Mammary Gland Of Dairy Cows Research Articles

Bioactive Compounds and Probiotics Mitigate Mastitis by Targeting NF-κB Signaling Pathway.

Mastitis is a significant inflammatory condition of the mammary gland in dairy cows. It is caused by bacterial infections and leads to substantial economic losses worldwide. The disease can be either clinical or sub-clinical and presents challenges such as reduced milk yield, increased treatment costs, and the need to cull affected cows. The pathogenic mechanisms of mastitis involve the activation of Toll-like receptors (TLRs), specifically TLR2 and TLR4. These receptors play crucial roles in recognizing pathogen-associated molecular patterns (PAMPs) and initiating immune responses through the NF-κB signaling pathway. Recent in vitro studies have emphasized the importance of the TLR2/TLR4/NF-κB signaling pathway in the development of mastitis, suggesting its potential as a therapeutic target. This review summarizes recent research on the role of the TLR2/TLR4/NF-κB signaling pathway in mastitis. It focuses on how the activation of TLRs leads to the production of proinflammatory cytokines, which, in turn, exacerbate the inflammatory response by activating the NF-κB signaling pathway in mammary gland tissues. Additionally, the review discusses various bioactive compounds and probiotics that have been identified as potential therapeutic agents for preventing and treating mastitis by targeting TLR2/TLR4/NF-κB signaling pathway. Overall, this review highlights the significance of targeting the TLR2/TLR4/NF-κB signaling pathway to develop effective therapeutic strategies against mastitis, which can enhance dairy cow health and reduce economic losses in the dairy industry.

Targeting PHB2-mediated mitophagy alleviates nonesterified fatty acid-induced mitochondrial dysfunction in bovine mammary epithelial cells

Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the immortalized bovine mammary epithelial cell line (MAC-T cells). First, mammary gland tissue and blood samples were collected from healthy cows (n = 15, BHB <0.6 mM) and cows with clinical ketosis (n = 15, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis exhibited lower DMI, milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in cows with clinical ketosis. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (MAP1LC3-II, also called LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in cows with clinical ketosis. The ATP content of mammary gland tissue in cows with clinical ketosis was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). The MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, and MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. The MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA-induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.

Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages

BackgroundCow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals.MethodThe present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA).ResultsA total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = − 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism.ConclusionsOur study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.

Effects of nanoselenium supplementation on lactation performance, nutrient digestion and mammary gland development in dairy cows

The objective of this experiment was to evaluate the influence of nanoselenium (NANO-Se) addition on milk production, milk fatty acid synthesis, the development and metabolism regulation of mammary gland in dairy cows. Forty-eight Holstein dairy cows averaging 720 ± 16.8 kg of body weight, 66.9 ± 3.84 d in milk (dry matter intake [DIM]) and 35.2 ± 1.66 kg/d of milk production were divided into four treatments blocked by DIM and milk yields. Treatments were control group, low-Se (LSe), medium-Se (MSe) and high-Se (HSe) with 0, 0.1, 0.2 and 0.3 mg Se, respectively, from NANO-Se per kg dietary dry matter (DM). Production of energy- and fat-corrected milk (FCM) and milk fat quadratically increased (p < 0.05), while milk lactose yields linearly increased (p < 0.05) with increasing NANO-Se addition. The proportion of saturated fatty acids (SFAs) linearly decreased (p < 0.05), while proportions of monounsaturated fatty acids (MUFAs) linearly increased and polyunsaturated fatty acids (PUFAs) quadratically increased. The digestibility of dietary DM, organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) quadratically increased (p < 0.05). Ruminal pH quadratically decreased (p < 0.01), while total VFA linearly increased (p < 0.05) with increasing NANO-Se addition. The acetic to propionic ratio decreased (p < 0.05) linearly due to the unaltered acetic molar percentage and a quadratical increase in propionic molar percentage. The activity of CMCase, xylanase, cellobiase and pectinase increased linearly (p < 0.05) following NANO-Se addition. The activity of α-amylase increased linearly (p < 0.01) with an increase in NANO-Se dosage. Blood glucose, total protein, estradiol, prolactin, IGF-1 and Se linearly increased (p < 0.05), while urea nitrogen concentration quadratically decreased (p = 0.04). Moreover, the addition of Se at 0.3 mg/kg from NANO-Se promoted (p < 0.05) mRNA and protein expression of PPARγ, SREBP1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2 and the ratios of p-ACACA/ACACA and BCL2/BAX4, but decreased (p < 0.05) mRNA and protein expressions of Bax, Caspase-3 and Caspase-9. The results suggest that milk production and milk fat synthesis increased by NANO-Se addition by stimulating rumen fermentation, nutrients digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

