Bovine Muscle Research Articles

Exploration of the growth pattern and stemness maintenance of bovine muscle stem cells in vitro culture

Cultured meat technology has rapidly advanced in recent years, offering promising prospects. However, a major challenge in industrializing this process is the procurement and expansion of seed cells, which frequently lose their proliferative potential during in vitro culture. In this study, high-purity bovine muscle stem cells were isolated using fluorescence-activated cell sorting, achieving a pax7 positivity rate of 90%. These cells demonstrated standard proliferation and differentiation capabilities in vitro. However, long-term culture led to decreased proliferation and differentiation capacities, as well as an accumulation of reactive oxygen species. To restore cellular function, we investigated the effects of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a powerful antioxidant. Treatment with 50 μM Trolox effectively reversed the decline in bovine muscle stem cells proliferation, decreased reactive oxygen species levels, and preserved cell stemness during extended culture. These results provide a foundation for muscle seed cells acquisition and preservation, improving their viability and functionality in vitro.

Effect of IGF1 on Myogenic Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells Through PI3K/AKT Signaling Pathway

Effect of IGF1 on Myogenic Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells Through PI3K/AKT Signaling Pathway

Influence of Bovine Myosin Heavy Chain Isoforms and Muscle Fiber Cross-Sectional Are<em>a o</em>n the Eating Quality and Connective Tissue Characteristics of 11 Different Beef Muscles

The objective of this study was to determine the impact of muscle fiber type, cross-sectional area (CSA), and diameter on the eating quality of 11 different beef muscles. Eleven different beef muscles were utilized in 2 separate studies. In the 2 studies, triceps brachii, rectus abdominus, rectus femoris, supraspinatus, gluteus medias, pectoralis profundi, semitendinosus, longissimus thoracis, longissimus lumborum, tensor fascia latae, and gastrocnemius were collected from 10 USDA Choice carcasses (N = 110). To determine muscle fiber type, myofibrillar proteins were extracted and separated via gel electrophoresis and immunoblot, while muscle fiber CSA and diameter were determined using a dystrophin antibody stain via fluorescence microscopy. Pearson correlation analysis was performed to determine the relationship between muscle fiber type, CSA, diameter, and the eating quality of the 11 beef cuts from previously reported studies. Muscles from both studies showed distinct differences in the relative percentage of type I and type IIA muscle fiber types, CSA, and diameter (P < 0.05). Correlation analysis from study 1 demonstrated positive correlations between type I fibers and many positive attributes of eating quality such as tenderness, juiciness, and lipid flavor intensity, while negative correlations were found between type IIA fibers and those attributes (P < 0.01). Interestingly, results from study 2 showed that increasing type I fiber percentage may also contribute to greater connective tissue content and collagen crosslink density (P < 0.01). Finally, a negative correlation was found between muscle fiber CSA and diameter with connective tissue amount (P < 0.05), and a positive correlation was found between muscle fiber CSA and diameter with tenderness measurements (P < 0.05) in both studies. Overall, muscles with greater type I fiber % delivered a more favorable eating experience than those with more glycolytic metabolism. Notably, increased CSA and fiber diameter did not diminish eating quality and were found to have a muscle-specific relationship with tenderness.

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.

Kinin B1- and B2-Receptor Subtypes Contract Isolated Bovine Ciliary Muscle: Their Role in Ocular Lens Function and Intraocular Pressure Reduction.

Background: Bradykinin is an endogenously produced nonapeptide with many physiological and pathological functions that are mediated by two pharmacologically defined receptor subtypes, B1- and B2-receptors. Current studies sought to characterize the functional bradykinin (BK) receptors present in freshly isolated bovine ciliary muscle (BCM) using an organ-bath tissue contraction system. Methods: Cumulative longitudinal isometric tension responses of BCM strips (4-5 mm) were recorded before and after the addition of test compounds to BCM strips hooked up to an isometric strain gauge transducer system. Results: BK and its analogs (7-11 concentrations) contracted BCM in a biphasic concentration-dependent manner. The first high affinity/potency phase accounted for 40-60% of the maximal contraction by each of BK (potency, EC50 = 0.9 ± 0.3 nM), Lys-BK (EC50 = 0.7 ± 0.1 nM), Met-Lys-BK (EC50 = 1 ± 0.1 nM), Hyp3-BK (EC50 = 1 ± 0.2 nM), RMP-7 (EC50 = 3.5 ± 0.5 nM), and Des-Arg9-BK (EC50 = 10 ± 0.4nM) (mean ± SEM, n = 3-8). The second lower activity phase of contraction potency values for these peptides ranged between 100 nM and 3 µM. In the presence of a selective B1-receptor antagonist (R715; 0.1-10 µM), the concentration-response curves to Des-Arg9-BK (B1-receptor agonist) were still observed, indicating activation of B2-receptors by this kinin. Likewise, when B2-receptors were completely blocked by using a B2-selective antagonist (WIN-64338; 1-10 µM), BK still induced BCM contraction, now by stimulating B1-receptors. Conclusions: This agonist/antagonist profile of BCM receptors indicated the presence of both B1- and B2-receptor subtypes, both being responsible for contracting this smooth muscle. The BCM kinin receptors may be involved in changing the shape of the ocular lens to influence accommodation, and since the ciliary muscle is attached to the trabecular meshwork through which aqueous humor drains, endogenously released kinins may regulate intraocular pressure.

