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BACKGROUND: Mastitis, a pervasive and detrimental disease in dairy farming, poses a significant challenge to the global dairy industry. Monitoring the milk somatic cell count (SCC) is vital for assessing the incidence of mastitis and the quality of raw cow's milk. However, existing SCC detection methods typically require large-scale instruments and specialized operators, limiting their application in resource-constrained settings such as dairy farms and small-scale labs. To address these limitations, this study introduces a novel, smartphone-based, on-site SCC testing method that leverages smartphone capabilities for milk somatic cell identification and enumeration, offering a portable and user-friendly testing platform. RESULTS: The central findings of our study demonstrate the effectiveness of the proposed method for counting milk somatic cells. Its on-site applicability, facilitated by the microfluidic chip, optical system, and smartphone integration, heralds a paradigm shift in point-of-care testing (POCT) for dairy farms and smaller laboratories. This approach bypasses complex processing and presents a user-friendly solution for real-time SCC monitoring in resource-limited settings. This device boasts several unique features: small size, low cost (<$1,000 total manufacturing cost and <$1 per test), and high accuracy. Remarkably, it delivers test results within just 2 min. Actual-sample testing confirmed its consistency with results from the commercial Bentley FTS/FCM cytometer, affirming the reliability of the proposed method. Overall, these results underscore the potential for transformative change in dairy farm management and laboratory testing practices. SIGNIFICANCE: In summary, this study concludes that the proposed smartphone-based method significantly contributes to the accessibility and ease of SCC testing in resource-limited environments. By fostering the use of POCT technology in food safety control, particularly in the dairy industry, this innovative approach has the potential to revolutionize the monitoring and management of mastitis, ultimately benefiting the global dairy sector.

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The production of milk by dairy cows far exceeds the nutritional needs of the calf and is vital for the economical use of dairy cattle. High milk yield is a unique production trait that can be effectively enhanced through traditional selection methods. The process of lactation in cows serves as an excellent model for studying the biological aspects of lactation with the aim of exploring the mechanistic base of this complex trait at the cellular level. In this study, we analyzed the milk transcriptome at the single-cell level by conducting scRNA-seq analysis on milk samples from two Holstein Friesian cows at mid-lactation (75 and 93 days) using the 10× Chromium platform. Cells were pelleted and fat was removed from milk by centrifugation. The cell suspension from each cow was loaded on separate channels, resulting in the recovery of 9313 and 14,544 cells. Library samples were loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument. After filtering at the cell and gene levels, a total of 7988 and 13,973 cells remained, respectively. We were able to reconstruct different cell types (milk-producing cells, progenitor cells, macrophages, monocytes, dendritic cells, T cells, B cells, mast cells, and neutrophils) in bovine milk. Our findings provide a valuable resource for identifying regulatory elements associated with various functions of the mammary gland such as lactation, tissue renewal, native immunity, protein and fat synthesis, and hormonal response.

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The present study focused on Sahiwal cows, a prominent milch breed in tropical India, to correlate udder temperature with physiological markers of stress and inflammation during subclinical mastitis (SCM). The primary goal was to assess the potential of udder infrared thermography for the early detection of SCM under the semi-intensive production. Cows were categorized based on milk somatic cell counts (SCC), with healthy (H) cows having SCC <2 × 105 cells/mL and no history of mastitis, and cows with subclinical mastitis (SCM) and initial stages of clinical mastitis (CM) having quarter milk SCC of 2-5 × 105 and >5 × 105 cells/mL, respectively. Firstly, udder thermograms were analysed for udder skin surface temperature (USST), teat skin surface temperature (TSST), and teat apex temperature (TAT) using Fluke software to determine the optimal site for temperature measurement during intramammary infection. Secondly, milk samples were collected for automatic estimation of compositional changes, electrical conductivity, and pH. Thirdly, milk whey was separated for quantifying stress and inflammatory indicators, including cortisol, prolactin, and acute-phase proteins (APPs): milk amyloid A and milk haptoglobin using bovine-specific ELISA kits. Significant increases (p < 0.01) in USST, TSST, TAT, cortisol, and APPs were observed in SCM and CM compared to healthy cows, while prolactin levels decreased (p < 0.01). The correlation matrix revealed strong positive correlations of SCC with USST (r = 0.84, p < 0.01). In ROC analysis, USST demonstrated cut-off values of 37.74 and 39.58 °C, with accuracy (p < 0.05) of 98% for SCM and 95% for CM, surpassing both TAT and TSST. Therefore, the combination of these non-invasive methods increases the reliability and accuracy of infrared thermography for early detection of SCM, providing valuable insights for the development of a protocol for routine screening and udder health monitoring in indigenous dairy cows.

