Strategic administration of antioxidant multiminerals and vitamins to transitional buffaloes augments antioxidant and udder defense mechanisms in early lactation.

The aim of the study was to examine the effects of repeated administrations of antioxidant multiminerals and vitamins in transition buffaloes on udder defense mechanism, antioxidant activity and occurrence of intramammary infection (IMI) in early lactation period. Forty clinically healthy pregnant buffaloes were enrolled 45 days before expected date of calving and randomly allocated into five different supplementation groups (n = 8): only basal ration (control), vitamin E and selenium (VES), multiminerals (MM), ascorbic acid (AA) and chromium (Cr) picolinate in basal diet. The udder defense mechanism was monitored by measuring phagocytic activity (PA), myeloperoxidase (MPO) and nitric oxide (NO) productions in milk leukocytes, antioxidant activity was evaluated by measuring total antioxidant capacity (TAC) in plasma and occurrence of IMI was assessed by milk cytology, bacterial count in milk and visible clinical signs of udder until day 28 post-calving. The results showed that the VES and MM supplementations exhibited significantly higher PA, MPO and NO productions of milk leukocytes till first week of lactation whereas, elevated mean TAC in plasma was maintained from day -7 to 1 of calving in MM supplementation group as compared to control group. Statistically, no significant difference in occurrences of subclinical or clinical IMI was noted across the groups until four weeks of lactation. Taken together, it is concluded that repeated administrations of VES and MM to transition buffaloes could be an effective strategy to maintain good udder health by augmenting milk leukocyte functions and antioxidant status and preventing incidence of IMI in early lactation.

Exploring the inhibitory impact of Mongolian medicinal He-Zi-3 soup on mammary gland hyperplasia in rats induced by estrogen and progestogen.

ETHNOPHARMACOLOGICAL RELEVANCE: Mammary gland hyperplasia, a prevalent benign breast condition, often serves as a precursor to various other breast diseases. He-Zi-3 soup (HZ-3), a traditional Mongolian remedy, is utilized for treating this condition. AIM OF THE STUDY: To explore the effect and underlying mechanism of HZ-3, a Mongolian medicinal preparation, on mammary gland hyperplasia. MATERIALS AND METHODS: This study aimed to assess the impact of different doses of HZ-3 in a rat model of mammary hyperplasia. The active components within HZ-3 drug serum were identified and analyzed through network pharmacology and target prediction. To elucidate the underlying mechanism of HZ-3 in addressing mammary hyperplasia, we conducted a series of investigations on estradiol-induced mammary hyperplasia in model rates. Assessments included measurements of papilla width and height, hematoxylin and eosin staining, Masson staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry. RESULTS: Our investigation revealed the identification of 21 compounds, primarily terpenoids, through serum medicinal chemistry screening. Utilizing network pharmacological analysis, we observed predominant regulation through the estrogen pathway, closely associated with key genes including esr1,esr2, ncoa1, krt 19, ctsd, ebag 9, and bcl-2. Assessments encompassing nipple height and width, histological examination, immunohistochemical analysis, and serum hormone levels via enzyme-linked immunosorbent assay demonstrated the inhibitory effect of HZ-3 on mammary hyperplasia in rat models. RT-qPCR and Western blot analyses corroborated these findings, affirming the suppression of mammary hyperplasia by HZ-3 through the activation of estrogen pathway signaling.

Factors affecting N-acetyl-ß-D-glucosaminidase as an indicator for mastitis detection in dairy sheep.

The study of new indirect methods for mastitis detection is of great relevance both at the economic level of the farm and dairies, and in terms of consumer health, and animal welfare. These methods help us to monitor the disease and speed up the decision-making process on treatment of the affected animal and the destination of the milk. The main aim of this work was to study the effect of intramammary infection and other non-infectious factors on the activity of the enzyme N-acetyl-ß-D-glucosaminidase (NAGase) in milk, in order to evaluate its use as an indicator for the early diagnosis of mastitis in sheep that could be less expensive, easier to measure and a better marker of inflammation or complementary to existing methods such as somatic cell count (SCC). Seven biweekly samplings were carried out, in which NAGase activity, SCC and milk were analyzed. Glands were classified according to their sanitary status based on the results of the SCC and bacteriological analysis. Non-infectious factors such as lactation stage, parity number and milking session had a statistically significant effect on NAGase values, finding the highest NAGase values at the onset and end of the study, in infectious mastitic glands of multiparous females and at morning milking. However, among the NAGase variation factors studied, the health status of the gland was the factor that caused the highest variation in enzyme levels, with infectious mastitic glands showing higher values than healthy glands. The predictive ability of NAGase was also studied by means of several logistic regression models, with the one that included NAGase together with lactation stage and parity obtaining the best results if sensitivity is to be prioritized, or the model that included NAGase, lactation stage, parity, milking and production if specificity is to be prioritized. From the results obtained, it can be concluded that the use of NAGase as an intramammary infection detection method in sheep can be useful when non-infectious factors that cause changes in the concentration of the enzyme are also considered.

