Increased milk levels of insulin-like growth factor 1 (IGF-1) for the identification of bovine somatotropin (bST) treated cows.

The present EU moratorium banning the use of bST to increase milk yield implies the need for official controls. Our study aimed to identify milk from bST treated cows via the induced increase of insulin-like growth factor 1 (IGF-1) concentrations. A non-extraction radioimmunoassay for IGF-1 was improved and thoroughly validated for milk. Accuracy was 99% recovery in a fortified sample material, the precision was 5.1% intra-assay variation and 13.4% inter-assay variation. Parallelism was proved by a dilution experiment which yielded a regression line with a slope (-0.7%) not significantly different from zero (P = 0.534). Naturally occurring milk IGF-1 levels were recorded in 5777 random milk samples from the Bavarian dairy cow population. In samples from lactation week 7 to 33, the effect of somatic cell count (SCC), protein content and parity could be quantified and corrected; thus a normal distribution (-0.068 mean +/- 0.440 s) of the corrected logarithmic IGF-1 levels (corr ln IGF-1) was obtained. IGF-1 concentrations occurring in milk from bST treated cows were recorded in 33 Brown Swiss cows treated once with rbST (POSILAC). Mean corr in IGF-1 levels increased by 0.828 and 0.477 in first parity and older cows, respectively. Thus 60% and 29%, respectively, of the positives could be detected at a 95% probability. If our results are confirmed in experiments with more bST treated cows and with prolonged treatment intervals. IGF-1 measurements might be useful to monitor for bST application in milk samples.     

The Improved Milk Quality and Enhanced Anti-Inflammatory Effect in Acetylserotonin-O-methyltransferase (ASMT) Overexpressed Goats: An Association with the Elevated Endogenous Melatonin Production

Background: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. Methods: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase (ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. Results: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. Conclusions: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.     

Calibration of somatic cell count in milk based on near-infrared spectroscopy

A PLS model for prediction of somatic cell count (SCC) based on near-infrared (NIR) spectra of unhomogenized milk is presented in the study. Samples of raw milk were collected from cows in the early lactation period (from 7th to 29th day after parturition). The NIR spectra were measured in the region 400–1100 nm. As reference method a fluoro-opto-electronic method was applied. Different preprocessing methods were investigated. The robust version of PLS regression was applied to handle outliers present in the dataset and the uninformative variable elimination–partial least squares (UVE–PLS) method was used to eliminate uninformative variables. The final model is acceptable for prediction of SCC in raw milk.     

Milk-Compositional Study of Metabolites and Pathogens in the Milk of Bovine Animals Affected with Subclinical Mastitis

Bovine milk is an important food component in the human diet due to its nutrient-rich metabolites. However, bovine subclinical mastitis alters the composition and quality of milk. In present study, California mastitis testing, somatic cell count, pH, and electrical conductivity were used as confirmatory tests to detect subclinical mastitis. The primary goal was to study metabolome and identify major pathogens in cows with subclinical mastitis. In this study, 29 metabolites were detected in milk using gas chromatography–mass spectrometry. Volatile acidic compounds, such as hexanoic acid, hexadecanoic acid, lauric acid, octanoic acid, n-decanoic acid, tricosanoic acid, tetradecanoic acid, and hypogeic acid were found in milk samples, and these impart good flavor to the milk. Metaboanalyst tool was used for metabolic pathway analysis and principal component estimation. In this study, EC and pH values in milk were significantly increased (p < 0.0001), whereas fat (p < 0.04) and protein (p < 0.0002) significantly decreased in animals with subclinical mastitis in comparison to healthy animals. Staphylococcus aureus was the predominant pathogen found (n = 54), followed by Escherichia coli (n = 30). Furthermore, antibiotic sensitivity revealed that Staphylococcus aureus was more sensitive to gentamicin (79.6%), whereas Escherichia coli showed more sensitivity to doxycycline hydrochloride (80%).     