MSI2 Modulates Unsaturated Fatty Acid Metabolism by Binding FASN in Bovine Mammary Epithelial Cells.

The regulation of fatty acid metabolism is crucial for milk flavor and quality. Therefore, it is important to explore the genes that play a role in fatty acid metabolism and their mechanisms of action. The RNA-binding protein Musashi2 (MSI2) is involved in the regulation of numerous biological processes and plays a regulatory role in post-transcriptional translation. However, its role in the mammary glands of dairy cows has not been reported. The present study examined MSI2 expression in mammary glands from lactating and dry milk cows. Experimental results in bovine mammary epithelial cells (BMECs) showed that MSI2 was negatively correlated with the ability to synthesize milk fat and that MSI2 decreased the content of unsaturated fatty acids (UFAs) in BMECs. Silencing of Msi2 increased triglyceride accumulation in BMECs and increased the proportion of UFAs. MSI2 affects TAG synthesis and milk fat synthesis by regulating fatty acid synthase (FASN). In addition, RNA immunoprecipitation experiments in BMECs demonstrated for the first time that MSI2 can bind to the 3'-UTR of FASN mRNA to exert a regulatory effect. In conclusion, MSI2 affects milk fat synthesis and fatty acid metabolism by regulating the triglyceride synthesis and UFA content through binding FASN.

The Quality of Dairy Cow Bedding and the Occurrence of Environmental Mastitis: Review

An excessive incidence of inflammation of the mammary glands of dairy cows has been observed in many dairy farms in the past, which has been demonstrably correlated with an increased incidence of microbial pathogens in livestock environments. The causes of mastitis can be infectious or non-infectious in nature, one of the main sources of this disease is litter material in the lager. The issue of mastitis has a significant impact on the economic value of dairy cows and therefore it is necessary to create effective measures that eliminate the appearance of pathogenic microorganisms. In this respect, litter material obtained by physical separation and thermal treatment of slurry may represent a suitable alternative ensuring inhibition of the development of dangerous bacteria while maintaining the comfort of dairy cows. Current evidence on the safety and benefits of such material is limited and data on effects on clinical and/or subclinical mastitis are insufficient. In this review, therefore, we would like to shed light on the onset of mastitis in dairy cows and point out possible procedures for treating slurry in practice.

Dietary supplementation of sodium butyrate enhances lactation performance by promoting nutrient digestion and mammary gland development in dairy cows

This experiment was to evaluate the influence of sodium butyrate (SB) addition on milk production, ruminal fermentation, nutrient digestion, and the development and metabolism regulation of the mammary gland in dairy cows. Forty Holstein dairy cows averaging 710 ± 18.5 kg body weight, 72.8 ± 3.66 d in milk (DIM), and 41.4 ± 1.42 kg/d milk production were divided into four treatments blocked by DIM and milk production. Treatments were control group, low SB, medium SB, and high SB with 0, 100, 200 and 300 g/d of SB addition per cow, respectively. The study lasted for 105 d. Production of milk, milk protein and lactose quadratically increased (P < 0.05), while fat-corrected milk, energy-corrected milk and milk fat yields linearly increased (P < 0.05) with increasing SB addition. The digestibility of dietary dry matter, organic matter, and crude protein linearly increased (P < 0.05), whereas the digestibility of ether extract, neutral detergent fibre, and acid detergent fibre quadratically increased (P < 0.05). Ruminal pH quadratically decreased (P = 0.04), while total volatile fatty acids (VFA) quadratically increased (P = 0.03) with increasing SB addition. The acetic acid to propionic acid ratio increased (P = 0.03) linearly due to the unaltered acetic acid molar percentage and a linear decrease in propionic acid molar percentage. Ruminal enzymatic activity of carboxymethyl-cellulase and α-amylase, populations of total bacteria, total anaerobic fungi, total protozoa, Ruminococcus albus, R. flavefaciens, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, and Ruminobacter amylophilus linearly increased (P < 0.05). Blood glucose, urea nitrogen, and non-esterified fatty acids linearly decreased (P < 0.05), while total protein concentration linearly increased (P = 0.04). Moreover, the addition of SB at 200 g/d promoted (P < 0.05) mRNA and protein expression of PPARγ, SREBF1, ACACA, FASN, SCD, CCNA2, CCND1, PCNA, Bcl-2, GPR41, and the ratios of p-Akt/Akt and p-mTOR/mTOR, but decreased (P < 0.05) mRNA and protein expressions of Bax, caspase-3, and caspase-9. The results suggest that milk production and milk fat synthesis increased with SB addition by stimulating rumen fermentation, nutrient digestion, gene and protein expressions concerned with milk fat synthesis and mammary gland development.