1,25 dihydroxyvitamin D3-mediated effects on bovine innate immunity and on biofilm-forming Staphylococcus spp. isolated from cattle with mastitis.

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host's immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0-200 nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72 h. Concentrations of 0-100 mM for 24 h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24 h decreased the internalization of S. aureus V329 into MAC-T cells (0-100 nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10 nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200 nM of the metabolite for 24 h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0-200 nM for 12 and 24 h, respectively). In contrast, the conditioned media (0-100 nM for 24 h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host's endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol's potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated ß-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells.

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucine-enriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced ß-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of ß-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Utilizing intramammary Melaleuca alternifolia as an organic internal sealant for dry-off therapy in Murrah buffaloes.

The effects of intramammary dry cow therapy based on the administration of 5% Melaleuca alternifolia tea tree essential oil (TTO) as an internal teat sealant to Murrah cows were evaluated. A longitudinal prospective and retrospective negative control study was performed using 12 buffaloes from a total of 20 Murrah buffaloes on an organic farm, with the cow used as a control for herself. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for treatments with pure oil (TTO) and medication containing 5% TTO (O5) were determined. The buffaloes were clinically examined, and the teats were evaluated using thermography and ultrasound. Udder health was monitored during the first 100 days in milk (DIM) using milk somatic cell count (SCC) and California mastitis test (CMT). Laboratory tests against standard strains Staphylococcus aureus ATCC®25,923™, Escherichia coli ATCC®25,922™, and wild bacterial strains showed maximum MIC values of 50 µL/mL for the TTO and O5 treatments. One wild-type S. aureus strain showed no MBC. No adverse effects were observed after the intramammary application of TTO. The CMT and SCC values were similar (P > 0.05) for all observations. The medication containing 5% TTO was effective in vitro and compatible with the intramammary tissue in vivo of Murrah buffaloes. TTO was safe, not inducing inflammatory processes or other modifications of the teat detectable by thermography or ultrasound. It was able to protect buffaloes during the dry period under field conditions, demonstrating potential use as a teat sealant for organic farms.

Stearic acid promotes lipid synthesis through CD36/Fyn/FAK/mTORC1 axis in bovine mammary epithelial cells.

Stearic acid (C18:0, SA) is a saturated long-chain fatty acid (LCFA) that has a prominent function in lactating dairy cows. It is obtained primarily from the diet and is stored in the form of triacylglycerol (TAG) molecules. The transmembrane glycoprotein cluster of differentiation 36 (CD36) is also known as fatty acid translocase, but whether SA promotes lipid synthesis through CD36 and FAK/mTORC1 signaling is unknown. In this study, we examined the function and mechanism of CD36-mediated SA-induced lipid synthesis in bovine mammary epithelial cells (BMECs). SA-enriched supplements enhanced lipid synthesis and the FAK/mTORC1 pathway in BMECs. SA-induced lipid synthesis, FAK/mTORC1 signaling, and the expression of lipogenic genes were impaired by anti-CD36 and the CD36-specific inhibitor SSO, whereas overexpression of CD36 effected the opposite results. Inhibition of FAK/mTORC1 by TAE226/Rapamycin attenuated SA-induced TAG synthesis, inactivated FAK/mTORC1 signaling, and downregulated the lipogenic genes PPARG, CD36, ACSL1, SCD, GPAT4, LIPIN1, and DGAT1 at the mRNA and protein levels in BMECs. By coimmunoprecipitation and yeast two-hybrid screen, CD36 interacted directly with Fyn but not Lyn, and Fyn bound directly to FAK; FAK also interacted directly with TSC2. CD36 linked FAK through Fyn, and FAK coupled mTORC1 through TSC2 to form the CD36/Fyn/FAK/mTORC1 signaling axis. Thus, stearic acid promotes lipogenesis through CD36 and Fyn/FAK/mTORC1 signaling in BMECs. Our findings provide novel insights into the underlying molecular mechanisms by which LCFA supplements promote lipid synthesis in BMECs.

