TNFα Altered Inflammatory Responses,Impaired Health and Productivity,but Did Not Affect Glucose or Lipid Metabolism in Early-Lactation Dairy Cows

Inflammation may be a major contributing factor to peripartum metabolic disorders in dairy cattle. We tested whether administering an inflammatory cytokine, recombinant bovine tumor necrosis factor-α (rbTNFα), affects milk production, metabolism, and health during this period. Thirty-three Holstein cows (9 primiparous and 24 multiparous) were randomly assigned to 1 of 3 treatments at parturition. Treatments were 0 (Control), 1.5, or 3.0 µg/kg body weight rbTNFα, which were administered once daily by subcutaneous injection for the first 7 days of lactation. Statistical contrasts were used to evaluate the treatment and dose effects of rbTNFα administration. Plasma TNFα concentrations at 16 h post-administration tended to be increased (P<0.10) by rbTNFα administration, but no dose effect (P>0.10) was detected; rbTNFα treatments increased (P<0.01) concentrations of plasma haptoglobin. Most plasma eicosanoids were not affected (P>0.10) by rbTNFα administration, but 6 out of 16 measured eicosanoids changed (P<0.05) over the first week of lactation, reflecting elevated inflammatory mediators in the days immediately following parturition. Dry matter and water intake, milk yield, and milk fat and protein yields were all decreased (P<0.05) by rbTNFα treatments by 15 to 18%. Concentrations of plasma glucose, insulin, β-hydroxybutyrate, non-esterified fatty acids, triglyceride, 3-methylhistidine, and liver triglyceride were unaffected (P>0.10) by rbTNFα treatment. Glucose turnover rate was unaffected (P = 0.18) by rbTNFα administration. The higher dose of rbTNFα tended to increase the risk of cows developing one or more health disorders (P = 0.08). Taken together, these results indicate that administration of rbTNFα daily for the first 7 days of lactation altered inflammatory responses, impaired milk production and health, but did not significantly affect liver triglyceride accumulation or nutrient metabolism in dairy cows.     

MiR-486 Regulates Lactation and Targets the PTEN Gene in Cow Mammary Glands

Mammary gland development is controlled by several genes. Although miRNAs have been implicated in mammary gland function, the mechanism by which miR-486 regulates mammary gland development and lactation remains unclear. We investigated miR-486 expression in cow mammary gland using qRT-PCR and ISH and show that miR-486 expression was higher during the high-quality lactation period. We found that miR-486 targets phosphoinositide signaling in the cow mammary gland by directly downregulating PTEN gene expression and by altering the expression of downstream genes that are important for the function of the mammary gland, such as AKT, mTOR. We analyzed the effect of β-casein, lactose and triglyceride secretion in bovine mammary gland epithelial cells (BMECs) transfected by an inhibitor and by mimics of miR-486. Our results identify miR-486 as a downstream regulator of PTEN that is required for the development of the cow mammary gland.     

Longitudinal Profiling of the Tissue-Specific Expression of Genes Related with Insulin Sensitivity in Dairy Cows during Lactation Focusing on Different Fat Depots

In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week −3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation.     

Effects of Glucose Availability on Expression of the Key Genes Involved in Synthesis of Milk Fat,Lactose and Glucose Metabolism in Bovine Mammary Epithelial Cells

As the main precursor for lactose synthesis, large amounts of glucose are required by lactating dairy cows. Milk yield greatly depends on mammary lactose synthesis due to its osmoregulatory property for mammary uptake of water. Thus, glucose availability to the mammary gland could be a potential regulator of milk production. In the present study, the effect of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in vitro was investigated. Bovine mammary epithelial cells (BMEC) were treated for 12 h with various concentrations of glucose (2.5, 5, 10 or 20 mmol/L). The higher concentrations of glucose (10–20 mmol/L) did not affect the mRNA expression of acetyl-CoA carboxylase, diacyl glycerol acyl transferase, glycerol-3 phosphate acyl transferase and α-lactalbumin, whereas fatty acid synthase, sterol regulatory element binding protein-1 and beta-1, 4-galactosyl transferase mRNA expression increased at 10 mmol/L and then decreased at 20 mmol/L. The content of lactose synthase increased with increasing concentration of glucose, with addition of highest value at 20 mmol/L of glucose. Moreover, the increased glucose concentration stimulated the activities of pyruvate kinase and glucose-6-phosphate dehydrogenase, and elevated the energy status of the BMEC. Therefore, it was deduced that after increasing glucose availability, the extra absorbed glucose was partitioned to entering the synthesis of milk fat and lactose by the regulation of the mRNA expression of key genes, promoting glucose metabolism by glycolysis and pentose phosphate pathway as well as energy status. These results indicated that the sufficient availability of glucose in BMEC may promote glucose metabolism, and affect the synthesis of milk composition.     