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.

Postmortem proteolysis and its indicators vary within bovine muscles: Novel insights in muscles that differ in their contractile, metabolic, and connective tissue properties

This study assessed postmortem proteolysis over 14 d in bovine Masseter (MS), Longissimus thoracis (LT), and Cutaneous trunci (CT) muscles. First, the metabolic, contractile, and connective tissue properties were characterized to establish their intrinsic differences. The MS contained the highest levels of oxidative markers and myosin heavy chain-I (MyHC-I), whereas the CT possessed the greatest glycolytic capacity, MyHC-IIx, and connective tissue proteins (P < 0.05). The LT had intermediate metabolic characteristics, a heterogeneous mixture of MyHC isoforms, and the lowest amount of connective tissue proteins (P < 0.05), confirming the muscles' intrinsic divergence. Proteolytic analysis revealed increased desmin and slow troponin-T (TT-slow) degradation, with a higher 110 kDa band intensity in the MS than in the CT (P < 0.05). In comparison, the CT exhibited greater TT-fast degradation and higher 30 kDa fragment intensity (P < 0.05). The LT demonstrated the greatest overall proteolysis, indicated by increased TT-fast and TT-slow degradation and the highest intensity of the 30 kDa band (P < 0.05). This is likely due to protease activity, as the LT and MS exhibited more calpain-1 autolysis and less calpastatin abundance than the CT (P < 0.05). However, caspase-3 activity was highest in the MS and lowest in the LT. A principal component analysis incorporating proteolytic indicators further demonstrated the distinct proteolytic profiles in the three muscles. Overall, findings suggest that the progression of postmortem proteolysis is muscle-specific and that a single proteolytic indicator does not sufficiently describe proteolysis when comparing muscles differing in contractile and metabolic properties.

In vitro proteolysis mirrors intact muscle maturation in beef carcasses

An in vitro assay was developed to study protease activity during the maturation of beef postmortem. Myofibrils were purified from the semitendinosus and used as a sentinel for assessing the activity of endogenous proteases in longissimus thoracis et lumborum (LTL) and the extensor carpi radialis (ER) over time postmortem in beef carcasses. Samples were collected from each muscle at 0, 1, 2, 7, and 14 d of aging and snap frozen. Samples were powdered and added to an in vitro proteolysis assay containing buffer and purified myofibrils. Aliquots were collected at 0, 2, 120, 480, and 1440 min of incubation, and intact desmin and troponin T were quantified. Digestions at 0 and 1 d using either muscle had little desmin degradation during the entire digestion period. In contrast, LTL muscle collected at 2, 7, and 14 d had the greatest proteolytic capacity as indicated by disappearance of intact desmin by 480 and 1440 min incubation. Though degradation ensued using powdered ER muscle, disappearance of intact proteins was limited. Degradation in vitro paralleled that observed in intact muscle. Addition of ethylene glycol tetra-acetic acid (EGTA), a cysteine protease inhibitor, and calpastatin inhibited proteolysis and suggest proteolytic activity observed in muscles and detected in our proteolysis assays is due to an active calpain protease. Collectively, our data show an active protease is minimal in bovine muscle until 48 h postmortem in the LTL muscle and suggest an in vitro assay containing purified myofibrils is a potential tool for studying temporal changes in proteolysis during the maturation and tenderization of beef across muscles.

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.

Mitochondrial Abundance and Function Differ Across Muscle Within Species.