The use of a homeopathic preparation in the treatment of subclinical form of mastitis in cows.

Background: Mastitis is a disease of productive cows, widespread throughout the world, and characterized by significant economic damage to the dairy industry. The subclinical form of this disease is aggravated by additional difficulties with its diagnosis and the lack of clear treatment protocols. Aim: Therefore, the study of the effectiveness of diagnostic studies and the search for new methods of treatment of latent forms of mastitis is an important direction in scientific research in countries with developed dairy cattle breeding. Methods: Studies conducted on the number of dairy cows of the production cooperative "Izhevsky" of the Akmola region of the Republic of Kazakhstan showed that when using rapid tests Kenotest, Somatest, Mastidine test, and Wideside test, the same results were obtained when the disease was detected in cows. The effectiveness of the tests was at the level of 60%-62% when using the settling sample as a control. Medical procedures were carried out using the Aquaton-2 microwave radiation apparatus and a homeopathic preparation. When using physiotherapy with microwave radiation, a decrease in the level of microbial contamination of milk from the treated part of the udder by 1.5-5 times was observed. Results: Biologically active substances of plant origin in the homeopathic preparation, due to the immunostimulating effect, made it possible to increase the level of γ-globulins in the blood serum of sick animals during the application. Conclusion: The complex use of both methods in the treatment of animals with a subclinical form of mastitis made it possible to reduce the level of somatic cells in the milk of the affected udder lobe to a level that cannot be determined using Kenotest in 4-6 days, which is 2-4 days faster than using these methods separately.

Effects of dietary folic acid supplementation on lactation performance and mammary epithelial cell development of dairy cows and its regulatory mechanism.

The experiment investigated the impacts of FA on the proliferation of bovine mammary gland epithelial cells (BMECs) and to investigate the underlying mechanisms. Supplementation of 10 µM FA elevated the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin A2 and cyclin D1, and protein expression of PCNA and Cyclin A1. The mRNA and protein expression of B-cell lymphoma-2 (BCL2) and the BCL2 to BCL2 associated X 4 (BAX4) ratio elevated, while that of BAX, Caspase-3 and Caspase-9 reduced by FA. Both Akt and mTOR signaling pathways were activated by FA. Moreover, the stimulation of BMECs proliferation, the alteration of proliferative genes and protein expression, the change of apoptotic genes and protein expression, and the activation of mTOR signaling pathway caused by FA were obstructed by Akt inhibitor. Suppression of mTOR with Rapamycin reversed the FA-modulated promotion of BMECs proliferation and change of proliferous genes and protein expression, with no impact on mRNA or proteins expression related to apoptosis and FA-activated Akt signaling pathway. Supplementation of rumen-protected FA in cow diets evaluated milk yields and serum insulin-like growth factor-1 and estradiol levels. The results implied that the proliferation of BMECs was stimulated by FA through the Akt-mTOR signaling pathway.

Docosahexaenoic Acid Alters Lipid Metabolism Processes via H3K9ac Epigenetic Modification in Dairy Goat.

Goat milk is increasingly recognized by consumers due to its high nutritional value, richness in short- and medium-chain fatty acids, and richness in polyunsaturated fatty acids (PUFA). Exogenous supplementation of docosahexaenoic acid (DHA) is an important approach to increasing the content of PUFA in goat milk. Several studies have reported benefits of dietary DHA in terms of human health, including potential against chronic diseases and tumors. However, the mechanisms whereby an increased supply of DHA regulates mammary cell function is unknown. In this study, we investigated the effect of DHA on lipid metabolism processes in goat mammary epithelial cells (GMEC) and the function of H3K9ac epigenetic modifications in this process. Supplementation of DHA promoted lipid droplet accumulation increased the DHA content and altered fatty acid composition in GMEC. Lipid metabolism processes were altered by DHA supplementation through transcriptional programs in GMEC. ChIP-seq analysis revealed that DHA induced genome-wide H3K9ac epigenetic changes in GMEC. Multiomics analyses (H3K9ac genome-wide screening and RNA-seq) revealed that DHA-induced expression of lipid metabolism genes (FASN, SCD1, FADS1, FADS2, LPIN1, DGAT1, MBOAT2), which were closely related with changes in lipid metabolism processes and fatty acid profiles, were regulated by modification of H3K9ac. In particular, DHA increased the enrichment of H3K9ac in the promoter region of PDK4 and promoted its transcription, while PDK4 inhibited lipid synthesis and activated AMPK signaling in GMEC. The activation of the expression of fatty acid metabolism-related genes FASN, FADS2, and SCD1 and their upstream transcription factor SREBP1 by the AMPK inhibitor was attenuated in PDK4-overexpressing GMEC. In conclusion, DHA alters lipid metabolism processes via H3K9ac modifications and the PDK4-AMPK-SREBP1 signaling axis in goat mammary epithelial cells, providing new insights into the mechanism through which DHA affects mammary cell function and regulates milk fat metabolism.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Ozone use in the treatment of subclinical mastitis in dairy cows.