A Study on Differential Biomarkers in the Milk of Holstein Cows with Different Somatic Cells Count Levels.

Dairy cow mastitis is one of the common diseases of dairy cows, which will not only endanger the health of dairy cows but also affect the quality of milk. Dairy cow mastitis is an inflammatory reaction caused by pathogenic microorganisms and physical and chemical factors in dairy cow mammary glands. The number of SCC in the milk of dairy cows with different degrees of mastitis will increase in varying degrees. The rapid diagnosis of dairy cow mastitis is of great significance for dairy cow health and farm economy. Based on the results of many studies on the relationship between mastitis and somatic cell count in dairy cows, microflora, and metabolites in the milk of Holstein cows with low somatic cell level (SCC less than 200,000), medium somatic cell level (SCC up to 200,000 but less than 500,000) and high somatic cell level (SCC up to 5000,00) were analyzed by microbiome and metabolic group techniques. The results showed that there were significant differences in milk microbiota and metabolites among the three groups (p < 0.05), and there was a significant correlation between microbiota and metabolites. Meanwhile, in this experiment, 75 differential metabolites were identified in the H group and L group, 40 differential metabolites were identified in the M group and L group, and six differential microorganisms with LDA scores more than four were found in the H group and L group. These differential metabolites and differential microorganisms may become new biomarkers for the diagnosis, prevention, and treatment of cow mastitis in the future.

CircRNA-02191 regulating unsaturated fatty acid synthesis by adsorbing miR-145 to enhance CD36 expression in bovine mammary gland

CD36 functions as a receptor for long-chain fatty acids, promoting the absorption and transport of long-chain unsaturated fatty acids. However, the regulatory influence of upstream circRNAs or miRNAs on its expression in cow mammary gland remains unclear. Herein, we performed high-throughput sequencing to screen for differentially expressed miRNAs and mRNAs in bovine mammary tissue during the late-lactation and the dry period to screen and conducted bioinformatics analysis to identify 420 miRNA/mRNA pairs, including miR-145/CD36. Experimental results indicate that miR-145 can directly target CD36 and inhibit its expression. Additionally, the circRNA-02191 sequence is predicted to contain a miR-145 binding site. As shown by dual luciferase reporter system detection, circRNA-02191 bound to miR-145 and its overexpression significantly reduced the expression of miR-145. Furthermore, the overexpression of miR-145 inhibited triglyceride accumulation, while circRNA-02191 enhanced the expression of the miR-145 target gene CD36. The above results indicate that circRNA-02191 can regulate triglyceride and fatty acid components by binding miR-145 and subsequently alleviating the inhibitory effect of miR-145 on the expression of CD36. Taken together, these findings present a novel approach to improve milk quality by analyzing the regulatory effect and mechanism regulating the circ02191/miR-145/CD36 pathway on fatty acid synthesis in the mammary gland of dairy cows.

