Extracellular vesicle miRNome during subclinical mastitis in dairy cows.

Bovine mastitis is one of the main inflammatory diseases that can affect the udder during lactation. Somatic cell counts and sometimes microbiological tests are routinely adopted during monitoring diagnostics in dairy herds. However, subclinical mastitis is challenging to identify, reducing the possibility of early treatments. The main aim of this study was to investigate the miRNome profile of extracellular vesicles isolated from milk as potential biomarkers of subclinical mastitis. Milk samples were collected from a total of 60 dairy cows during routine monitoring tests. Small RNA sequencing technology was applied to extracellular vesicles of milk samples collected from cows classified according to the somatic cell count to identify differences in the miRNome between mastitic and healthy cows. A total of 1997 miRNAs were differentially expressed between both groups. Among them, 68 miRNAs whose FDRs were < 0.05 were mostly downregulated, with only one upregulated miRNA (i.e., miR-361). Functional analysis revealed that miR-455-3p, miR-503-3p, miR-1301-3p and miR-361-5p are involved in the regulation of several biological processes related to mastitis, including immune system-related processes. This study suggests the involvement of extracellular vesicle-derived miRNAs in the regulation of mastitis. Moreover, these findings provide evidence that miRNAs from milk extracellular vesicles can be used to identify biomarkers of mastitis. However, further studies must be conducted to validate these miRNAs, especially for subclinical diagnosis.

Inflammatory Signaling via PEIZO1 Engages and Enhances the LPS-Mediated Apoptosis during Clinical Mastitis.

Bovine clinical mastitis is characterized by inflammation and immune responses, with apoptosis of mammary epithelial cells as a cellular reaction to infection. PIEZO1, identified as a mechanotransduction effector channel in nonruminant animals and sensitive to both mechanical stimuli or inflammatory signals like lipopolysaccharide (LPS). However, its role in inflammatory processes in cattle has not been well-documented. The aim of this study was to elucidate the in situ expression of PIEZO1 in bovine mammary gland and its potential involvement in clinical mastitis. We observed widespread distribution and upregulation of PIEZO1 in mammary epithelial cells in clinical mastitis cows and LPS-induced mouse models, indicating a conserved role across species. In vitro studies using mammary epithelial cells (MAC-T) revealed that LPS upregulates PIEZO1. Notably, the effects of PIEZO1 artificial activator Yoda1 increased apoptosis and NLRP3 expression, effects mitigated by PIEZO1 silencing or NLRP3 inhibition. In conclusion, the activation of the PIEZO1-NLRP3 pathway induces abnormal apoptosis in mammary epithelial cells, potentially serving as a regulatory mechanism to combat inflammatory responses to abnormal stimuli.

Genotyping of Holstein Cows by SELL, MX1 and CXCR1 Gene Loci Associated With Mastitis Resistance.

Mastitis is a significant factor that decreases milk production in cows of different breeds in Kazakhstan. The objective of this study was to determine the genetic makeup of Holstein cows by analysing specific gene loci (SELL, MX1, CXCR1+291C>T and CXCR1+1093C>T) that are linked to resistance against mastitis. The goal was to identify cows with favourable genotypes that are less prone to udder diseases. At the SELL gene locus c.567T>C, all three genetic variants were identified in the control population with the respective frequencies: TT (0.20), CT (0.44), and CC (0.36). Genetic variation was also detected at the MX1 gene c.567T>C, CXCR1 c.+291C>T and CXCR1+1093C>T loci. Deviation from the expected Hardy-Weinberg equilibrium was observed for two gene loci, MX1 g.143182088 and CXCR1+1093C>T, with increased chi-square values of 10.6261 and 9.7137, respectively. The analysis of subclinical mastitis incidence indicates that cows carrying the heterozygous CT genotype at the L-selectin gene locus exhibit greater resistance to the disease. Animals carrying the CCCCCT genotype at the MX1 c.567T>C, CXCR1 c.+291C>T and CXCR1+1093C>T gene loci were discovered to have a significant likelihood of developing subclinical mastitis. This suggests that these genes could serve as potential indicators of susceptibility to the condition. The practical significance of this study lies in determining the frequency of genotypes linked to mammary gland morbidity in Holstein breeding farms in Kazakhstan.

