulsion in dairy cows.