A high-concentrate diet induces mitochondrial dysfunction by activating the MAPK signaling pathway in the mammary gland of dairy cows

Subacute rumen acidosis can lead to mastitis in dairy cows. Mitochondrial dysfunction is closely related to the inflammatory response. This experiment was conducted to investigate the effects of a high-concentrate diet on mammary gland inflammation and mitochondrial damage in dairy cows. Twelve Holstein dairy cows in mid-lactation were randomly divided into 2 groups and fed a 40% concentrate (low concentrate, LC) diet or a 60% concentrate (high concentrate, HC) diet. Cows were fed individually, and the experiment lasted for 3 wk. After the experiment, mammary gland tissue, blood, and rumen fluid were collected. Compared with the LC diet, the HC diet significantly decreased rumen pH; the pH was <5.6 for more than 3 h. The HC diet also increased the concentration of LPS in the blood (7.17 ± 1.25 µg/mL vs. 12.12 ± 1.26 µg/mL), which indicated that feeding the HC diet successfully induced subacute rumen acidosis. The HC diet also increased the concentration of Ca2+ (34.80 ± 4.23 µg/g vs. 46.87 ± 7.24 µg/g) in the mammary gland and upregulated the expression of inflammatory factors IL-6 (1,128.31 ± 147.53 pg/g vs. 1,538.42 ± 241.38 pg/g), IL-1β (69.67 ± 5.86 pg/g vs. 90.13 ± 4.78 pg/g), and tumor necrosis factor-α (91.99 ± 10.43 pg/g vs. 131.75 ± 17.89 pg/g) in mammary venous blood. The HC diet also increased the activity of myeloperoxidase (0.41 ± 0.05 U/g vs. 0.71 ± 0.11 U/g) and decreased the content of ATP (0.47 ± 0.10 µg/mL vs. 0.32 ± 0.11 µg/mL) in the mammary gland. In addition, phosphorylation of JNK (1.00 ± 0.21 vs. 2.84 ± 0.75), ERK (1.00 ± 0.20 vs. 1.53 ± 0.31), and p38 (1.00 ± 0.13 vs. 1.47 ± 0.41) and protein expression of IL-6 (1.00 ± 0.22 vs. 2.21 ± 0.27) and IL-8 (1.00 ± 0.17 vs. 1.96 ± 0.26) were enhanced in cows of the HC group, indicating that the mitogen-activated protein kinase (MAPK) signaling pathway was activated. Compared with the LC diet, the HC diet reduced the protein expression of mitochondrial biogenesis-related proteins PGC-1α (1.00 ± 0.17 vs. 0.55 ± 0.12), NRF1 (1.00 ± 0.17 vs. 0.60 ± 0.10), TFAM (1.00 ± 0.10 vs. 0.73 ± 0.09), and SIRTI (1.00 ± 0.44 vs. 0.40 ± 0.10). The HC diet promoted mitochondrial fission and inhibited mitochondrial fusion by reducing protein expression of MFN1 (1.00 ± 0.31 vs. 0.49 ± 0.09), MFN2 (1.00 ± 0.19 vs. 0.69 ± 0.13), and OPA1 (1.00 ± 0.08 vs. 0.72 ± 0.07), and by increasing that of DRP1 (1.00 ± 0.09 vs. 1.39 ± 0.10), MFF (1.00 ± 0.15 vs. 1.89 ± 0.12), and TTC1/FIS1 (1.00 ± 0.08 vs. 1.76 ± 0.14), leading to mitochondrial dysfunction. The HC diet increased mitochondrial permeability by upregulating the protein expression of VDAC1 (1.00 ± 0.42 vs. 1.90 ± 0.44), ANT (1.00 ± 0.22 vs. 1.27 ± 0.17), and CYPD (1.00 ± 0.41 vs. 1.82 ± 0.43). Taken together, these results indicated that feeding the HC diet induced mitochondrial damage via the MAPK signaling pathway in the mammary gland of dairy cows.

Progress in Research on Key Factors Regulating Lactation Initiation in the Mammary Glands of Dairy Cows.

Lactation initiation refers to a functional change in the mammary organ from a non-lactating state to a lactating state, and a series of cytological changes in the mammary epithelium from a non-secreting state to a secreting state. Like the development of the mammary gland, it is regulated by many factors (including hormones, cytokines, signaling molecules, and proteases). In most non-pregnant animals, a certain degree of lactation also occurs after exposure to specific stimuli, promoting the development of their mammary glands. These specific stimuli can be divided into two categories: before and after parturition. The former inhibits lactation and decreases activity, and the latter promotes lactation and increases activity. Here we present a review of recent progress in research on the key factors of lactation initiation to provide a powerful rationale for the study of the lactation initiation process and mammary gland development.