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Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae-infected versus healthy; (2) Prototheca-infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae-infected versus Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae-infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca-infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations.

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Bovines infected by bovine leukemia virus (BLV) are characterized by presenting low proviral load (LPL) or high proviral load (HPL). It is reported that animals with HPL in peripheral blood mononuclear cells (PBMCs) present a decrease in apoptosis, an increase in viability and the proliferation rate, while animals that maintain an LPL have an intrinsic ability to control the infection, presenting an increased apoptosis rate of their PBMCs. However, there is little information on the effect of BLV on these mechanisms when the virus infects somatic milk cells (SC). This study investigates the mechanisms underlying apoptosis in milk and blood from BLV-infected animals with HPL and LPL. Relative levels of mRNA of tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNF-RI), TNF receptor 2 (TNF-RII), anti-apoptotic B-cell lymphoma 2 protein (Bcl-2), and pro-apoptotic Bcl-2-like protein 4 (Bax) were measured in SC and PBMCs using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. A significant decrease in the expression of TNF-α in SC from HPL animals vs non-infected bovines was observed, but the infection in SC with BLV did not show a modulation on the expression of TNF receptors. A significant increase in TNF-RI expression in PBMCs from HPL bovines compared to LPL bovines was observed. No significant differences in PBMCs between HPL and LPL compared to non-infected animals concerning TNF-α, TNF-RI, and TNF-RII expression were found. There was a significant increase of both Bcl-2 and Bax in SC from LPL compared to non-infected bovines, but the Bcl-2/Bax ratio showed an anti-apoptotic profile in LPL and HPL bovines compared to non-infected ones. Reduced mRNA expression levels of Bax were determined in the PBMCs from HPL compared to LPL subjects. In contrast, BLV-infected bovines did not differ significantly in the mRNA expression of Bax compared to non-infected bovines. Our data suggest that the increased mRNA expression of Bax corresponds to the late lactation state of bovine evaluated and the exacerbated increase of mRNA expression of Bcl-2 may be one of the mechanisms for the negative apoptosis regulation in the mammary gland induced by BLV infection. These results provide new insights into the mechanism of mammary cell death in HPL and LPL BLV-infected bovine mammary gland cells during lactation.

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BACKGROUND: Subclinical intramammary infection (IMI) represents a significant problem in maintaining dairy cows' health. Disease severity and extent depend on the interaction between the causative agent, environment, and host. To investigate the molecular mechanisms behind the host immune response, we used RNA-Seq for the milk somatic cells (SC) transcriptome profiling in healthy cows (n = 9), and cows naturally affected by subclinical IMI from Prototheca spp. (n = 11) and Streptococcus agalactiae (S. agalactiae; n = 11). Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) was used to integrate transcriptomic data and host phenotypic traits related to milk composition, SC composition, and udder health to identify hub variables for subclinical IMI detection. RESULTS: A total of 1,682 and 2,427 differentially expressed genes (DEGs) were identified when comparing Prototheca spp. and S. agalactiae to healthy animals, respectively. Pathogen-specific pathway analyses evidenced that Prototheca's infection upregulated antigen processing and lymphocyte proliferation pathways while S. agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle, and carbohydrate and lipid metabolism. The integrative analysis of commonly shared DEGs between the two pathogens (n = 681) referred to the core-mastitis response genes, and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells (r2 = 0.72), followed by the udder health (r2 = 0.64) and milk quality parameters (r2 = 0.64). Variables with r ≥ 0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cytohubba plug-in. The genes in common between DIABLO and cytohubba (n = 10) were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals (sensitivity > 0.89, specificity > 0.81, accuracy > 0.87, and precision > 0.69). Among these genes, CIITA could play a key role in regulating the animals' response to subclinical IMI. CONCLUSIONS: Despite some differences in the enriched pathways, the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response. The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

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Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection.