Focus: Allergic Diseases and Type II Immunity: Expression of Leucine-rich Repeat-containing Protein 32 Following Lymphocyte Stimulation in Patients with Non-IgE-mediated Gastrointestinal Food Allergies

The lymphocyte stimulation test (LST) facilitates the diagnosis of non-IgE-mediated gastrointestinal food allergies (non-IgE-GI-FAs). However, LSTs require large volumes of blood and prolonged culture durations. Recently, we found that IL2RA mRNA expression in peripheral blood mononuclear cells (PBMCs) of patients with non-IgE-GI-FAs increased after a 24 h stimulation with milk proteins. We designated this gene expression test as the instant peripheral blood allergen stimulation test (iPAST). In this study, we investigated whether other activated T cell-associated genes are superior to IL2RA in the iPAST for the supplementary diagnosis of non-IgE-GI-FAs. After incubating PBMCs with milk proteins for 24 h, the mRNA levels of three genes, LRRC32, TNFRSF4, and CD69, were assessed using quantitative RT-PCR. The diagnostic significance of the mRNA expression was evaluated by analyzing the receiver operating characteristic (ROC) curve. Upon stimulation with α-casein, κ-casein, α-lactalbumin, or a mixture of four milk protein components (Pmix), LRRC32 expression in the PBMCs of 16 patients with non-IgE-GI-FAs was found to be higher than that in their 17 control counterparts, whereas TNFRSF4 and CD69 levels remained unaltered. Except for β-lactoglobulin and cow’s milk (CM), the area under the ROC curve (AUC) for LRRC32 mRNA expression upon stimulation was >0.7, which validated the diagnostic ability of this test. Notably, α-casein and Pmix had higher AUC scores of 0.820 and 0.842, respectively, than other antigens. iPAST assessed by LRRC32 as well as IL2RA may be useful for the supplementary diagnosis of non-IgE-GI-FAs as an alternative to LSTs and provide insight into the pathogenesis of non-IgE-GI-FAs.     

Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S₁-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.     

Pten对奶牛乳腺上皮细胞活力及乳蛋白合成的影响

Pten作为抑癌基因,参与调控细胞生长、粘附、凋亡以及其它细胞活动.目前,国内外关于Pten在奶牛乳腺发育过程中表达及调节的研究鲜有报道.为了揭示Pten的表达与奶牛乳腺发育与泌乳之间的关系,本研究应用qRT-PCR技术检测Pten在不同泌乳时期和不同乳品质的奶牛乳腺组织中的表达差异,进而应用脂质体转染方法,通过siRNA介导的RNA干扰技术改变Pten基因在奶牛乳腺上皮细胞中的表达量,CASY法检测细胞活力,用ELISA试剂盒检测细胞分泌β-酪蛋白的含量,采用qRT-PCR、Western 印迹等技术检测Pten对奶牛乳腺上皮细胞中乳蛋白相关信号通路基因表达的影响.结果显示,泌乳期高乳品质奶牛乳腺组织中Pten表达水平显著低于泌乳期低乳品质及干乳期奶牛;Pten基因沉寂后,细胞活力提高,β-酪蛋白质量浓度增加,CSN2、AKT、MTOR、STAT5表达量增加.研究表明,Pten可通过抑制细胞活力和乳蛋白分泌而影响泌乳.     

Peripheral Serotonin Regulates Maternal Calcium Trafficking in Mammary Epithelial Cells during Lactation in Mice

Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood) into the ductal lumen (milk). Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT) is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1), which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2) and basolateral (CaSR, ORAI-1) membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2). Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK_1/2) are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation.     