High-concentrate diet elevates histone lactylation mediated by p300/CBP through the upregulation of lactic acid and induces an inflammatory response in mammary gland of dairy cows

High-concentrate diet can cause metabolic diseases, such as subacute ruminal acidosis (SARA), and secondary mastitis. To investigate the effect of SARA induced by high-concentrate diet on the lysine lactylation (Kla) and inflammatory responses in the mammary gland of dairy cows and the mechanism between them, we selected twelve mid-lactation Holstein cows with similar body conditions for modelling. They were randomly divided into two groups, fed a low-concentrate diet (LC) and a high-concentrate diet (HC) for 21 days. Our results showed that high-concentrate diet feeding significantly reduced ruminal pH, and the pH was below 5.6 for more than 3 h per day, indicating successful induction of the SARA model. Lactic acid concentrations in mammary gland and plasma were higher in the HC group than that in the LC group. HC diet feeding significantly up-regulated the expression levels of the Pan Kla, H3K18la, p300/CBP and monocarboxylate transporter 1 (MCT1) in the mammary gland. In addition, the mRNA expression levels of inflammatory factors were significantly regulated, including IL-1β, IL-1α, IL-6, IL-8, SAA3, and TNF-α, while the anti-inflammatory factor IL-10 was down-regulated. The mammary gland of HC group was structurally disorganized with incomplete glandular vesicles, with a large number of detached mammary epithelial cells and inflammatory cells infiltration. The up-regulation of TLR4, TNF-α, p-p65, and p-IκBα indicated that the TLR4/NF-κB signaling pathway was activated. In conclusion, this study found that HC diet feeding can induce SARA and increase the concentration of lactic acid in mammary gland and plasma. Then, lactic acid could be transported into cells by MCT1 and up-regulate the expression level of histone lactylation mediated by p300/CBP, and subsequently promote the activation of TLR4/NF-κB signaling pathway, ultimately causing inflammatory responses in the mammary gland.

MiR-145 Modulates Fatty Acid Metabolism by Targeting FOXO1 to Affect SERBP1 Activity in Bovine Mammary Epithelial Cells

MicroRNA-mediated gene regulation is important for the regulation of fatty acid metabolism and synthesis. Our previous study uncovered that the miR-145 expression is higher in the lactating mammary gland of dairy cows than in the dry-period, but the underlying molecular mechanism is incompletely understood. In this study, we have investigated the potential role of miR-145 in bovine mammary epithelial cells (BMECs). We found that the expression of miR-145 gradually increased during lactation. CRISPR/Cas9-mediated knockout (KO) of miR-145 in BMECs results in the downregulated expression of fatty acid metabolism-associated genes. Further results revealed that miR-145 KO reduced total triacylglycerol (TAG) and cholesterol (TC) accumulation and altered the composition of intracellular fatty acids (C16:0, C18:0, and C18:1). Conversely, miR-145 overexpression had the opposite effect. Bioinformatics online program predicted that miR-145 targets the 3'-UTR of the Forkhead Box O1 (FOXO1) gene. Subsequently, FOXO1 was identified as a direct target of miR-145 by qRT-PCR, Western blot analysis, and luciferase reporter assay. Furthermore, siRNA-mediated silencing of FOXO1 promoted fatty acid metabolism and TAG synthesis in BMECs. Additionally, we observed the involvement of FOXO1 in the transcriptional activity of the sterol regulatory element-binding protein 1 (SREBP1) gene promoter. Overall, our findings indicated that miR-145 relieves the inhibitory effect of FOXO1 on SREBP1 expression by targeting FOXO1 and subsequently regulating fatty acid metabolism. Thus, our results provide valuable information on the molecular mechanisms for improving milk yield and quality from the perspective of miRNA-mRNA networks.

Tebuconazole Induces ER-Stress-Mediated Cell Death in Bovine Mammary Epithelial Cell Lines.