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Somatic cell count (SCC) is a fundamental approach for determining the quality of cattle and bovine milk. So far, different classification and recognition methods have been proposed, all with certain limitations. In this study, we introduced a new deep learning tool, i.e., an improved ResNet50 model constructed based on the residual network and fused with the position attention module and channel attention module to extract the feature information more effectively. In this paper, macrophages, lymphocytes, epithelial cells, and neutrophils were assessed. An image dataset for milk somatic cells was constructed by preprocessing to increase the diversity of samples. PolyLoss was selected as the loss function to solve the unbalanced category samples and difficult sample mining. The Adam optimization algorithm was used to update the gradient, while Warm-up was used to warm up the learning rate to alleviate the overfitting caused by small sample data sets and improve the model's generalization ability. The experimental results showed that the classification accuracy, precision rate, recall rate, and comprehensive evaluation index F value of the proposed model reached 97%, 94.5%, 90.75%, and 92.25%, respectively, indicating that the proposed model could effectively classify the milk somatic cell images, showing a better classification performance than five previous models (i.e., ResNet50, ResNet18, ResNet34, AlexNet andMobileNetv2). The accuracies of the ResNet18, ResNet34, ResNet50, AlexNet, MobileNetv2, and the new model were 95%, 93%, 93%, 56%, 37%, and 97%, respectively. In addition, the comprehensive evaluation index F1 showed the best effect, fully verifying the effectiveness of the proposed method in this paper. The proposed method overcame the limitations of image preprocessing and manual feature extraction by traditional machine learning methods and the limitations of manual feature selection, improving the classification accuracy and showing a strong generalization ability.

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Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes - the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

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Despite the known benefits of breastfeeding for both infant and mother, clinical support for problems such as benign inflammation of the lactating breast remain a research frontier. Breast pain associated with inflammation is a common reason for premature weaning. Multiple diagnoses are used for benign inflammatory conditions of the lactating breast which lack agreed or evidence-based aetiology, definitions, and treatment. This article is the second in a three-part series. This second review analyses the heterogeneous research literature concerning benign lactation-related breast inflammation from the perspectives of the mechanobiological model and complexity science, to re-think classification, prevention, and management of lactation-related breast inflammation. Benign lactation-related breast inflammation is a spectrum condition, either localized or generalized. Acute benign lactation-related breast inflammation includes engorgement and the commonly used but poorly defined diagnoses of blocked ducts, phlegmon, mammary candidiasis, subacute mastitis, and mastitis. End-stage (non-malignant) lactation-related breast inflammation presents as the active inflammations of abscess, fistula, and septicaemia, and the inactive condition of a galactocoele. The first preventive or management principle of breast inflammation is avoidance of excessively high intra-alveolar and intra-ductal pressures, which prevents strain and rupture of a critical mass of lactocyte tight junctions. This is achieved by frequent and flexible milk removal. The second preventive or management principle is elimination of the mechanical forces which result in high intra-alveolar pressures. This requires elimination of conflicting vectors of force upon the nipple and breast tissue during milk removal; avoidance of focussed external pressure applied to the breast, including avoidance of lump massage or vibration; and avoidance of other prolonged external pressures upon the breast. Three other key preventive or management principles are discussed. Conservative management is expected to be effective for most, once recommendations to massage or vibrate out lumps, which worsen micro-vascular trauma and inflammation, are ceased.