Melatonin prevents magnetic-field-induced neurite outgrowth in a subline of pheochromocytoma cells,PC12D

Exposure to extremely low frequency (ELF) magnetic fields (MFs) has been reported to affect several cellular processes, including cell growth and differentiation. Other research has demonstrated that the pineal gland and its hormone melatonin have a wide spectrum of effects in cells and organs and can exert modulatory actions on cell proliferation and cell differentiation. Since ELF electric and magnetic fields have been shown to influence pineal activity and melatonin synthesis and/or function, it has been suggested that some of the reported effects of ELF MFs could be a consequence of the direct action of these fields on the pineal gland and/or melatonin function. Possible interactions between MFs and melatonin effects are in an early stage of investigation. In this study, we have investigated the influence of melatonin on the in vitro response of a subline of pheochromocytoma cells, PC12D, to a MF. Cells were exposed to the combined action of a physiological concentration (10⁻¹⁰ M) of melatonin and a vertical, 50 Hz, 40 mG rms MF for 23 h. At the end of the treatment, the percentages of neurite-bearing cells were determined by microscopic examination and compared with those from samples treated with the field alone or with melatonin alone. MF exposure alone significantly increased the neurite outgrowth when compared with negative controls, supporting previous results by Blackman and coworkers; this effect was not observed when melatonin was present in the medium from the onset of the exposure. Although the mechanisms of action of melatonin and ELF MFs at the cellular level remain unknown, the present data suggest that physiological levels of melatonin can prevent PC12D cells from responding to the MF stimulus.     

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk

In the dairy industry one of the most common frauds is mixing high-value milk (sheep’s and goats’) with milk of lower value (cows’). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows’ milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows’ and goats’ milk and cows’ and sheep’s milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows’ milk in sheep’s and goats’ milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

The Influence of Bacteria Causing Subclinical Mastitis on the Structure of the Cow’s Milk Microbiome

Mastitis is the most expensive disease of dairy cattle across the world and is the main reason for the use of antibiotics in animal husbandry. The aim of this study was to analyze the microbiome of raw milk obtained from a semi-subsistence farm located in the Kuyavian–Pomeranian Voivodeship in Poland. Milk from healthy cows and from cows with subclinical mastitis was analyzed. The following pathogenic bacteria were found in milk from individuals with subclinical mastitis: Escherichia coli or Streptococcus agalactiae. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on the physiological state of the cows. The microbiome of milk from healthy cows differed significantly from the milk from cows with subclinical mastitis. Two phyla dominated in the milk from healthy cows: Firmicutes and Proteobacteria, in equal amounts. On the contrary, in the milk from cows with diagnosed subclinical mastitis, one of the types dominated: either Firmicutes or Proteobacteria, and was largely predominant. Moreover, the milk microflora from the ill animals were characterized by lower values of the determined biodiversity indicators than the milk from healthy cows. The presence of pathogenic bacteria in the milk resulted in a significant reduction in the share of lactic acid bacteria in the structure of the population of microorganisms, which are of great importance in the production technology of regional products.     

Liquid chromatographic determination of tylosin in mastitic cow's milk following therapy

Liquid chromatographic analysis of milk samples from 6 cows treated with tylosin in a veterinary practice indicated that tylosin persisted in milk for more than 3 days after the final treatment. The concentration of tylosin was not below the stated maximum residue limit (0.05 mg/kg). The milk from 3 cows being treated for mastitis catarrhalis chronica contained tylosin residues for 3.5 days after the last withdrawal time (72 h). No residue was detected in the milk of any animal 6 days after cessation of therapy.     