This research communication paper addresses the hypothesis that the use of therapeutic alternatives for mastitis, such as intramammary ozone, can cure the disease with lower costs and without harmful residues for human consumption and without formation of microbial resistance like the ones caused by indiscriminate use of antibiotics in dairy farms. The study was performed in 36 mammary quarters from 12 dairy cows with subclinical mastitis grade three. The experimental units were randomly assigned into four groups and each group received a treatment. Treatments comprised (a) 20 µg/ml ozone gas; (b) 40 µg/ml ozone gas; (c) negative control treatment of 12.5 µg/ml ozonated saline and (d) positive control treatment of 100 mg of cephalexin + 100 mg of neomycin + 10 mg of prednisolone, all by intramammary injection. In all quarters, milk was collected before and after the application of treatments for California mastitis test and evaluation of milk composition, somatic cell count, and bacterial cultures. The results indicated that the use of intramammary ozone did have a therapeutic effect, and whilst this was less than that of antibiotics, ozone does confer some advantages. Treated milk had a good composition, the treatment cost was low, milk withdrawal may not be necessary and there is no risk of antibiotic resistance.

Immune defenses of the mammary gland epithelium of dairy ruminants.

The epithelium of the mammary gland (MG) fulfills three major functions: nutrition of progeny, transfer of immunity from mother to newborn, and its own defense against infection. The defense function of the epithelium requires the cooperation of mammary epithelial cells (MECs) with intraepithelial leucocytes, macrophages, DCs, and resident lymphocytes. The MG is characterized by the secretion of a large amount of a nutrient liquid in which certain bacteria can proliferate and reach a considerable bacterial load, which has conditioned how the udder reacts against bacterial invasions. This review presents how the mammary epithelium perceives bacteria, and how it responds to the main bacterial genera associated with mastitis. MECs are able to detect the presence of actively multiplying bacteria in the lumen of the gland: they express pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) released by the growing bacteria. Interactions with intraepithelial leucocytes fine-tune MECs responses. Following the onset of inflammation, new interactions are established with lymphocytes and neutrophils recruited from the blood. The mammary epithelium also identifies and responds to antigens, which supposes an antigen-presenting capacity. Its responses can be manipulated with drugs, plant extracts, probiotics, and immune modifiers, in order to increase its defense capacities or reduce the damage related to inflammation. Numerous studies have established that the mammary epithelium is a genuine effector of both innate and adaptive immunity. However, knowledge gaps remain and newly available tools offer the prospect of exciting research to unravel and exploit the multiple capacities of this particular epithelium.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

Effects of glucose availability on αS1-casein synthesis in bovine mammary epithelial cells.

Glucose has been demonstrated to affect milk protein synthesis in dairy cows. However, its potential mechanisms has not been thoroughly studied. The objective of this study was to investigate the effects of glucose availability on αS1-casein synthesis, glucose uptake, metabolism, and the expression of proteins involved in AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells (BMEC). BMEC were treated for 24 h with different concentrations of glucose (0, 7, 10.5, 14, 17.5, and 21 mM). The results showed that 10.5 and 14 mM glucose supply increased the expression of αS1-casein, glucose uptake, cellular ATP content, and the phosphorylation of mTOR and P70S6K, but repressed AMPK phosphorylation in BMEC. Compared with 10.5 and 14 mM glucose supply, 17.5 and 21 mM glucose decreased the expression of αS1-casein, P70S6K phosphorylation as well as the activity of hexokinase (HK) and pyruvate kinase (PK), but increased the activity of glucose-6-phosphate dehydrogenase (G6PD). These results indicate that 10.5 to 14 mM glucose supply is the proper range for αS1-casein synthesis, and the promotion effects may be related to the increase of glucose uptake, ATP content and the changes of key proteins' phosphorylation in AMPK/mTOR signaling pathway. However, the inhibition of the expression of αS1-casein by 17.5 and 21 mM glucose may be associated with the changes of key enzymes' activity involved in glucose metabolism.

