Bovine Cells Research Articles

Chromium and Methionine Effects on Amino Acid Uptake, Gene Expression, and Protein Abundance in Bovine Satellite Cells

The objective of the study was to evaluate the effects of chromium (Cr) and methionine on myogenic gene expression, GLUT4 and insulin receptor (IR) synthesis, and amino acid uptake in differentiated bovine satellite cells. Cells were treated with 0, 1, or 10 μM of chromium acetate (CrAc) and 0, 1, or 10 mg/mL of methionine, with cells utilized for the amino acid assay receiving 0.1 μM instead of 1 μM of CrAc. The addition of 10 μM of Cr tended to downregulate AMPKα mRNA gene expression (P < 0.1). Myogenin was impacted by methionine and CrAc with high doses and low doses upregulating expression (P < 0.01). The ratio and protein levels of AMPKα to pAMPKα, GLUT4, and IR abundance were not affected (P > 0.1). Increase in time decreased concentrations of leucine, isoleucine, threonine, methionine, phenylalanine, glutamine, ornithine, lysine, and cysteine (P < 0.01) in conditioned media. Over time, alanine increased (P < 0.01). Methionine inclusion increased alanine, proline, glutamic acid, ornithine, and tryptophan concentrations, as well as decreased leucine, isoleucine, and cysteine (P < 0.01). CrAc only affected glutamine and tryptophan, which both increased with CrAc dose, whereas alanine decreased (P < 0.01). Methionine decreased over time within the 10 mg/mL group (P < 0.01). Tryptophan was generally unaffected but decreased in the 0 mg/mL group at 48 h. Cysteine decreased with a decreased dosage of CrAc at 24 h (P < 0.01). Lysine was generally unaffected by this interaction (P > 0.1), with the exception of the 0.1 μM treatment group at 24 h being higher (P < 0.01). CrAc affected the uptake of alanine, glutamine, and tryptophan (P < 0.01). Methionine inclusion increased alanine, proline, glutamic acid, ornithine, and tryptophan, as well as decreased leucine, isoleucine, and cystine (P < 0.01). Increased time saw a significant decrease in leucine, isoleucine, threonine, methionine, phenylalanine, glutamine, ornithine, lysine, and cystine (P < 0.01) and an increase in alanine (P < 0.01).

Molecular cloning of TPM3 gene in qinchuan cattle and its effect on myoblast proliferation and differentiation

Tropomyosin 3 (TPM3) plays a significant role as a regulatory protein in muscle contraction, affecting the growth and development of skeletal muscles. Despite its importance, limited research has been conducted to investigate the influence of TPM3 on bovine skeletal muscle development. Therefore, this study revealed the role of TPM3 in bovine myoblast growth and development. This research involved conducting a thorough examination of the Qinchuan cattle TPM3 gene using bioinformatics tools to examine its sequence and structural characteristics. Furthermore, TPM3 expression was evaluated in various bovine tissues and cells using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the coding region of TPM3 spans 855 bp, with the 161st base being the T base, encoding a protein with 284 amino acids and 19 phosphorylation sites. This protein demonstrated high conservation across species while displaying a predominant α-helix secondary structure despite being an unstable acidic protein. Notably, a noticeable increase in TPM3 expression was observed in the longissimus dorsi muscle and myocardium of calves and adult cattle. Expression patterns varied during different stages of myoblast differentiation. Functional studies that involved interference with TPM3 in Qinchuan cattle myoblasts revealed a very significantly decrease in S-phase cell numbers and EdU-positive staining (P < 0.01), and disrupted myotube morphology. Moreover, interference with TPM3 resulted in significantly (P < 0.05) or highly significantly (P < 0.01) decreased mRNA and protein levels of key proliferation and differentiation markers, indicating its role in the modulation of myoblast behavior. These findings suggest that TPM3 plays an essential role in bovine skeletal muscle growth by influencing myoblast proliferation and differentiation. This study provides a foundation for further exploration into the mechanisms underlying TPM3-mediated regulation of bovine muscle development and provides valuable insights that could guide future research directions as well as potential applications for livestock breeding and addressing muscle-related disorders.

Bta-miR-181d and Bta-miR-196a mediated proliferation, differentiation, and apoptosis in Bovine Myogenic Cells.