Decoding epigenetic markers: implications of traits and genes through DNA methylation in resilience and susceptibility to mastitis in dairy cows.

Cattle farming faces challenges linked to intensive exploitation and climate change, requiring the reinforcement of animal resilience in response to these dynamic environments. Currently, genetic selection is used to enhance resilience by identifying animals resistant to specific diseases; however, certain diseases, such as mastitis, pose difficulties in genetic prediction. This study introduced the utilization of enzymatic methyl sequencing (EM-seq) of the blood genomic DNA from twelve dairy cows to identify DNA methylation biomarkers, with the aim of predicting resilience and susceptibility to mastitis. The analysis uncovered significant differences between cows resilient and susceptible to mastitis, with 196,275 differentially methylated cytosines (DMCs) and 1,227 Differentially Methylated Regions (DMRs). Key genes associated with the immune response and morphological traits, including ENOPH1, MYL10 and KIR2DL5A, were identified by our analysis. Quantitative trait loci (QTL) were also highlighted and the body weight trait was the most targeted by DMCs and DMRs. Based on our results, the risk of developing mastitis can potentially be estimated with as few as fifty methylation biomarkers, paving the way for early animal selection. This research sets the stage for improved animal health management and economic yields within the framework of agricultural sustainability through early selection based on the epigenetic status of animals.

Association of polymorphic variants of prolactin (PRL) and beta-lactoglobulin (BLG) genes with resistance/susceptibility to mastitis in holstein cows.

The work aims to analyze the associations of polymorphic variants of the PRL and BLG genes with resistance and susceptibility to mastitis in Holstein cows. The experimental study consisted of the selection of biomaterial samples from 250 heads of Holstein cows aged 3 years divided into two groups (healthy and with a confirmed diagnosis of mastitis). The determination of animal genotypes was carried out using polymerase chain reaction and restriction fragment length polymorphism. The study of the nature of the association of polymorphic variants of the PRL and BLG gene with resistance/increased risk of mastitis established a significant deviation from the theoretically expected distribution of bBLG-HaeIII genotypes in the group of animals suffering from mastitis (the value of χ2 was 0.24). The bBLG-HaeIIIBB genotype can act as a marker of an increased risk of developing mastitis in Holstein cows; its frequency in the group of sick animals exceeds the frequency in the control group by more than 2 times (44.0 compared to 17.0%, respectively). The bBLG-HaeIIIAB genotype is significantly associated with mastitis resistance in Holstein cows; its frequency is 2 times lower than in the control group (28.0 compared to 54.0%).

AMP-activated protein kinase mediates (-)-epigallocatechin-3-gallate (EGCG) to promote lipid synthesis in mastitis cows.

Mastitis, a serious threat to the health and milk production function of dairy cows decreases milk quality. Blood from three healthy cows and three mastitis cows were collected in this study and their transcriptome was sequenced using the Illumina HiSeq platform. Differentially expressed genes (DEGs) were screened according to the |log2FoldChange| > 1 and P-value < 0.05 criteria. Pathway enrichment and functional annotation were performed through KEGG and GO analyses. Finally, the mechanism of the AMP-activated protein kinase (AMPK) mediation of (-)-epigallocatechin-3-gallate (EGCG) to promote lipid metabolism in mastitis cows was analyzed in bovine mammary epithelial cells (BMECs). Transcriptome analysis revealed a total of 825 DEGs, with 474 genes showing increased expression and 351 genes showing decreased expression. The KEGG analysis of DEGs revealed that they were mainly linked to tumour necrosis factor, nuclear factor-κB signalling pathway, and lipid metabolism-related signalling pathway, whereas GO functional annotation found that DEGs were enriched in threonine and methionine kinase activity, cellular metabolic processes, and cytoplasm. AMPK expression, which is involved in several lipid metabolism pathways, was downregulated in mastitis cows. The results of in vitro experiments showed that the inhibition of AMPK promoted the expression of lipid synthesis genes in lipopolysaccharide-induced BMECs and that EGCG could promote lipid synthesis by decreasing the expression of AMPK and downregulating the expression of inflammatory factors in inflammatory BMECs. In conclusion, our study demonstrated that AMPK mediated EGCG to inhabit of inflammatory responses and promote of lipid synthesis in inflammatory BMECs.