Glucose play an important role in milk protein synthesis in dairy cows. But the effects of glucose availability on casein synthesis and its underlying mechanisms has not been thoroughly studied. To elucidate the underlying mechanisms of glucose availability affecting casein synthesis, the effects of glucose availability on αS1-casein synthesis, glucose uptake, metabolism, and the expression of proteins involved in AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells were measured. We found that the expression of αS1-casein increased with 10.5 and 14 mM glucose supplementation, which may be associated with the increase of glucose uptake, ATP content and the changes of key proteins' phosphorylation in AMPK/mTOR signaling pathway. The inhibition of αS1-casein expression with 17.5 and 21 mM glucose supplementation may be related to the changes of key enzymes' activity involved in glucose metabolism. This study provided an insight into the potential mechanisms of glucose availability affecting milk protein synthesis.

Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells.

Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37 °C in customized Dulbecco's modified Eagle medium containing 5 mmol/L D-glucose, 1 mmol/L L-phenylalanine, L-[ring-2,4-3H]phenylalanine, 0.1 (control), 0.25, 0.5, 1, or 2 mmol/L L-leucine, L-isoleucine or L-valine or an equimolar mixture of the three BCAAs. The culture medium also contained physiological concentrations of other amino acids found in the plasma of lactating sows. Proliferation, protein synthesis, proteolysis, ß-casein production, the mechanistic target of rapamycin (mTOR) signaling, and the ubiquitin-proteasome pathway were determined for PMECs. Cell proliferation and abundances of phosphorylated mTOR, eukaryotic translation initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase ß-1 proteins increased (P < 0.05), but abundances of ubiquitinated protein and 20S proteasome decreased (P < 0.05) when extracellular concentrations of L-leucine, L-isoleucine, L-valine, or an equimolar mixture of BCAAs were increased from 0.1 to 2 mmol/L. Compared with the control, 0.25, 0.5, 1 or 2 mmol/L BCAAs enhanced (P < 0.01) protein (including ß-casein) synthesis, while decreasing (P < 0.05) proteolysis in PMECs in a dose-dependent manner. Collectively, our results indicate that physiological concentrations of BCAAs regulate protein turnover in mammary epithelial cells to favor net protein synthesis through stimulating the mTOR signaling pathway and inhibiting the ubiquitin-proteasome pathway.

Symposium review: Environmental effects on mammary immunity and health.

Environmental effects on pathogen abundance and access are precursors to mastitis. Indeed, high heat and humidity, and unsanitary housing and equipment, are associated with greater pathogen load and exposure. Although less is known about effects of environment on a cow's ability to resist infection, several indicators suggest that it can affect pathogen responses. Mastitis incidence and bulk tank somatic cell count vary with season, typically peaking in summer. Recent controlled studies have revealed that heat stress exposure results in changes in the microbiome of the cow and her environment, which may relate to negative effects on milk quality and cow health. Alternatively, specific pathogen loads may vary based on housing dynamics rather than associations with physical environment. Indeed, housing-related stressors, such as overcrowding and social group challenge, influence secretion of glucocorticoids, thus affecting pathogen resistance in the cow. Two key seasonal variables are photoperiod and temperature, specifically the heat stress consequent to elevated temperature and humidity. Shifts in light duration regulate immune function in other species, but apparently have limited effect on udder health of lactating cows. In contrast, in dry cows, short days increase peripheral blood mononuclear cell number and are associated with lower somatic cell count in the next lactation, compared with long days. With heat stress, elevated body temperature directly affects expression of immune-related genes in mammary tissue. Responses depend on duration of exposure and feature acute upregulation of immune-signaling pathways, followed by enrichment of other immune-related pathways after prolonged exposure. Most responses are transient and recover within 1 wk. Functionally, heat stress impairs some aspects of acquired immunity in dry cows, including antigen responses and lymphocyte proliferation, but apparently not innate immune function. However, heat stress in late gestation reduces neutrophil phagocytosis and killing in vitro, and neutrophils in circulation are reduced in vivo as are responses to pathogen challenge in the subsequent lactation. A holistic understanding of the complex interplay of environment, pathogens, and host is needed to inform advances in this area.