Tebuconazole (TEB) is a triazole fungicide used to increase crop production by controlling fungi, insects, and weeds. Despite their extensive use, people are concerned about the health risks associated with pesticides and fungicides. Numerous studies have defined the cellular toxicity of triazole groups in pesticides, but the mechanisms of TEB toxicity in bovine mammary gland epithelial cells (MAC-T cells) have not yet been studied. Damage to the mammary glands of dairy cows directly affects milk production. This study investigated the toxicological effects of TEB on MAC-T cells. We found that TEB decreases both cell viability and proliferation and activates apoptotic cell death via the upregulation of pro-apoptotic proteins, such as cleaved caspases 3 and 8 and BAX. TEB also induced endoplasmic reticulum (ER) stress via the upregulation of Bip/GRP78; PDI; ATF4; CHOP; and ERO1-Lα. We found that TEB induced mitochondria-mediated apoptotic MAC-T cell death by activating ER stress. This cell damage eventually led to a dramatic reduction in the expression levels of the milk-protein-synthesis-related genes LGB; LALA; CSN1S1; CSN1S2; and CSNK in MAC-T cells. Our data suggest that the exposure of dairy cows to TEB may negatively affect milk production by damaging the mammary glands.

Methionine alleviates heat stress-induced ferroptosis in bovine mammary epithelial cells through the Nrf2 pathway

Heat stress (HS) triggers mammary gland degradation, accompanied by apoptosis and autophagy in bovine mammary epithelial cells, negatively affecting milk performance and mammary gland health. Ferroptosis is iron-mediated regulated cell death caused by over production of lipid peroxides, however, the relationship between ferroptosis and HS in bovine mammary epithelial cells has not been clarified. Methionine (Met) plays a notable role in alleviating HS affecting the mammary glands in dairy cows, but the underlying mechanisms require further exploration. Therefore, we evaluated the regulatory effect and mechanism of Met in alleviating HS-induced ferroptosis by using bovine mammary epithelial cell line (MAC-T) as an in vitro model. The results showed that Met improved cell vitality, restored mitochondrial function; reduced the content of various reactive oxygen species (ROS), especially hydrogen peroxide (H2O2) and superoxide anion (O2·-); had positive effects on antioxidant enzyme activity, namely glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). More importantly, Met reduced labile iron protein (LIP) levels; increased iron storage and simultaneously decreased the levels of lipid reactive oxygen species (lipid ROS) and malondialdehyde (MDA), which all caused by HS in MAC-T. Mechanistically, Met increased the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11) and ferritin heavy chain 1 (FTH1) by activating nuclear factor E2-related factor 2 (Nrf2) expression. Additionally, the protection effect of Met was cut off in MAC-T cells after interference with Nrf2, manifesting in decresing the protein expression levels of GPX4, SLC7A11 and FTH1,and increasing the levels of LIP and lipid ROS. Our findings indicate that Met eases HS-induced ferroptosis in MAC-T through the Nrf2 pathway, revealing that Met produces a marked effect on easing HS-induced bovine mammary gland injury in dairy cows.

High concentrate diet induced inflammatory response and tight junction disruption in the mammary gland of dairy cows.

This study aimed to investigate the effect and mechanism of a high concentrate (HC) diet on the inflammatory response and cellular tight junctions (TJs) in the mammary gland of dairy cows. Twelve lactating Holstein dairy cows were randomly assigned into low concentrate (LC) and HC groups (n=6), which were fed with LC diet and HC diet respectively for 3 weeks. The HC diet lead to subacute ruminant acidosis with a rumen pH<5.6 more than 3h daily. The HC diet triggered an inflammatory response with increased levels of inflammatory cytokines in the lacteal vein, upregulated expression of inflammation-related genes, elevated activity of myeloperoxidase, and inflammatory cells infiltration in the mammary gland. Furthermore, the HC diet induced the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways with enhanced phosphorylation ratios of NF-κB P65, inhibitor of NF-κB (IκB), P38 and extracellular signal-regulated kinase 1/2 (ERK1/2) as well as decreased ratios of DNA methylation and chromatin compaction of genes coding for proinflammatory cytokines, which contributed to the upregulation of proinflammatory cytokine expression. The HC diet also destroyed the integrity of TJ with discontinuous and decreased expression levels of zonula occludens-1, Occludin, Claudin-4 and increased expression level of Claudin-1 in the mammary epithelial cells compared with LC group. Conclusively, the HC diet induced the activation of NF-κB and MAPK signaling pathways and epigenetic modifications, promoted the transcription of proinflammatory cytokines, and finally caused inflammatory response and TJ disruption in the mammary gland of dairy cows.

