Milk characteristics are affected by heat stress, but very little information is available on changes of milk protein fractions and their relationship with cheesemaking properties of milk. The main objective of the study was to evaluate the effect of hot season on milk protein fractions and cheesemaking properties of milk for Grana Padano cheese production. The study was carried out in a dairy farm with a cheese factory for transforming the milk to Grana Padano cheese. The study was carried out from June 2012 to May 2013. Temperature and relative humidity of the inside barn were recorded daily during the study period using 8 electronic data loggers programmed to record every 30 min. Constant managerial conditions were maintained during the experimental periods. During the experimental period, feed and diet characteristics, milk yield, and milk characteristics were recorded in summer (from June 29 to July 27, 2012), winter (from January 25 to March 8, 2013), and spring (from May 17 to May 31, 2013). Milk yield was recorded and individual milk samples were taken from 25 cows selected in each season during the p.m. milking. Content of fat, proteins, caseins (CN), lactose and somatic cell count (SCC), titratable acidity, and milk rennet coagulation properties were determined on fresh samples. Milk protein fraction concentrations were determined by the sodium dodecyl sulfate-PAGE. Data were tested for nonnormality by the Shapiro-Wilk test. In case of nonnormality, parameters were normalized by log or exponential transformation. The data were analyzed with repeated measures ANOVA using a mixed model procedure. For all the main milk components (fat, protein, total solids, and solids-not-fat), the lowest values were observed in the summer and the greatest values were observed in the winter. Casein fractions, with the exception of γ-CN, showed the lowest values in the summer and the greatest values in the winter. The content of IgG and serum albumin was greater in summer than in the winter and spring. A mild effect of season was observed for milk SCC, with greater values in summer than in the winter and spring. A worsening of milk coagulation properties was observed in summer season. The alteration of cheesemaking properties during hot season seems strictly linked with changes of milk protein fractions mainly with the decrease of αS-CN and β-CN and the increase of undefined proteins.
Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 μM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of HO exposure was assessed to evaluate and to compare the potential protection of different FA against HO-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by HO compared with other FA.
Fatty acids are important modulators of inflammatory responses, in particular, n-3 and n-6 essential fatty acids and CLA have received particular attention for their ability to modulate inflammation. The objectives of this study were to compare the effects of CLA and essential fatty acids on the expression of pro and anti- inflammatory cytokines and their protective efficacy against inflammatory status in mammary gland by an in vitro model based on bovine mammary epithelial cells (BME-UV1). Bovine mammary epithelial cells were treated with complete medium containing either 50 µM of cis-9, trans-11 CLA (c9,t11 CLA) or trans-10, cis-12 CLA (t10,c12 CLA) or (α)-linolenic acid (aLnA) or (γ)-linolenic acid (gLnA) or linoleic acid (LA). After 48 h by fatty acids administration the cells were treated for 3 h with 20 µM of lipopolysaccharide (LPS) to induce inflammatory stimulus. Reactive oxygen species (ROS) production after treatments was assessed to verify and to compare the potential protection of different fatty acids against LPS-induced oxidative stress. The messenger RNA abundance of bovine pro and anti-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukine-10 (IL-10)) and peroxisome proliferator receptor-α/γ (PPARγ/α) were determined in BME-UV1 by real-time PCR. The results showed that cells treated with fatty acids and LPS increased ROS production compared with control cells. Among treatments, cells treated with c9,t11 CLA and t10,c12 CLA isomers revealed significant lower levels of ROS production compared with other fatty acids. All fatty acids reduced the gene expression of pro- and anti-inflammatory cytokines. Among fatty acids, t10,c12 CLA, LA and gLnA showed an homogeneous reduction of the three pro-inflammatory cytokines and this may correspond to more balanced and efficient physiological activity and may trigger a better protective effect. The PPARγ gene expression was significantly greater in cells treated with t10,c12 CLA, aLnA and LA, whereas the PPARα gene expression levels were significantly lower in cells treated with all different fatty acids, compared with the control. These results suggest that fatty acids inhibited the transcription of pro-inflammatory cytokines by the upregulation of PPARγ expression.