Background: Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods: Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and oxygen consumption. Results: Bovine oxidative muscle mitochondria contain greater mtDNA (p < 0.05), protein (succinate dehydrogenase, SDHA, p < 0.01; citrate synthase, CS, p < 0.01; complex I, CI, p < 0.05), and oxygen consumption (p < 0.01) than their glycolytic counterpart. Likewise, porcine oxidative muscle contains greater mtDNA (p < 0.01), mitochondrial proteins (SDHA, p < 0.05; CS, p < 0.001; CI, p < 0.01) and oxidative phosphorylation capacity (OXPHOS, p < 0.05) in comparison to glycolytic muscle. However, avian oxidative skeletal muscle showed no differences in absolute mtDNA, SDHA, CI, complex II, lactate dehydrogenase, or glyceraldehyde 3 phosphate dehydrogenase compared to their glycolytic counterpart. Even so, avian mitochondria isolated from oxidative muscles had greater OXPHOS capacity (p < 0.05) than glycolytic muscle. Conclusions: These data show avian mitochondria function is independent of absolute mtDNA content and protein abundance, and argue that multiple levels of inquiry are warranted to determine the wholistic role of mitochondria in skeletal muscle.

Identification of Phospholipids Relevant to Cancer Tissue Using Differential Ion Mobility Spectrometry

Phospholipids are the main building components of cell membranes and are also used for cell signaling and as energy storages. Cancer cells alter their lipid metabolism, which ultimately leads to an increase in phospholipids in cancer tissue. Surgical energy instruments use electrical or vibrational energy to heat tissues, which causes intra- and extracellular water to expand rapidly and degrade cell structures, bursting the cells, which causes the formation of a tissue aerosol or smoke depending on the amount of energy used. This gas phase analyte can then be analyzed via gas analysis methods. Differential mobility spectrometry (DMS) is a method that can be used to differentiate malignant tissue from benign tissues in real time via the analysis of surgical smoke produced by energy instruments. Previously, the DMS identification of cancer tissue was based on a ‘black box method’ by differentiating the 2D dispersion plots of samples. This study sets out to find datapoints from the DMS dispersion plots that represent relevant target molecules. We studied the ability of DMS to differentiate three subclasses of phospholipids (phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine) from a control sample using a bovine skeletal muscle matrix with a 5 mg addition of each phospholipid subclass to the sample matrix. We trained binary classifiers using linear discriminant analysis (LDA) and support vector machines (SVM) for sample classification. We were able to identify phosphatidylcholine, -inositol, and -ethanolamine with SVM binary classification accuracies of 91%, 73%, and 66% and with LDA binary classification accuracies of 82%, 74%, and 72%, respectively. Phosphatidylcholine was detected with a reliable classification accuracy, but ion separation setups should be adjusted in future studies to reliably detect other relevant phospholipids such as phosphatidylinositol and phosphatidylethanolamine and improve DMS as a microanalysis method and identify other phospholipids relevant to cancer tissue.

Comparative Transcriptome Analysis of Bovine, Porcine, and Sheep Muscle Using Interpretable Machine Learning Models.

The growth and development of muscle tissue play a pivotal role in the economic value and quality of meat in agricultural animals, garnering close attention from breeders and researchers. The quality and palatability of muscle tissue directly determine the market competitiveness of meat products and the satisfaction of consumers. Therefore, a profound understanding and management of muscle growth is essential for enhancing the overall economic efficiency and product quality of the meat industry. Despite this, systematic research on muscle development-related genes across different species still needs to be improved. This study addresses this gap through extensive cross-species muscle transcriptome analysis, combined with interpretable machine learning models. Utilizing a comprehensive dataset of 275 publicly available transcriptomes derived from porcine, bovine, and ovine muscle tissues, encompassing samples from ten distinct muscle types such as the semimembranosus and longissimus dorsi, this study analyzes 113 porcine (n = 113), 94 bovine (n = 94), and 68 ovine (n = 68) specimens. We employed nine machine learning models, such as Support Vector Classifier (SVC) and Support Vector Machine (SVM). Applying the SHapley Additive exPlanations (SHAP) method, we analyzed the muscle transcriptome data of cattle, pigs, and sheep. The optimal model, adaptive boosting (AdaBoost), identified key genes potentially influencing muscle growth and development across the three species, termed SHAP genes. Among these, 41 genes (including NANOG, ADAMTS8, LHX3, and TLR9) were consistently expressed in all three species, designated as homologous genes. Specific candidate genes for cattle included SLC47A1, IGSF1, IRF4, EIF3F, CGAS, ZSWIM9, RROB1, and ABHD18; for pigs, DRP2 and COL12A1; and for sheep, only COL10A1. Through the analysis of SHAP genes utilizing Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, relevant pathways such as ether lipid metabolism, cortisol synthesis and secretion, and calcium signaling pathways have been identified, revealing their pivotal roles in muscle growth and development.