The separation of major components of the casein of bovine milk by electrophoresis in a density gradient

1. Zonal electrophoresis in a column stabilized by a density gradient has been applied to the small-scale fractionation of the proteins of the casein complex of cow's milk. 2. The α_s- and β-fractions from the milk of individual Ayrshire cows have been shown to behave as single homogeneous proteins on electrophoresis at two pH values in starch gels. 3. The α_s-fraction has been found to be indistinguishable from α_s-casein prepared by Ca²⁺ fractionation of the same milk samples. 4. On the evidence of their electrophoretic behaviour in starch gels and their elementary analyses, α₁- and α_s-casein are concluded to be substantially the same protein.     

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.     

The Membrane-Associated Form of αs1-Casein Interacts with Cholesterol-Rich Detergent-Resistant Microdomains

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α_s1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α_s1-casein in rat mammary epithelial cells. Using metabolic labelling we show that α_s1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α_s1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α_s1-casein. These experiments reveal that the insolubility of α_s1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α_s1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.     

Feeding a High Concentrate Diet Down-Regulates Expression of ACACA,LPL and SCD and Modifies Milk Composition in Lactating Goats

High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase α (ACACA, ACCα) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.     

Effect of Milk Proteins on Adhesion of Bacteria to Stainless Steel Surfaces

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.     

Mapping and Analysis of Dairy Cattle Quantitative Trait Loci by Maximum Likelihood Methodology Using Milk Protein Genes as Genetic Markers

Maximum likelihood methodology was used to estimate effects of both a marker gene and a linked quantitative trait locus (QTL) on quantitative traits in a segregating population. Two alleles were assumed for the QTL. In addition to the effects of genotypes at both loci on the mean of the quantitative trait, recombination frequency between the loci, frequency of the QTL alleles and the residual standard deviation were also estimated. Thus six parameters were estimated in addition to the marker genotype means. The statistical model was tested on simulated data, and used to estimate direct and linked effects of the milk protein genes, β-lactoglobulin, κcasein, and β-casein, on milk, fat, and protein production and fat and protein percent in the Dutch dairy cattle population. β-Lactoglobulin had significant direct effects on milk yield and fat percent. κ-Casein had significant direct effects on milk yield, protein percent and fat yield. β-Casein had significant direct effects on milk yield, fat and protein percent and fat and protein yield. Linked QTL with significant effects on fat percent were found for κ-casein and β-casein. Since the β-casein and κ-casein genes are closely linked, it is likely that the same QTL was detected for those two markers. Further, a QTL with a significant effect on fat yield was found to be linked to κ-casein and a QTL with a significant effect on protein yield was linked to β-lactoglobulin.     

Effects of Isoflavone-Enriched Feed on the Rumen Microbiota in Dairy Cows

In this study, we compared the effects of two diets containing different isoflavone concentrations on the isoflavone transfer from feed into milk and on the rumen microbiota in lactating dairy cows. The on-farm experiment was conducted on twelve lactating Czech Fleckvieh x Holstein cows divided into two groups, each with similar mean milk yield. Twice daily, cows were individually fed a diet based on maize silage, meadow hay and supplemental mixture. Control group (CTRL) received the basal diet while the experimental group (EXP) received the basal diet supplemented with 40% soybean isoflavone extract. The average daily isoflavone intake in the EXP group (16 g/day) was twice as high as that in the CTRL group (8.4 g/day, P<0.001). Total isoflavone concentrations in milk from the CTRL and EXP groups were 96.89 and 276.07 μg/L, respectively (P<0.001). Equol concentrations in milk increased from 77.78 μg/L in the CTRL group to 186.30 μg/L in the EXP group (P<0.001). The V3-4 region of bacterial 16S rRNA genes was used for metagenomic analysis of the rumen microbiome. The experimental cows exhibited fewer OTUs at a distance level of 0.03 compared to control cows (P<0.05) and reduced microbial richness compared to control cows based on the calculated Inverse Simpson and Shannon indices. Non-metric multidimensional scaling analysis showed that the major contributor to separation between the experimental and control groups were changes in the representation of bacteria belonging to the phyla Bacteroidetes, Proteobacteria, Firmicutes, and Planctomycetes. Surprisingly, a statistically significant positive correlation was found only between isoflavones and the phyla Burkholderiales (r = 0.65, P<0.05) and unclassified Betaproteobacteria (r = 0.58, P<0.05). Previous mouse and human studies of isoflavone effects on the composition of gastrointestinal microbial populations generally report similar findings.