Lactococcus lactis subsp lactis CRL1655 and Schleiferilactobacillus perolens CRL1724 inhibit the adherence of common bovine mastitis pathogens to mammary gland cells, without causing histological changes in the mammary gland.

AIMS: The present work assessed the ability of two selected lactic acid bacteria (LAB) strains (Schleiferilactobacillus perolens CRL1724 and Lactococcus lactis subsp. lactis CRL1655) to inhibit the adherence of bovine mastitis pathogens to mammary epithelial cells (MAC-T) and their effects (if any) on the structure of the gland after intramammary inoculation at dry-off. METHODS AND RESULTS: Established bovine mammary epithelial cells (MAC-T) were used to assess the LAB strains' ability to inhibit the adherence of bovine mastitis pathogens. Monolayers of MAC-T cells were co-cultured with the LABs and then individual pathogen was added. Both strains prevented the adherence of S. aureus RC108, S. chromogenes, S. uberis UT102 and E. coli ATCC 35218. Adherence of the latter two pathogens was inhibited most strongly in vitro. To evaluate the effect of the LAB on the structure of the bovine udders, quarters were intramammary inoculated with the LAB mixture at dry-off. After slaughtering, the teats were dissected and histopathologically analysed. No modifications were identified post-inoculation in the structure of the epithelial, subepithelial and connective tissues of the mammary gland. CONCLUSIONS: Probiotic strains L. lactis subsp lactis CRL1655 and S. perolens CRL1724 were both able to inhibit the adherence of a number of bovine mastitis pathogens in vitro, and that the intramammary inoculation of these strains at the established dose and concentration did not cause significant alterations in the mammary epithelium nor had undesirable effects on tissues, and may therefore be considered harmless. SIGNIFICANCE AND IMPACT OF STUDY: The promising findings demonstrated in this work support the potential of probiotic micro-organisms as a natural and effective alternative to prevent bovine mastitis during the dry-off period.

Comparison of effect of parenteral and oral supplementation of Selenium and vitamin E on selected antioxidant parameters and udder health of dairy cows.

The aim of this study was to compare the effect of parenteral and oral supplementation of Selenium (Se) and vitamin E (VTE) on selected antioxidant parameters in blood and colostrum as well as their effect on the incidence of mastitis in dairy cows during the final phase of gravidity (6 weeks) and first two weeks after calving. For the practical part of the study 36 dairy cows of Slovak pied breed in the second to fourth lactation-gestation cycle were selected. The animals weredivided into three groups: the control (C) and two experimental groups (D1 and D2). The selected groups were treated as follows: in group D1 products containing Se (Selevit inj.) and vitamin E (Erevit sol. inj.) were administered intramuscularly twice, six and three weeks prior to parturition; in group D2 a vitamin-minerals supplement in the form of sodium selenite (Na2SeO3) and dl-α-tocopherol acetate were supplemented orally for six weeks calving. The blood samples were collected from the vena jugularis in dairy cows approximately 42 days before calving (control sampling), on parturition day, and the 14th day after calving. Higher concentrations of Se and VTE were found in the blood plasma samples of both experimental groups collected on the day of parturition. In addition, the orally supplemented group (D2) showed higher Se and α-tocopherol concentrations in blood plasma on the14th day after calving as well a reduction of occurrence of mastitis by about 25 % compared to the control group. The relationship between inflammatory response and oxidative stress was also confirmed. The concentrations of milk malondialdehyde indicating lipid peroxidation during mastitis were significantly higher in milk samples from infected cows than in milk samples from healthy animals in each monitored group. In order to prevent oxidative stress and moderate inflammatory response in dairy cows it is very important to optimally balance their nutritive needs with an appropriate ratio of Se and VTE supplements. Therefore we still recommend supplementation of the cows' postpartum dietwith 0.5 mg of Se/kg dry matter (DM) and 102 mg of dl-α-tocopherol acetate/kg DM to stabilize their optimal blood levels, stimulate the activity of glutathione peroxidase and reduce the incidence of mastitis.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions.

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 µM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 µM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 µM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.