Some studies have shown the protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation in animal models, but no information is available about CLA and changes in oxidative status of the bovine mammary gland. The objectives of the study were to assess in vitro the effect of CLA on the cellular antioxidant response of bovine mammary cells, to examine whether CLA isomers could play a role in cell protection against the oxidative stress, and to study the molecular mechanism involved. For the study, BME-UV1 cells, a bovine mammary epithelial cell line, were used as the experimental model. The BME-UV1 cells were treated with complete medium containing 50 µM cis-9,trans-11 CLA (c9,t11 CLA), trans-10,cis-12 CLA (t10,c12 CLA), and CLA mixture (1:1, cis-9,trans-11: trans-10,cis-12 CLA). To monitor cellular uptake of CLA isomers, cells and culture medium were collected at 0, 3, and 48 h from CLA addition for lipid extraction and fatty acid analyses. To assess the cellular antioxidant response, glutathione (GSH/GSSH), NADPH, and γ-glutamyl-cysteine ligase activity was measured after 48 h from addition of CLA. Cytoplasmic superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase activities and mRNA were also determined. Intracellular reactive oxygen species and thiobarbituric acid reactive substance production were assessed in cells supplemented with CLA isomers. Cell viability after 3h to H2O2 exposure was assessed to evaluate and to compare the potential protection of different CLA isomers against H2O2-induced oxidative stress. Mammary cells readily picked up all CLA isomers, their accumulation was time dependent, and main metabolites at 48 h are two 18:3 isomers. The CLA treatment induced an intracellular GSH increase, matched by high concentration of NADPH, and an increase of γ-glutamyl-cysteine ligase activity mainly in cells treated with the t10,c12 CLA isomer. The CLA isomer treatment of bovine mammary cells increased superoxide dismutase, glutathione peroxidase, and glutathione S-transferase activity and decreased glutathione reductase activity, but no changes in gene expression of these antioxidant enzymes were observed. Cells supplemented with CLA isomers showed a reduction in intracellular reactive oxygen species and thiobarbituric acid reactive substance levels. All CLA isomers were able to enhance cell resistance against H2O2-induced oxidative stress. These suggest an antioxidant role of CLA, in particular of t10,c12 CLA, by developing a significantly high redox status in cells.
(-)-Epigallocatechin-3-gallate (EGCG), the major phenolic compound of green tea, and hydroxytyrosol (HTyr), a phenol found in olive oil, have received attention due to their wide-ranging health benefits. To date, there are no studies that report their effect in bovine mammary gland. Therefore, the aim of this study was to evaluate the anti-oxidative and anti-inflammatory effects of EGCG and HTyr in bovine mammary epithelial cell line (BME-UV1) and to compare their antioxidant and anti-inflammatory in vitro efficacy. Sample of EGCG was obtained from a commercially available green tea extract while pure HTyr was synthetized in our laboratories. The mammary oxidative stress and inflammatory responses were assessed by measuring the oxidative stress biomarkers and the gene expression of inflammatory cytokines. To evaluate the cellular antioxidant response, glutathione (GSH/GSSH), γ-glutamylcysteine ligase activity, reactive oxygen species and malondialdehyde (MDA) production were measured after 48-h incubation of 50 µM EGCG or 50 µM of HTyr. Reactive oxygen species production after 3 h of hydrogen peroxide (50 µM H2O2) or lipopolysaccharide (20 µM LPS) exposure was quantified to evaluate and to compare the potential protection of EGCG and HTyr against H2O2-induced oxidative stress and LPS-induced inflammation. The anti-inflammatory activity of EGCG and HTyr was investigated by the evaluation of pro and anti-inflammatory interleukins (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10) messenger RNA abundance after treatment of cells for 3 h with 20 µM of LPS. Data were analyzed by one-way ANOVA. (-)-Epigallocatechin-3-gallate or HTyr treatments induced higher concentrations of intracellular GSH compared to control cells, matched by an increase of γ-glutamylcysteine ligase activity mainly in cells treated with HTyr. Interestingly, EGCG and HTyr prevented oxidative lipid damage in the BME-UV1 cells by a reduction of intracellular MDA levels. (-)-Epigallocatechin-3-gallate and HTyr were able to enhance cell resistance against H2O2-induced oxidative stress. It was found that EGCG and HTyr elicited a reduction of the three inflammatory cytokines TNF-α, IL-1β, IL-6 and an increase of the anti-inflammatory cytokine IL-10. Hydroxytyrosol has proved to be a strong antioxidant compound, and EGCG has shown mainly an anti-inflammatory profile. These results indicated that EGCG and HTyr may provide dual protection because they were able to attenuate oxidative stress and inflammatory responses, suggesting that these phenolic compounds are potential natural alternatives to be used in dairy cattle as feed supplement for reducing the development of oxidative and inflammatory processes related to parturition or as topical treatments for the control of bovine intramammary inflammation.
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