Meta-analysis of six dairy cattle breeds reveals biologically relevant candidate genes for mastitis resistance.

BACKGROUND: Mastitis is a disease that incurs significant costs in the dairy industry. A promising approach to mitigate its negative effects is to genetically improve the resistance of dairy cattle to mastitis. A meta-analysis of genome-wide association studies (GWAS) across multiple breeds for clinical mastitis (CM) and its indicator trait, somatic cell score (SCS), is a powerful method to identify functional genetic variants that impact mastitis resistance. RESULTS: We conducted meta-analyses of eight and fourteen GWAS on CM and SCS, respectively, using 30,689 and 119,438 animals from six dairy cattle breeds. Methods for the meta-analyses were selected to properly account for the multi-breed structure of the GWAS data. Our study revealed 58 lead markers that were associated with mastitis incidence, including 16 loci that did not overlap with previously identified quantitative trait loci (QTL), as curated at the Animal QTLdb. Post-GWAS analysis techniques such as gene-based analysis and genomic feature enrichment analysis enabled prioritization of 31 candidate genes and 14 credible candidate causal variants that affect mastitis. CONCLUSIONS: Our list of candidate genes can help to elucidate the genetic architecture underlying mastitis resistance and provide better tools for the prevention or treatment of mastitis, ultimately contributing to more sustainable animal production.

Long non-coding RNA (CMR) involved in autoprotection in S. aureus mastitis in dairy cows by regulating miR-877/FOXM1.

Long non-coding RNAs (LncRNAs) are dysregulated in a variety of human diseases and are highly involved in the development and progression of tumors. Studies on lncRNAs associated with cow mastitis have been lagging behind compared to humans or model animals, therefore, the aim of this study was to explore the mechanism of LncRNAs (CMR) involved in autoprotection against S. aureus mastitis in Bovine Mammary Epithelial Cells (BMECs). First, qRT-PCR was used to examine the relative expression of CMR in a S. aureus mastitis model of BMECs. Then, cell proliferation and apoptosis were detected by EdU and apoptosis assay. Finally, the targeting relationship between miRNAs and mRNA/LncRNAs was determined by dual luciferase reporter gene, qRT-PCR and western blotting techniques. The results showed that CMR was upregulated in the S. aureus mastitis model of BMECs and promoted the expression of inflammatory factors, and SiRNA-mediated CMR inhibited the proliferation of mammary epithelial cells and induced apoptosis. Mechanistically, CMR acts as a competitive endogenous RNA (ceRNA) sponge miR-877, leading to upregulation of FOXM1, a target of miR-877. Importantly, either miR-877 overexpression or FOXM1 inhibition abrogated CMR knockdown-induced apoptosis promoting cell proliferation and reducing inflammatory factor expression levels. In summary, CMR is involved in the regulation of autoprotection against S. aureus mastitis through the miR-877/FOXM1 axis in BMECs and induces immune responses in mammary tissues and cells of dairy cows, providing an important reference for subsequent prevention and control of cow mastitis and the development of targeted drugs.

Considering potential roles of selected MicroRNAs in evaluating subclinical mastitis and Milk quality in California mastitis test (+) and infected bovine milk.