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Despite the known benefits of breastfeeding for both infant and mother, clinical support for problems such as inflammation of the lactating breast remains a research frontier. Breast pain associated with inflammation is a common reason for premature weaning. Multiple diagnoses are used for inflammatory conditions of the lactating breast, such as engorgement, blocked ducts, phlegmon, mammary candidiasis, subacute mastitis, mastitis and white spots, which lack agreed or evidence-based aetiology, definitions and treatment. This is the first in a series of three articles which review the research literature concerning benign lactation-related breast inflammation. This article investigates aetiological models. A complex systems perspective is applied to analyse heterogeneous and interdisciplinary evidence elucidating the functional anatomy and physiology of the lactating breast; the mammary immune system, including the human milk microbiome and cellular composition; the effects of mechanical forces during lactation; and the interactions between these. This analysis gives rise to a mechanobiological model of breast inflammation, in which very high intra-alveolar and intra-ductal pressures are hypothesized to strain or rupture the tight junctions between lactocytes and ductal epithelial cells, triggering inflammatory cascades and capillary dilation. Resultant elevation of stromal tension exerts pressure on lactiferous ducts, worsening intraluminal backpressure. Rising leucocyte and epithelial cell counts in the milk and alterations in the milk microbiome are signs that the mammary immune system is recruiting mechanisms to downregulate inflammatory feedback loops. From a complex systems perspective, the key mechanism for the prevention or treatment of breast inflammation is avoidance of excessively high intra-alveolar and intra-ductal pressures, which prevents a critical mass of mechanical strain and rupture of the tight junctions between lactocytes and ductal epithelial cells.

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In dairy sheep industry, milk production dictates the value of a ewe. Milk production is directly related to the morphology and physiology of the mammary gland; both being designated targets of breeding strategies. Although within a flock breeding parameters are mutual, large differences in milk production among individual ewes are usually observed. In this work, we tested two of the most productive dairy sheep breeds reared intensively in Greece, one local the Chios breed and one foreign the Lacaune breed. We used transcriptome sequencing to reveal molecular mechanisms that render the mammary gland highly productive or not. While highly expressed genes (caseins and major whey protein genes) were common among breeds, differences were observed in differentially expressed genes. ENSOARG00000008077, as a member of ribosomal protein 14 family, together with LPCAT2, CCR3, GPSM2, ZNF131, and ASIP were among the genes significantly differentiating mammary gland's productivity in high yielding ewes. Gene ontology terms were mainly linked to the inherent transcriptional activity of the mammary gland (GO:0005524, GO:0030552, GO:0016740, GO:0004842), lipid transfer activity (GO:0005319) and innate immunity (GO:0002376, GO:0075528, GO:0002520). In addition, clusters of genes affecting zinc and iron trafficking into mitochondria were highlighted for high yielding ewes (GO:0071294, GO:0010043). Our analyses provide insights into the molecular pathways involved in lactation between ewes of different performances. Results revealed management issues that should be addressed by breeders in order to move toward increased milk yields through selection of the desired phenotypes. Our results will also contribute toward the selection of the most resilient and productive ewes, thus, will strengthen the existing breeding systems against a spectrum of environmental threats.

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Polymorphisms of milk protein genes have been proposed as candidate markers for dairy production traits in cattle. In the present study, a polymorphism was detected in the 5'-flanking (promoter) region of the bovine alpha-lactalbumin (LALBA) gene, a T/C transition located at nucleotide -1,001 relative to the transcription start site g.-1001T > C (NC_037332.1:g.31183170T > C), which is recognizable with PstI restriction endonuclease. In silico analyses showed that this mutation created novel retinoid X receptor alpha and vitamin D receptor transcription factor binding sites. Real-time PCR found that cows with different genetic variants of the promoter demonstrated different levels of expression of LALBA mRNA in milk somatic cells (MSCs). The TT genotype cows demonstrated low expression, whereas those with CT demonstrated much higher expression (P < 0.05). ELISA analysis found milk LALBA protein levels also differed between the TT and CT cows (P < 0.05) and that these levels were not correlated with the mRNA abundance in MSC. Association analysis found that the g.-1001T > C polymorphism in the promoter region of the LALBA gene influenced milk production traits in Polish Holstein-Friesian cows. High daily milk yield and dry matter yield, and high lactose yield and concentration were associated with the TT genotype. The TT genotype cows also had a lower number of somatic cells in the milk, considered as an indicator of udder health status. Therefore, the TT genotype could be more desirable from the breeder's perspective.