Skeletal muscle is an important component of livestock and poultry organisms. The proliferation and differentiation of myoblasts are highly coordinated processes, which rely on the regulation of miRNA. MiRNAs are widely present in organisms and play roles in various biological processes, including cell proliferation, differentiation, and apoptosis. MiR-181d and miR-196a, identified as tumor suppressors, have been found to be involved in cell proliferation, apoptosis, directed differentiation, and cancer cell invasion. However, their role in beef cattle skeletal muscle metabolism remains unclear. In this study, we discovered that overexpression of bta-miR-181d and bta-miR-196a in Qinchuan cattle myoblasts inhibited proliferation and apoptosis while promoting myogenic differentiation through EDU staining, flow cytometry analysis, immunofluorescence staining, and Western blotting. RNA-seq analysis of differential gene expression revealed that after overexpression of bta-miR-181d and bta-miR-196a, the differentially expressed genes were mainly enriched in the PI3K-Akt and MAPK signaling pathways. Furthermore，the phosphorylation levels of key proteins p-AKT in the PI3K signaling pathway and p-MAPK in the MAPK signaling pathway were significantly decreased after overexpression of bta-miR-181d and bta-miR-196a. Overall，this study provides preliminary evidence that bta-miR-181d and bta-miR-196a may regulate proliferation，apoptosis，and differentiation processes in Qinchuan cattle myoblasts by affecting the phosphorylation status of key proteins in PI3K-Akt and MAPK-ERK signaling pathways.

Inhibitory Effect of Puerarin on Lipopolysaccharide-triggered Inflammatory Responses of Bovine Kidney Cells.

Puerarin (Pue), a flavonoid compound, possesses cytoprotective effects and LPS has been reported to induce renal inflammatory injury in bovine. However, whether Pue inhibits lipopolysaccharide (LPS)-induced inflammatory damage of bovine kidney cells remains unknown. Based on an in vitro model with Madin-Darby bovine kidney (MDBK) cell line, it has found that Pue attenuated LPS-induced damage of MDBK cells, as evidenced by cell viability and lactic dehydrogenase (LDH) release rescued by Pue (P < 0.05). Additionally, the real-time quantitative PCR (qPCR) and enzyme linked immunosorbent assay (ELISA) showed that LPS elevated the levels of pro-inflammatory factors interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α, which was reversed by pretreatment of Pue (P < 0.05). Besides, Pue reduced the expression of Toll like receptor 4 (TLR4) and phosphorylated nuclear factor kappa B (p-NF-κB) of LPS-exposed MDBK cells (P < 0.05). Collectively, these results showed that Pue suppresses LPS-evoked inflammatory damage of bovine kidney cells, suggesting Pue a potential compound for intervention of bovine inflammation.

Nicotinamide Supplementation Mitigates Oxidative Injury of Bovine Intestinal Epithelial Cells through Autophagy Modulation.

The small intestine is important to the digestion and absorption of rumen undegradable nutrients, as well as the barrier functionality and immunological responses in ruminants. Oxidative stress induces a spectrum of pathophysiological symptoms and nutritional deficits, causing various gastrointestinal ailments. Previous studies have shown that nicotinamide (NAM) has antioxidant properties, but the potential mechanism has not been elucidated. The aim of this study was to explore the effects of NAM on hydrogen peroxide (H2O2)-induced oxidative injury in bovine intestinal epithelial cells (BIECs) and its potential mechanism. The results showed that NAM increased the cell viability and total antioxidant capacity (T-AOC) and decreased the release of lactate dehydrogenase (LDH) in BIECs challenged by H2O2. The NAM exhibited increased expression of catalase, superoxide dismutase 2, and tight junction proteins. The expression of autophagy-related proteins was increased in BIECs challenged by H2O2, and NAM significantly decreased the expression of autophagy-related proteins. When an autophagy-specific inhibitor was used, the oxidative injury in BIECs was not alleviated by NAM, and the T-AOC and the release of LDH were not affected. Collectively, these results indicated that NAM could alleviate oxidative injury in BIECs by enhancing antioxidant capacity and increasing the expression of tight junction proteins, and autophagy played a crucial role in the alleviation.

Inactivation of Myostatin Delays Senescence via TREX1-SASP in Bovine Skeletal Muscle Cells.