Effects of rumen metabolite butyric acid on bovine skeletal muscle satellite cells proliferation, apoptosis and transcriptional states during myogenic differentiation

Butyric acid, a pivotal short-chain fatty acid in rumen digestion, profoundly influences animal digestive and locomotor systems. Extensive research indicates its direct or indirect involvement in the growth and development of muscle and fat cells. However, the impact of butyric acid on the proliferation and differentiation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. This study aimed to elucidate the effects of butyrate on SMSCs proliferation and differentiation. After isolating, SMSCs were subjected to varying concentrations of sodium butyrate (NaB) during the proliferation and differentiation stages. Optimal treatment conditions (1 mM NaB for 2 days) were determined based on proliferative force, cell viability, and mRNA expression of proliferation and differentiation marker genes. Transcriptome sequencing was employed to screen for differential gene expression between 1 mM NaB-treated and untreated groups during SMSCs differentiation. Results indicated that lower NaB concentrations (≤1.0 mM) inhibited proliferation while promoting differentiation and apoptosis after a 2-day treatment. Conversely, higher NaB concentrations (≥2.0 mM) suppressed proliferation and differentiation and induced apoptosis. Transcriptome sequencing revealed differential expression of genes(ND1, ND3, CYTB, COX2, ATP6, MYOZ2, MYOZ3, MYBPC1 and ATP6V0A4,etc.) were associated with SMSCs differentiation and energy metabolism, enriching pathways such as Oxidative phosphorylation, MAPK, and Wnt signaling. These findings offer valuable insights into the molecular mechanisms underlying butyrate regulation of bovine SMSCs proliferation and differentiation, as well as muscle fiber type conversion in the future study.

A multiple x-ray-source array (MXA) system with a planar two-dimensional source distribution for digital breast tomosynthesis.

Digital breast tomosynthesis (DBT) has outpaced digital mammography in clinical adoption in the United States; however, substantial technological limitations remain to image quality in DBT, including undersampling from a one-dimensional (1D) scan geometry, x-ray source motion during acquisition, and patient motion artifacts from long exam times. A thermionic cathode x-ray system employing two-dimensional (2D, planar) multiple x-ray-source arrays (MXA) is proposed to improve DBT image quality. A 1D MXA, consisting of a linear array of thermionic cathodes was used to simulate a 2D MXA. The 1D MXA included 11 focal spots separated by a distance of =23mm. The 11 cathodes were paired with 11 molybdenum 50mm diameter anode disks, mounted on a rotating shaft within a single vacuum enclosure. Image quality was investigated as a function of MXA configuration by integrating the 1D MXA with a 200 × 250 mm2 flat panel detector at a source-to-detector distance of 630mm, resulting in a 20° tomographic arc. To simulate a 2D MXA, the detector (with phantom) was translated orthogonally to the linear array by a distance ( ) ranging from =0mm (conventional 1D) to =57mm. All sources operated at 30kV with 80mA and 4.5 mAs/pulse, yielding ∼100 mAs per DBT dataset. DBT reconstructions involved 22 projections and used filtered backprojection with a ramp and Hann apodization filter. Volumetric reconstructions for each source were weighted by sampling differences between sources, and averaged. Image quality was assessed in terms of contrast-to-noise ratio (CNR), background clutter noise and power spectrum, and slice sensitivity profile (SSP) using a set of physical phantoms, including: (i) contrast-detail signals coupled to spherical clutter (PMMA in air); (ii) an SSP phantom; (iii) a commercial "breast" phantom (CIRS BR3D, Sun Nuclear, Norfolk, VA); and (iv) bovine muscle. Background clutter noise amplitude reduced monotonically from the 1D MXA (σclutter=5.9 A.U., =0mm) and 2D MXA arrays with increasing , with statistical significance between the 1D MXA and 2D MXA with =57mm (σclutter=5.0 A.U., p<0.001). The contrast-detail/clutter phantom demonstrated CNR from the 2D MXA (δ=57mm) outperforming the 1D MXA in all combinations of contrast and detail. 2D power spectrum analysis of clutter demonstrated a pronounced Fourier domain null cone for the 1D MXA in the anterior field-of-view (away from the 1D MXA position), whereas the 2D MXA geometry (δ=57mm) did not exhibit the null cone. The SSP was 15%-50% narrower (FWHM) for the 2D versus the 1D geometry, across all reconstruction setups. The advantages of a 2D source geometry for DBT imaging were demonstrated quantitatively compared to a conventional 1D line of x-ray sources. The improvement in the 2D geometry was attributed both to improved Fourier domain sampling and reduced SSP. We conclude that 2D MXA sources have the potential to substantially improve DBT imaging in comparison to existing commercial DBT systems.