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Flow cytometry is a powerful technology used in many fields of cell biology. It is also used as a routine method to count somatic cells in milk and to characterize bovine milk leukocytes. In this study, we used flow cytometry to simultaneously assess the viability, the percentage of the single subsets of leukocytes and to quantify the expression of CD11b, an immunological marker of cell activation status. Immunological markers were then related with on farm recorded parameters as milk electrical conductivity (MEC) and average milk flow rate (MFR). Composite milk samples were collected from 43 cows, nine of which had naturally infected udders and 34 of which had no infected udders. First, the milk samples were classified according to bacteriological test in positive and negative. The results showed that the negative samples to bacteriological test had: (i) significantly higher percentages of live lymphocytes; (ii) significantly lower percentages of CD11b+ leukocytes; (iii) significantly lower MEC and higher MFR values. Then, samples were classified in three groups according to somatic cell count (SCC): Group A (n = 15), samples with SCC ≤ 100,000 cells/mL, all negative to bacteriological analysis; Group B (n = 11), samples with 100,000 < SCC < 300,000 cells/m, with four samples positive to bacteriological analysis; Group C (n = 17), samples with SCC ≥ 300,000 cell/mL with five samples positive to bacteriological analysis. Multivariate discriminant analysis was used to verify which flow cytometry immunological markers and on farm recorded parameters could better discriminate among the different groups of SCCs. Linear discriminant analysis (LDA) indicated that 5 of the 10 parameters could best be used to reveal the differences between positive and negative samples among the considered groups of SCCs. Furthermore, the Canonical discriminant analysis (CDA) indicated that composite milk samples with different SCC and infection status were clearly separated from each other in a two-dimensional space. In conclusion, the study highlighted that: (1) the conventional flow cytometry analysis applied on milk samples is a useful tool to investigate immunological parameters as potential indicators of udder health; (2) the combined evaluation of live milk leukocytes and recorded farm parameters could be useful to assess udder health status in dairy cows. The results obtained from this pilot study on few data require new and larger trials to be confirmed.

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The identification of genes involved in phenotypes related to milk quality is important for both economic and health aspects in livestock production. The aim of this study was to assess the level of gelsolin gene expression in two breeds of dairy sheep - Sarda and Gentile - with pronounced differences in quantitative and qualitative milk traits. Gelsolin, a type of actin-modulating proteins is involved in the processes of actin remodeling during cell growth and apoptosis; therefore a role of this protein in mammary changes during lactation was here hypothesized. Individual milk samples were collected three times during lactation from 26 ewes of the two breeds. The differential gene expression of gelsolin in the two breeds and the three lactation times was estimated by quantitative PCR on RNA extracted from milk somatic cells. Correlations of gelsolin gene expression with milk yield and quality and days of lactation were also estimated. The results showed that gelsolin gene expression was significantly higher in the Sarda compared to the Gentile at each lactation stage, in agreement with the longer lactation duration and the higher daily milk yield of the first breed. Significant correlations of gelsolin gene expression were found with milk fat content in Sarda breed (-0.46, P<0.05). Gelsolin expression analysis confirmed the link between gelsolin gene function and milk fat content of sheep.

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This study analyzed effects of vegetable oils fed to dairy cows on abundance of genes related to lipid metabolism in milk somatic cells (MSC). During 63 days, 15 cows were allocated to 3 treatments: a control diet with no added lipid the same diet supplemented with olive oil (OO, 30 g/kg DM) or hydrogenated vegetable oil (HVO, 30 g/kg DM). On days 21, 42 and 63, MSC were obtained from all cows. Relative abundance of genes involved in lipid metabolism in MSC from cows fed control on days 42 and 63 was compared with relative abundance at day 21 to evaluate fold-changes. Those genes without changes over the time were selected to analyze effects of OO and HVO. Compared with control, on day 42, PLIN2 and THRSP were upregulated by OO. Compared with control, on day 21, HVO up regulated ACACA, down regulated FABP3, and on day 63 THRSP and FABP4 were down regulated. Dietary oil supplementation (3% DM) had a modest nutrigenomic effect on different biological functions such as acetate and FA activation and intra-cellular transport, lipid droplet formation, and transcription regulation in MSC.