代谢组学方法研究奶牛注射氯霉素后牛奶中的生物标志物

建立了高效液相色谱-质谱(HPLC-MS)技术研究牛奶代谢组学的方法.样品用乙腈提取,经浓缩富集,采用HPLC-MS技术研究了注射氯霉素后牛奶中内源性代谢物随时间的变化情况.结果表明,注射氯霉素后,牛奶的代谢指纹图谱中8个谱峰的变化随给药及休药时间呈现一定升降规律;采用偏最小二乘判别分析法(PLS-DA)可将给药组与空白组样品完全分开;鉴定了对组间差异贡献较大的化合物为单羟基十八碳二烯酸(HODE),HODE可以作为注射氯霉素后奶牛体内代谢应激产生的内源性生物标志物.     

Reference system for somatic cell counting in milk

With some analytical parameters, certified reference materials are lacking and the reference method shows limited performance. Somatic cell counting in milk is a clear example. It is one of the most frequently performed measurements, estimated at over 500 000 000?tests/year world wide. It serves as an indicator for the udder health status of lactating animals, is relevant in food legislation, in payment of raw milk, and also has a considerable impact for farm management and animal-breeding programs. The analytical performance of nowadays fluoro-opto-electronic routine methods in terms of precision is superior to the reference method based on microscopy. Laboratories have therefore adopted various solutions for anchoring their counting level. It is there that a reference system approach can serve to optimally safeguard comparability of routine testing results in laboratories world wide. A reference system is characterized as a systematically developed anchoring system that is fed by different types of information from various sources, that is, measurements on reference materials, reference method analysis, and routine method results. A joint Project Group of the International Dairy Federation and the International Committee on Animal Recording has been given the task to design a workable global reference system for somatic cell counting in milk. This paper describes the structure and the functioning of such a reference system, a plan for the implementation and the responsibilities of the involved stakeholders in safeguarding its functioning. After approval in IDF and ICAR, the resulting proposal is to be offered for adoption by concerned governmental and non-governmental bodies worldwide.     

Validation study of the BetaStar plus lateral flow assay for detection of beta-lactam antibiotics in milk

A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration, Center for Veterinary Medicine (FDA/CVM) was conducted for a receptor and antibody-based, immunochromatographic method (BetaStar Plus) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels, but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments (NCIMS). Results of the part I (internal) and part II (independent laboratory) dose-response studies employing spiked samples were in close agreement. The test was able to detect all six drugs at the approximate 90/95% sensitivity levels when presented as incurred residues in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing of 1031 control milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar Plus assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.     

Melatonin Reverses the Warburg-Type Metabolism and Reduces Mitochondrial Membrane Potential of Ovarian Cancer Cells Independent of MT1 Receptor Activation

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.     

The Involvement of PDE4 in the Protective Effects of Melatonin on Cigarette-Smoke-Induced Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a significant disease threatening human health. Currently, roflumilast, a phosphodiesterase (PDE)4 inhibitor, is recommended as a therapeutic agent for COPD. In this study, we investigated the therapeutic effects of melatonin against COPD, focusing on determining whether it is a PDE4 inhibitor via in vivo and in vitro experiment using cigarette smoke (CS) and cigarette smoke condensate (CSC), respectively. In the in vivo experiments, melatonin treatment reduced inflammatory responses, including inflammatory cell counts. Melatonin treatment also suppressed the CS-exposure-induced upregulation of cytokine and matrix metalloproteinase (MMP)-9, reduced the PDE4B expression, and elevated cAMP levels. In addition, these effects were synergistic, as melatonin and roflumilast cotreatment eventually ameliorated the CS-exposure-induced worsening of lung function. In the CSC-stimulated NCI-H292 cells, melatonin inhibited elevation in the levels of inflammatory cytokines, MMP-9, and PDE4, and elevated cAMP levels. Furthermore, melatonin and roflumilast cotreatment was more effective on inflammatory responses than only melatonin or roflumilast treatment. Our results indicate that melatonin relieves inflammatory response and loss of lung function in COPD, which is associated with decreased PDE4 expression. Therefore, we suggest that melatonin is a putative candidate for the treatment of COPD.     