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.

Grifola frondosa extract as a fetal bovine serum supplement for the culture of bovine muscle satellite cells under low serum conditions

Expensive fetal bovine serum (FBS) is a major obstacle to the production of cultivated meat. However, because FBS substitutes do not sufficiently induce cell proliferation, a good alternative is to reduce the amount of FBS and use ingestible additives to promote cell proliferation. In this study, Grifola frondosa extract (GFE) was used to investigate its potential as an additive to promote myogenesis of bovine muscle satellite cells from Hanwoo cattle under low serum conditions (10 % FBS). GFE treated with 10 % FBS only during the proliferation period not only increased cell proliferation and related biomarkers in a concentration-dependent manner (0.78–12.5 μg/mL), but also increased cell differentiation. Additionally, differentiation was promoted when cells were with GFE treated only during the differentiation period. Especially GFE at 12.5 µg/mL induced significantly higher proliferation and differentiation rates than 20 % FBS medium. In particular, compared to treatment alone in the proliferation or differentiation periods, GFE treatment in both periods contributed to an increase in the differentiation rate and significantly enhanced total protein production. The integration of GFE into cultivated meat production presents a promising approach to reducing FBS dependence, lowering costs, and enhancing scalability, aligning with sustainability and consumer acceptance goals.

Smear Layer Removing and Pulp Dissolving Ability of Sodium Hypochlorite Mixed with Two Etidronate-Containing Irrigants in Continuous Chelation Technique

The study aimed to assess the effect of two etidronate-containing irrigants and EDTA on the ability of sodium hypochlorite (NaOCl) to remove the smear layer and dissolve organic tissues. This study evaluated the following solutions: distilled water, 3% NaOCl, 3% NaOCl + EDTA, and 3% NaOCl mixed with etidronate powder from two manufacturers [Dual Rinse, (DR); IsraDent, (ID)] to obtain 9%, 15%, and 18% solutions. To assess the proteolytic activity, bovine muscle tissue samples (56.1 ± 5.4 mg; n = 10 per group) were immersed in the tested solutions for 15 min. Absolute weight changes and percentages of weight changes (wt%) were calculated. To assess smear layer removal, the root canals of human wisdom teeth were instrumented, irrigated with the tested solutions (n = 10 per group), and evaluated using SEM. Statistical analysis employed an ANOVA with a post-hoc Tukey test and paired t-test, the Kruskal–Wallis test with a pairwise Wilcoxon rank sum test, and the Friedman test with a pairwise Wilcoxon signed-rank test. The mean weight loss in the NaOCl group comprised 17.3 mg (32 wt%). Sequential use of NaOCl and EDTA resulted in a significant increase in proteolytic activity of the former (57 wt%), while mixing these solutions led to a reduction of proteolytic activity (17 wt%). All NaOCl/DR groups exhibited a significantly greater dissolution activity than that of NaOCl alone, with the mean weight loss ranging from 23.3 mg (40 wt%) to 24 mg (41 wt%). ID9% and ID15% significantly decreased the proteolytic activity of NaOCl. In most groups, the apical thirds of the root canals demonstrated a significantly heavier smear layer compared to the middle and coronal thirds. The most effective smear layer removal was observed after irrigation with NaOCl combined with EDTA or DR (all concentrations); however, in the NaOCl + EDTA and DR18% groups, it was accompanied by moderate to severe erosion. Irrigation with ID did not result in smear layer removal or dentin erosion. In conclusion, the smear layer removal and pulp dissolving effects of continuous chelation using etidronate were manufacturer/composition-dependent. DR9% combined with NaOCl demonstrated the most promising results.

Exploring the Effects of S-Nitrosylation on Caspase-3 Modification and Myofibril Degradation of Beef In Vitro.