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Supplying dietary zinc in excess of traditional requirements has clear impacts on the gut epithelium, but little research has explored whether similar impacts on the mammary epithelium may occur. Our objective was to determine the effects of supplemental Zn sources, in excess of minimal requirements, on markers of mammary epithelial integrity in blood and in milk as well as the heat stability of milk in mid-lactation cows. Twelve multiparous Holstein cows (132 ± 21 days in milk and 51 ± 3 kg/day milk) were blocked according to milk yield and enrolled in a replicated 3 × 3 Latin square experiment. Experimental periods were 21 days, with 17 days allowed for diet adaptation and 4 days for sampling. Treatment sequences were randomly assigned to animals and treatments were as follows: (1) 0.97 g Zn/day provided as ZnSO4 (34.5 mg supplemental Zn/kg diet DM; 30-ZS), (2) 1.64 g Zn/day provided as ZnSO4 (56.5 mg supplemental Zn/kg diet DM; 60-ZS), and (3) 0.55 g Zn/day provided as ZnSO4 plus 1.13 g Zn/day provided as a zinc-methionine complex (58.2 mg supplemental Zn/kg diet DM; 60-ZM). Treatments were administered once daily as an oral bolus containing all supplemental trace minerals. Rumen-bypass methionine was also included in the 30-ZS and 60-ZS boluses to provide metabolizable methionine equivalent to that provided in 60-ZM rations. Milk samples were assessed for electrolytes, somatic cell transcript abundance of genes related to zinc metabolism, and heat coagulation time. Whole blood samples were analyzed for Na and K concentrations, and plasma samples were analyzed for lactose concentration. Cows fed 60-ZS or 60-ZM had greater zinc intake compared to 30-ZS. Dry matter intake and milk fat content tended to be greater in 60-ZS and 60-ZM cows compared to 30-ZS. Somatic cell linear score was similar among treatments. Treatments neither affected markers of mammary epithelial integrity in blood nor in milk of cows, including plasma concentration of lactose, milk concentrations of Na+ and K+, and SLC30A2 and CLU transcript abundance. Treatments had no effect on milk N fractions or heat coagulation time. This study provided no evidence that supplemental Zn above the established requirements can improve blood-milk epithelial barrier or heat stability of milk in healthy mid-lactation dairy cows.

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ABSTRACT: This study vised to detect possible changes in chemical and cellular composition of cow's milk at different milking sessions and to test if proportional or composite milk sampling can be used instead of separate milk analysis. Two experiments were conducted: one in cows milked thrice daily and the other one in cows milked twice daily. In both experiments, three milk samples were collected from each cow in each milking session in order to determine milk composition at each milking; to have a composite sample, i.e., a single sample from the same aliquots of milk from each milking session; and to have a proportional sample, i.e., a single sample from each milking session in a volume proportional to milk volume yielded. Fat content and fat to protein ratio were higher in the afternoon on both experiments. Protein content was larger in the afternoon on cows milked twice daily, but not on cows milked thrice daily. Lactose level was not different across the milking sessions in any of the experiments. The urea level was higher in the afternoon and evening milking on cows milked thrice daily, and in the morning milking on cows milked twice daily. Somatic cell count did not differ between milking in any experiment. No difference in milk composition was noted between proportional and composite samples on cows milked thrice or twice daily. The collection of proportional and composite samples is recommended in twice or thrice daily milking sessions so that the analysis of milk components becomes more easy and cheap.