Melatonin Induces Autophagy via Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress Pathway in Colorectal Cancer Cells

Even though an increasing number of anticancer treatments have been discovered, the mortality rates of colorectal cancer (CRC) have still been high in the past few years. It has been discovered that melatonin has pro-apoptotic properties and counteracts inflammation, proliferation, angiogenesis, cell invasion, and cell migration. In previous studies, melatonin has been shown to have an anticancer effect in multiple tumors, including CRC, but the underlying mechanisms of melatonin action on CRC have not been fully explored. Thus, in this study, we investigated the role of autophagy pathways in CRC cells treated with melatonin. In vitro CRC cell models, HT-29, SW48, and Caco-2, were treated with melatonin. CRC cell death, oxidative stress, and autophagic vacuoles formation were induced by melatonin in a dose-dependent manner. Several autophagy pathways were examined, including the endoplasmic reticulum (ER) stress, 5′–adenosine monophosphate-activated protein kinase (AMPK), phosphoinositide 3-kinase (PI3K), serine/threonine-specific protein kinase (Akt), and mammalian target of rapamycin (mTOR) signaling pathways. Our results showed that melatonin significantly induced autophagy via the ER stress pathway in CRC cells. In conclusion, melatonin demonstrated a potential as an anticancer drug for CRC.     

Natural Cryoprotective and Cytoprotective Agents in Cryopreservation: A Focus on Melatonin

Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the cryopreservation process by examining its drawbacks and the role of cytoprotective agents in protecting cell physiology. Natural cryoprotective and cytoprotective agents, such as antifreeze proteins, sugars and natural deep eutectic systems, have been compared with synthetic ones, addressing their mechanisms of action and efficacy of protection. The final part of this article focuses melatonin, a hormonal substance with antioxidant properties, and its emerging role as a cytoprotective agent for somatic cells and gametes, including ovarian tissue, spermatozoa and spermatogonial stem cells.     

Determination of fat, protein, casein, total solids, and somatic cell count in goat's milk by near-infrared reflectance spectroscopy

Analysis using near-infrared reflectance spectroscopy (NIRS) was investigated as a means of predicting quality parameters of goat's milk. Calibration equations were developed with samples from individual goats of Malague?a and Murciano-Granadina dairy breeds at different stages of lactation to obtain a wide range of variation in milk composition. Prediction equations for milk fat, protein, casein, total solids, and somatic cell count (SCC) were developed with 2 sample presentations, liquid drawer (homogenized milk) and aluminium cup (unhomogenized milk), to measure absorbance values, and accuracy of measurements was compared. The multiple correlation coefficient (R) values for homogenized milk were 0.98, 0.96, 0.91, 0.94, and 0.79 and for unhomogenized milk were 0.98, 0.95, 0.92, 0.95, and 0.74 for fat, protein, casein, total solid, and SCC, respectively. To validate the calibrations, an independent set of samples was used. The best simple correlation coefficient (r) values were 0.97, 0.95, 0.91, 0.93, and 0.72 for homogenized milk and 0.97, 0.95, 0.92, 0.95, and 0.79 for unhomogenized milk. The study showed that NIRS is a useful technique for the prediction of fat, protein, casein, total solids, and SCC in unhomogenized goat's milk.     

On-chip diagnosis of subclinical mastitis in cows by electrochemical measurement of neutrophil activity in milk

Subclinical mastitis is a common infectious disease affecting dairy cows. To develop an early diagnostic device for this disease, we focused on measuring an increase in the number of neutrophils in raw milk of mastitic cows. Superoxide anions (O(2)(-)), secreted by neutrophils, can be a good indicator of neutrophil concentration, and therefore, the seriousness of the mastitis. In this study, neutrophils in raw milk samples were separated from fat globules in a flow channel using differences in specific gravity and specific adhesion of neutrophils to P-selectin. Neutrophils trapped in the flow channel were subsequently concentrated in an array of micropillars of a working electrode modified with P-selectin and superoxide dismutase. The O(2)(-) secreted from the trapped neutrophils was electrochemically detected. A difference in the detection current was observed between normal and mastitic milk samples. A clear linear relationship between the electric current and cell density was observed.     