The myostatin (MSTN) gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between MSTN and age-related diseases. It is unclear how MSTN and age-associated muscle loss in other animals are related. In this study, we utilized MSTN gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to MSTN and muscle cell senescence. The expression of MSTN was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, MSTN was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that MSTN influenced three prime repair exonuclease 1 (TREX1) expression through the SMAD2/3 complex. The downregulation of MSTN contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of MSTN in cell senescence in large animals.

Exosomes-mediated transmission of standard bovine viral diarrhea strain OregonC24Va in bovine trophoblast cells

Bovine viral diarrhoea virus (BVDV) can infect cows on days 30-110 of gestation and crossing the placental barrier, resulting in persistently infected (PI) and causing significant economic losses to dairy farming. Bovine placental trophoblast cells (BTCs) are the major cells in the early chorionic tissue of the placenta and play important roles in placental resistance to viral transmission. In this study, we have confirmed that BTCs is among a groups of cell types those could be infected by BVDV in vivo, and BVDV infection stimulates the autophagic responses in BTCs and promotes the release of exosomes. Meanwhile, the exosomes derived from BTCs can be used by BVDV to spread between placental trophoblast cells, and this mode of transmission cannot be blocked by antibodies against the BVDV E2 protein, whereas the replication and spread of BVDV in BTCs can be blocked by inhibiting autophagy and exosomogenesis. Our study provides a theoretical and practical basis for scientific prediction and intervention of reproductive disorders caused by BVDV infection in cows of different gestation periods from a novel perspective.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line via impairing mTOR-mediated autophagy

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 μM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy.

Protective Effects of Growth Hormone-Releasing Hormone Antagonists in Interferon-γ-induced Endothelial Barrier Dysfunction

Growth Hormone - Releasing Hormone (GHRH) regulates the secretion of Growth Hormone from the anterior pituitary gland. An emerging body of evidence suggests that the activities of that neuropeptide are not limited to the GH /Insulin-like Growth Factor 1 axis, but expand towards the mediation of inflammatory processes. GHRH antagonists (GHRHAnt) were developed to oppose the activities of GHRH in malignancies, and have been associated with strong anti-inflammatory and anti-oxidative effects in a diverse variety of tissues, including the lungs. In the present study we report that GHRHAnt oppose interferon gamma (IFN-γ)-induced paracellular hyperpermeability and reactive oxygen species generation in bovine pulmonary endothelial cells. Moreover, those peptides suppress the activation of IFN-γ-triggered signal transducer and activator of transcription 3, cofilin and extracellular signal - regulated kinase 1/2. Our observations substantiate previous findings on the protective effects of GHRHAnt in endothelial inflammation, and suggest their potential application in disorders related to barrier dysfunction. Dr. Barabutis' research is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P2O GM103424-21. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries

Estrus detection of PE goats using the ASM application and confirmed by vagina cytology

The estrus cycle is the initial stage of increasing the milk production of PE goats. Estrus detection in PE goats is generally done manually by looking for signs of sound, restlessness, and aggression. The use of voice recording applications to detect estrus has been carried out, one of which is ASM. However, to increase the accuracy of detection based on ASM audio-visual, it is necessary to confirm the cytological status of bovine vaginal cells. This study aimed to detect estrus using audio-visual and vaginal cytology of goats. Eight female PE goats were observed on small breeders for their reproductive cycle, behavior, vocalization with ASM, and cell cytology sampling through vaginal mucus. They are processing data using Spearman rank correlation (SD). The results show aggressive behavior, frequent voices, and signs of clear discharge, and the tail is often moved. ASM detected the sound of goats in estrus at 430.0 Hz – 494.3 Hz and confirmed the superficial cell status, which predominated in PE goats. It can be concluded that the audio of goats in the 472 Hz range recorded using the ASM application can be used as a reference that goats are in estrus and ready to be mated.

Microdiversity of Salmonella Kentucky During Long-Term Colonization of a Dairy Herd.