RESUMO: Este estudo visou detectar possíveis mudanças na composição química e celular do leite de vaca em diferentes sessões de ordenha, e testar se a amostragem de leite proporcional ou composta pode ser usada em vez de uma análise separada do leite. Dois experimentos foram conduzidos: um em vacas ordenhadas três vezes ao dia e outro em vacas ordenhadas duas vezes ao dia. Em ambos os experimentos, três amostras de leite foram coletadas de cada vaca em cada ordenha: para determinar a composição do leite em cada ordenha; ter uma amostra composta, isto é, uma única amostra das mesmas alíquotas de leite de cada sessão de ordenha; e ter uma amostra proporcional, ou seja, uma única amostra de cada sessão de ordenha em um volume proporcional ao volume de leite produzido. O conteúdo de gordura e a relação gordura:proteína foram maiores no período da tarde nos dois experimentos. O conteúdo de proteína foi maior à tarde nas vacas ordenhadas duas vezes ao dia, mas não nas vacas ordenhadas três vezes ao dia. O nível de lactose não foi diferente nas sessões de ordenha em nenhum dos experimentos. O nível de ureia foi maior à tarde e à noite, nas vacas ordenhadas três vezes ao dia e, pela manhã, nas vacas ordenhadas duas vezes ao dia. A contagem de células somáticas não diferiu entre as ordenhas em nenhum experimento. Não houve diferença na composição do leite entre as amostras proporcionais e compostas em vacas ordenhadas três vezes ao dia. Recomenda-se a coleta de amostras proporcionais e compostas em duas ou três sessões diárias de ordenha, para que a análise dos componentes do leite se torne mais fácil e barata.

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The objective of this study was to demonstrate the usefulness of an immunomagnetic method to purify subpopulations of milk somatic cells. The experiment was conducted on milk samples collected from healthy cows (n = 17) and from cows with clinical mastitis (n = 24) due to a Staphylococcus aureus natural infection. A two-step immunomagnetic purification was applied to simultaneously separate three somatic cell subpopulations from the same milk sample. Total RNA was extracted and qPCR was performed to determinate mRNA levels of innate immunity target genes in purified somatic cell subpopulations. Good quality and quantity of RNA allowed the reference gene analysis in each cell subpopulation. An up-regulation of the main genes involved in innate immune defence was detected in separated polymorphonuclear neutrophilic leucocytes-monocytes and lymphocytes of mastitic milk. These results and flow cytometric analysis suggest that the immunomagnetic purification is an efficient method for the isolation of the three populations from milk, allowing the cells to be studied separately.

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BACKGROUND: The identification of genetic variation underlying desired phenotypes is one of the main challenges of current livestock genetic research. High-throughput transcriptome sequencing (RNA-Seq) offers new opportunities for the detection of transcriptome variants (SNPs and short indels) in different tissues and species. In this study, we used RNA-Seq on Milk Sheep Somatic Cells (MSCs) with the goal of characterizing the genetic variation within the coding regions of the milk transcriptome in Churra and Assaf sheep, two common dairy sheep breeds farmed in Spain. RESULTS: A total of 216,637 variants were detected in the MSCs transcriptome of the eight ewes analyzed. Among them, a total of 57,795 variants were detected in the regions harboring Quantitative Trait Loci (QTL) for milk yield, protein percentage and fat percentage, of which 21.44% were novel variants. Among the total variants detected, 561 (2.52%) and 1,649 (7.42%) were predicted to produce high or moderate impact changes in the corresponding transcriptional unit, respectively. In the functional enrichment analysis of the genes positioned within selected QTL regions harboring novel relevant functional variants (high and moderate impact), the KEGG pathway with the highest enrichment was "protein processing in endoplasmic reticulum". Additionally, a total of 504 and 1,063 variants were identified in the genes encoding principal milk proteins and molecules involved in the lipid metabolism, respectively. Of these variants, 20 mutations were found to have putative relevant effects on the encoded proteins. CONCLUSIONS: We present herein the first transcriptomic approach aimed at identifying genetic variants of the genes expressed in the lactating mammary gland of sheep. Through the transcriptome analysis of variability within regions harboring QTL for milk yield, protein percentage and fat percentage, we have found several pathways and genes that harbor mutations that could affect dairy production traits. Moreover, remarkable variants were also found in candidate genes coding for major milk proteins and proteins related to milk fat metabolism. Several of the SNPs found in this study could be included as suitable markers in genotyping platforms or custom SNP arrays to perform association analyses in commercial populations and apply genomic selection protocols in the dairy production industry.

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