Determination of oxytocin in milk of cows administered oxytocin

To address people's concerns of exogenous oxytocin (OT) administration to lactating bovines, a study was undertaken to (a) establish an enzyme immunoassay (EIA) for OT determination in milk, (b) quantify OT in milk of cows administered OT, and (c) study influence of pasteurization on OT stability in milk. A sensitive EIA validated according to the criteria of European Union—Decision 2002/657/EC was developed for OT in skim milk in an analytical range of 10-250 pg mL⁻¹ with a decision limit (CCα) of 30 pg mL⁻¹ and detection capability (CCβ) of 41.5 pg mL⁻¹. Milk samples collected from cows (n = 38) administered either 25 or 50 IU OT prior to milking were investigated for the presence of OT. There was no significant difference among both groups with the mean concentrations of OT being 15.8 and 14.9 pg mL⁻¹ for cows subjected to 25 and 50 IU OT administration, respectively. The OT levels in skim milk of control cows (n = 30; untreated) were basal (around 10 pg mL⁻¹). All the analyzed milk samples were below the CCα value of 30 pg mL⁻¹. Pasteurization of OT spiked milk samples at different temperature and sample holding conditions reduced the immunological activity of OT to 43% at 110 °C. However, no further decline occurred in the immunological activity with increased pasteurization temperature and time. It was concluded that the milk OT concentrations after OT administrations were minimal and below the assay decision limit. However, OT was quite stable to pasteurization in OT spiked milk.     

Effects of diazepam administration on melatonin synthesis in the rat pineal gland in vivo.

The effect of diazepam (DZP) on melatonin synthesis in rat pineal gland was investigated in vivo. Subcutaneous injection of DZP (3 mg/kg) 1 h before the start of darkness significantly suppressed nocturnal elevations of pineal N-acetylserotonin (NAS) and melatonin contents in rats, and caused a 2-h delay in reaching the maximum melatonin level in the dark phase. DZP treatment also markedly suppressed the dark-induced increase of pineal N-acetyltransferase activity, which catalyzes the rate-limiting step in melatonin synthesis, but had no effect on hydroxyindole-O-methyltransferase activity, which catalyzes the final step of melatonin formation. Pineal norepinephrine and dopamine contents, in contrast, were not altered by DZP injection. The distribution rate of DZP to the brain reached the highest level 30 min after a single injection, while that to the pineal gland was observed 5 h later (i.e., 4 h after the start of darkness). It is clear that the inhibitory effect of DZP on melatonin synthesis in rat pineal gland appears concomitantly with the increase in the distribution volume of DZP into this gland. These results suggest that the inhibitory effect of DZP on melatonin synthesis results from the drug's direct action on the rat pineal gland.     

NMR-based metabolomics to evaluate the milk composition from Friesian and autochthonous cows of Northern Italy at different lactation times

It is well established that different factors affect milk composition in cows and that milk composition, in turn, affect both technological and nutritional qualities. In this respect the comprehension of the metabolic variability of milk composition in relation to the lactation time as well as to the genetic background may be of paramount importance for the agri-food industries. In the present study we investigated the variations of the metabolic profiles during lactation in milks obtained from Friesian and autochthonous races from Northern Italy by ¹H NMR metabolomics. Furthermore, the external factors influencing the milk composition were minimized: the cows were breeded in the same farm, were fed with the same diet and were paired for the lactation interval and lactation stage. Our results showed a difference in milk composition between races and in relation to late lactation. The PLS-DA analysis permitted to distinguish the Friesian and autochthonous cow milks at the investigated different lactation times. Interestingly, the metabolites significantly involved into the discrimination between races appeared to be also technological property parameters, highlighting the importance of maintaining the biodiversity of cow breeds. Therefore, NMR-based metabolomics of milk could represent an informative tool to identify metabolites involved in milk quality both from a nutritional and industrial perspective.