This study aimed to explore the effects of S-nitrosylation on caspase-3 modification and its subsequent effects on beef myofibril degradation in vitro. Recombinant caspase-3 was reacted with different concentrations of S-nitrosoglutathione (GSNO, nitric oxide donor) at 37 °C for 30 min and subsequently incubated with purified myofibrillar protein from bovine semimembranosus muscle. Results indicated that the activity of caspase-3 was significantly reduced after GSNO treatments (P < 0.05) and showed a dose-dependent inhibitory effect, which was attributed to the increased S-nitrosylation extent of caspase-3. LC-MS/MS analysis revealed that caspase-3 was S-nitrosylated at cysteine sites 116, 170, 184, 220, and 264. Moreover, the degradation of desmin and troponin-T was notably suppressed by S-nitrosylated caspase-3 (P < 0.05). To conclude, protein S-nitrosylation could modify the cysteine residues of caspase-3, which accounts for the reduced caspase-3 activity and further represses its proteolytic ability on beef myofibrillar protein.

PSLBI-11 Relevance of mitochondrial-mediated apoptotic processes in color and oxidative stabilities of five different beef muscles

Abstract Fresh meat color stands as one of the most critical quality indicators influencing consumer perception of meat freshness and purchase decision. Variation in color and color stability among different muscle types exists due to differences in chemical compositions, including lipids and metabolites, and other redox stability-related biochemical properties. Understanding biochemical mechanisms governing color stability in different muscles will offer insights for the industry to develop practical strategies aimed at reducing food waste attributed to discoloration. The objective of the current study was to identify metabolites and lipid compounds potentially linked to oxidative stabilities and relevant biochemical properties across different bovine muscles. Five different muscles [longissimus lumborum (LL), gluteus medius (GM), biceps femoris (BF), semitendinosus (ST), and infraspinatus (IF)] were collected from 16 beef carcasses (USDA Choice) at 2-d postmortem. Multiple cuts were made from each muscle for meat color measurements including instrumental and sensory color evaluations during display, and biochemical attributes, including total reducing activity (TRA), mitochondria-swelling, mitochondrial-lipid oxidation, cytochrome c redox ability (CytC). For the lipidome and metabolome analyses, beef samples were extracted and profiled using non-targeted UPLC-MS analyses. Data analysis was conducted with Metaboanalyst 6.0. The correlation analysis was conduct with Pearson’s correlation and considered the significance level at P &amp;lt; 0.05. In brief, LL and ST maintained greater color stability, redox stability, and lipid/protein oxidative stabilities compared with BF and GM, with IF showing the least (P &amp;lt; 0.05). Also, IF had the greatest levels of mitochondrial-swelling, mitochondrial-lipid oxidation, and oxidized CytC compared with LL (P &amp;lt; 0.05). The ANOVA results found 1,410 significant features in the metabolome analysis and 531 in the lipidome analysis of beef samples. Utilizing partial least square-discriminant analysis (PLS-DA), scatter plots grouped the five muscle types into three distinct clusters: LL and ST, BF and GM, and IF. Pro-apoptotic factors such as phosphatidylserine and lysophosphatidylserine were greater in IF compared with LL and ST (P &amp;lt; 0.05). From the metabolome results, anti-apoptotic factors like creatine and glutathione were increased in LL and ST compared with IF, whereas pro-apoptotic spermidine and adenosine monophosphate (AMP) were greatest in IF (P &amp;lt; 0.05). CIE a* showed a positive correlation with phospholipids and mitochondria-swelling (P &amp;lt; 0.05). Chroma exhibited a positive correlation with TRA, creatine, cyclic AMP, and GMP (P &amp;lt; 0.05). The results suggest that variations in meat color stability among the five beef muscles were likely attributed to differences in the abundance of lipids and metabolites associated with redox and oxidative stabilities, as well as mitochondrial-mediated apoptotic pathways. Specifically, the varying abundance of pro-/anti-apoptotic factors across muscles indicates potential mechanistic biochemical linkages between the apoptosis and meat color. Further research is warranted to investigate the effects of extended aging periods on changes in apoptotic features and color stability of beef muscles.

Multi-Residue Analysis of Thyreostats in Animal Muscle Tissues by Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry: A Thorough Chromatographic Study

Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H2O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method’s LODs ranged between 2.8 ng g−1 (6-phenyl-2-thiouracil) and 4.1 ng g−1 (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results.