Diagnostic Screening of Bovine Mastitis Using MALDI-TOF MS Direct-Spotting of Milk and Machine Learning

Novel strategies for diagnostic screening of animal and herd health are crucial to contain disease outbreaks, maintain animal health, and maximize production efficiency. Mastitis is an inflammation of the mammary gland in dairy cows, often resulting from infection from a microorganism. Mastitis outbreaks result in loss of production, degradation of milk quality, and the need to isolate and treat affected animals. In this work, we evaluate MALDI-TOF mass spectrometry as a diagnostic for the culture-less screening of mastitis state from raw milk samples collected from regional dairies. Since sample preparation requires only minutes per sample using microvolumes of reagents and no cell culture, the technique is promising for rapid sample turnaround and low-cost diagnosis. Machine learning algorithms have been used to detect patterns embedded within MALDI-TOF spectra using a training set of 226 raw milk samples. A separate scoring set of 100 raw milk samples has been used to assess the specificity (spc) and sensitivity (sens) of the approach. Of machine learning models tested, the gradient-boosted tree model gave global optimal results, with the Youden index of J = 0.7, sens = 0.89, and spc = 0.81 achieved for the given set of conditions. Random forest models also performed well, achieving J > 0.63, with sens = 0.83 and spc = 0.81. Naïve Bayes, generalized linear, fast large-margin, and deep learning models failed to produce diagnostic results that were as favorable. We conclude that MALDI-TOF MS combined with machine learning is an alternative diagnostic tool for detection of high somatic cell count (SCC) and subclinical mastitis in dairy herds.

Evaluation of mammary gland health in dairy cows treated by pegylated granulocyte colony-stimulating factor

The study aimed to determine the effect of the treatment by pegbovigrastim on the health of the mammary gland in dairy cows. Experimental animals were successively treated with 15 mg of pegylated bovine granulocyte colony-stimulating factor (bG-CSF, pegbovigrastim, PEG, Imrestor, Elanco) 10 ± 3 days before expected parturition and 1 day after actual parturition. Cows in the control group remained without treatment. The occurrence of clinical and subclinical mastitis in dairy cows during the first three months postpartum, and bacteriological findings in milk and milk yield were evaluated after preventive pegbovigrastim treatment. The influence of pegbovigrastim, as an additional treatment of mastitis caused by Streptococcus uberis in dairy cows on the standard course of treatment, was evaluated. The average number of antimicrobial (AML) interventions necessary for healing, the proportion of cows with 1, 2, 3 and more AML interventions, milk somatic cell count before treatment, 1 and 2 months after treatment and average milk withdrawal time were evaluated between groups. The results of the study did not find positive effects of pegbovigrastim on any evaluated variables.

MICROBIOLOGICAL ASPECTS OF RECYCLED MANURE USED IN DAIRY COWS BEDDING – REVIEW

The economic and social value of cattle, especially dairy cows, is continuously increasing and is defined by the number of lactations during the production period or the milk yield of the individual itself. A significant influence on dairy production of dairy cows has housing comfort and therefore maintaining the quality parameters of the dairy farm is essential. The decreasing availability and increasing costs of traditional underlining materials have increased interest in finding and using alternative materials for underlaying. In this review, we focus on the separated fraction of livestock manure, which, after hygienization, can be a suitable bedding material for dairy cows. We identify possible negative impacts and risks in the context of human or animal health. This article also identifies pathogenic microorganisms that can initiate inflammation of the mammary glands in dairy cows and thus reduce the quality of final food products. Farmers using recycled livestock manure as bedding, reduce the total amount of nutrients which become part of the manure stream due to no net addition of nutrients in the form of bedding, thus increasing potential compliance with environmental regulations.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.