Salmonella enterica subsp. enterica serovar Kentucky was repeatedly isolated from a commercial dairy herd that was enrolled in a longitudinal study where feces of asymptomatic dairy cattle were sampled intensively over an 8-year period. The genomes of 5 Salmonella Kentucky isolates recovered from the farm 2 years before the onset of the long-term colonization event and 13 isolates collected during the period of endemicity were sequenced. A phylogenetic analysis inferred that the Salmonella Kentucky strains from the farm were distinct from poultry strains collected from the same region, and three subclades (K, A1, and A2) were identified among the farm isolates, each appearing at different times during the study. Based on the phylogenetic analysis, three separate lineages of highly similar Salmonella Kentucky were present in succession on the farm. Genomic heterogeneity between the clades helped identify regions, most notably transcriptional regulators, of the Salmonella Kentucky genome that may be involved in competition among highly similar strains. Notably, a region annotated as a hemolysin expression modulating protein (Hha) was identified in a putative plasmid region of strains that colonized a large portion of cows in the herd, suggesting that it may play a role in asymptomatic persistence within the bovine intestine. A cell culture assay of isolates from the three clades with bovine epithelial cells demonstrated a trend of decreased invasiveness of Salmonella Kentucky isolates over time, suggesting that clade-specific interactions with the animals on the farm may have played a role in the dynamics of strain succession. Results of this analysis further demonstrate an underappreciated level of genomic diversity within strains of the same Salmonella serovar, particularly those isolated during a long-term period of asymptomatic colonization within a single dairy herd.

Saturated fatty acids inhibit unsaturated fatty acid induced glucose uptake involving GLUT10 and aerobic glycolysis in bovine granulosa cells

Fatty acids have been shown to modulate glucose metabolism in vitro and in vivo. However, there is still a need for substantial evidence and mechanistic understanding in many cell types whether both saturated and unsaturated fatty acids (SFAs and UFAs) pose a similar effect and, if not, what determines the net effect of fatty acid mixes on glucose metabolism. In the present study, we asked these questions by treating granulosa cells (GCs) with the most abundant non-esterified fatty acid species in bovine follicular fluid. Results revealed that oleic and alpha-linolenic acids (UFAs) significantly increased glucose consumption compared to palmitic and stearic acids (SFAs). A significant increase in lactate production, extracellular acidification rate, and decreased mitochondrial activity indicate glucose channeling through aerobic glycolysis in UFA treated GCs. We show that insulin independent glucose transporter GLUT10 is essential for UFA driven glucose consumption, and the induction of AKT and ERK signaling pathways necessary for GLUT10 expression. To mimic the physiological conditions, we co-treated GCs with mixes of SFAs and UFAs. Interestingly, co-treatments abolished the UFA induced glucose uptake and metabolism by inhibiting AKT and ERK phosphorylation and GLUT10 expression. These data suggest that the net effect of fatty acid induced glucose uptake in GCs is determined by SFAs under physiological conditions.

Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment—with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Supplementation of Methionine Dipeptide Enhances the Milking Performance of Lactating Dairy Cows.

Methionine dipeptide (Met-Met) could improve milk protein synthesis in bovine epithelia mammary cells and lactating mice, while the effects of Met-Met on lactation performance, rumen fermentation and microbiota profile in lactating dairy cows have not been explored. For this reason, 60 Chinese lactating Holstein cows were allocated into three treatment groups: control group (CON), 6 g/d methionine dipeptide group (MM), and 6.12 g/d rumen-protected methionine dipeptide group (RPMM). The experiment lasted for 10 weeks to monitor lactation performance, plasma amino acid profile and rumen fermentation parameters and microbiota profile. Results showed that MM increased the energy-corrected milk (ECM), and RPMM increased both milk yield and ECM (p < 0.05). The milk protein concentration and yield were increased by MM and RPMM (p < 0.05). The rumen fermentation showed that RPMM increased total volatile fatty acids, acetate and valerate concentrations (p < 0.05). The relative abundance of Firmicutes, including Succiniclasticum, Selenomonas and Clostridium_XlVa, were enriched and the Prevotella was decreased by RPMM (p < 0.05). In summary, daily supplementing with 6 g of MM or RPMM in lactating dairy cows could improve milk yield and both percentage and yield of milk protein, and RPMM benefited the rumen fermentation and altered the bacterial composition. These results provided the first evidence that Met-Met supplementation can improve lactation performance of dairy cows.

Cultivation of Bovine Mesenchymal Stem Cells on Plant-Based Scaffolds in a Macrofluidic Single-Use Bioreactor for Cultured Meat.

Reducing production costs, known as scaling, is a significant obstacle in the advancement of cultivated meat. The cultivation process hinges on several key components, e.g., cells, media, scaffolds, and bioreactors. This study demonstrates an innovative approach, departing from traditional stainless steel or glass bioreactors, by integrating food-grade plant-based scaffolds and thermoplastic film bioreactors. While thermoplastic films are commonly used for constructing fluidic systems, conventional welding methods are cost-prohibitive and lack rapid prototyping capabilities, thus inflating research and development expenses. The developed laser welding technique facilitates contamination-free and leakproof sealing of polyethylene films, enabling the efficient fabrication of macrofluidic systems with various designs and dimensions. By incorporating food-grade plant-based scaffolds, such as rice seeded with bovine mesenchymal stem cells, into these bioreactors, this study demonstrates sterile cell proliferation on scaffolds within macrofluidic systems. This approach not only reduces bioreactor prototyping and construction costs but also addresses the need for scalable solutions in both research and industrial settings. Integrating single-use bioreactors with minimal shear forces and incorporating macro carriers such as puffed rice may further enhance biomass production in a scaled-out model. The use of food-grade plant-based scaffolds aligns with sustainable practices in tissue engineering and cultured-meat production, emphasizing its suitability for diverse applications.

Production of Plant-Based, Film-Type Scaffolds Using Alginate and Corn Starch for the Culture of Bovine Myoblasts.

Natural scaffolds have been the cornerstone of tissue engineering for decades, providing ideal environments for cell growth within extracellular matrices. Previous studies have favored animal-derived materials, including collagen, gelatin, and laminin, owing to their superior effects in promoting cell attachment, proliferation, and differentiation compared to non-animal scaffolds, and used immortalized cell lines. However, for cultured meat production, non-animal-derived scaffolds with edible cells are preferred. Our study represents the first research to describe plant-derived, film-type scaffolds to overcome limitations associated with previously reported thick, gel-type scaffolds completely devoid of animal-derived materials. This approach has been employed to address the difficulties of fostering bovine muscle cell survival, migration, and differentiation in three-dimensional co-cultures. Primary bovine myoblasts from Bos Taurus Coreanae were harvested and seeded on alginate (Algi) or corn-derived alginate (AlgiC) scaffolds. Scaffold functionalities, including biocompatibility and the promotion of cell proliferation and differentiation, were evaluated using cell viability assays, immunofluorescence staining, and reverse transcription-quantitative polymerase chain reaction. Our results reveal a statistically significant 71.7% decrease in production time using film-type scaffolds relative to that for gel-type scaffolds, which can be maintained for up to 7 days. Film-type scaffolds enhanced initial cell attachment owing to their flatness and thinness relative to gel-type scaffolds. Algi and AlgiC film-type scaffolds both demonstrated low cytotoxicity over seven days of cell culture. Our findings indicated that PAX7 expression increased 16.5-fold in alginate scaffolds and 22.8-fold in AlgiC from day 1 to day 3. Moreover, at the differentiation stage on day 7, MHC expression was elevated 41.8-fold (Algi) and 32.7-fold (AlgiC), providing initial confirmation of the differentiation potential of bovine muscle cells. These findings suggest that both Algi and AlgiC film scaffolds are advantageous for cultured meat production.

Phenotypic and Functional Characterization of Bovine Adipose-Derived Mesenchymal Stromal Cells.

Mesenchymal stem cells (MSCs) are increasingly trialed in cellular therapy applications in humans. They can also be applied to treat a range of diseases in animals, particularly in cattle to combat inflammatory conditions and aging-associated degenerative disorders. We sought to demonstrate the feasibility of obtaining MSCs from adipose tissue and characterizing them using established assays. Bovine adipose MSCs (BvAdMSCs) were isolated using in-house optimized tissue digestion protocols and characterized by performing a colony formation assay, cell growth assessments, cell surface marker analysis by immunocytochemistry and flow cytometry, osteogenic and adipogenic differentiation, and secretion of indoleamine 2,3-dioxygenease (IDO). Our results demonstrate the feasibility of successful MSC isolation and culture expansion from bovine adipose tissues with characteristic features of colony formation, in vitro multilineage differentiation into osteogenic and adipogenic lineages, and cell surface marker expression of CD105, CD73, CD90, CD44, and CD166 with negative expression of CD45. BvAdMSCs secreted significant amounts of IDO with or without interferon-gamma stimulation, indicating ability for immunomodulation. We report a viable approach to obtaining autologous adipose-derived MSCs that can be applied as potential adjuvant cell therapy for tissue repair and regeneration in cattle. Our methodology can be utilized by veterinary cell therapy labs for preparing MSCs for disease management in cattle.

Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.