This study investigates the relationships between subclinical mastitis and milk quality with selected microRNAs in cow milk. California Mastitis Test (CMT)-positive (n = 20) and negative (n = 20) samples were compared (Experiment I). Additionally, samples with CMT-positive but microbiological-negative, as well as positive for only Staphylococcus subspecies (Staph spp.) and only Streptococcus subspecies (Strep spp.) were examined (Experiment II). Four groups were formed in Experiment II: Group I (CMT and microbiological-negative) (n = 20), Group II (CMT-positive but microbiological-negative) (n = 10), Group III (Staph spp.) (n = 5), Group IV (Strep spp.) (n = 5). While electrical conductivity, somatic cell count (SCC), malondialdehyde (MDA) increased, miR-27a-3p and miR-223 upregulated and miR-125b downregulated in the CMT-positive group in Experiment I. SCC and MDA were higher in CMT-positive groups. miR-27a-3p and miR-223 upregulated in Groups III and IV. While miR-155 is upregulated, miR-125b downregulated in Group IV. Milk fat is positively correlated with miR-148a and miR-223. As miR-27a-3p positively correlated with SCC and MDA, miR-125b negatively correlated with electrical conductivity and SCC. miR-148a and MDA were positively correlated. miR-155 was correlated with fat-free dry matter, protein, lactose, and freezing point. miR-223 was positively correlated with SCC and miR-148a. Results particularly highlight miR-27a-3p and miR-223 as potential biomarkers in subclinical mastitis, especially those caused by Staph spp. and Strep spp., while miR-148a, miR-155, and miR-223 stand out in determining milk quality.

An Explainable Deep Learning Classifier of Bovine Mastitis Based on Whole-Genome Sequence Data-Circumventing the p >> n Problem.

The serious drawback underlying the biological annotation of whole-genome sequence data is the p >> n problem, which means that the number of polymorphic variants (p) is much larger than the number of available phenotypic records (n). We propose a way to circumvent the problem by combining a LASSO logistic regression with deep learning to classify cows as susceptible or resistant to mastitis, based on single nucleotide polymorphism (SNP) genotypes. Among several architectures, the one with 204,642 SNPs was selected as the best. This architecture was composed of two layers with, respectively, 7 and 46 units per layer implementing respective drop-out rates of 0.210 and 0.358. The classification of the test data resulted in AUC = 0.750, accuracy = 0.650, sensitivity = 0.600, and specificity = 0.700. Significant SNPs were selected based on the SHapley Additive exPlanation (SHAP). As a final result, one GO term related to the biological process and thirteen GO terms related to molecular function were significantly enriched in the gene set that corresponded to the significant SNPs. Our findings revealed that the optimal approach can correctly predict susceptibility or resistance status for approximately 65% of cows. Genes marked by the most significant SNPs are related to the immune response and protein synthesis.

Genetic parameters for mid-infrared-spectroscopy-predicted mastitis phenotypes and related traits.

Genetic improvement of udder health in dairy cows is of high relevance as mastitis is one of the most prevalent diseases. Since it is known that the heritability of mastitis is low and direct data on mastitis cases are often not available in large numbers, auxiliary traits, such as somatic cell count (SCC) are used for the genetic evaluation of udder health. In previous studies, models to predict clinical mastitis based on mid-infrared (MIR) spectral data and a somatic cell count-derived score (SCS) were developed. Those models can provide a probability of mastitis for each cow at every test-day, which is potentially useful as an additional auxiliary trait for the genetic evaluation of udder health. Furthermore, MIR spectral data were used to estimate contents of lactoferrin, a glycoprotein positively associated with immune response. The present study aimed to estimate heritabilities (h2) and genetic correlations (ra) for clinical mastitis diagnosis (CM), SCS, MIR-predicted mastitis probability (MIRprob), MIR + SCS-predicted mastitis probability (MIRSCSprob) and lactoferrin estimates (LF). Data for this study were collected within the routine milk recording and health monitoring system of Austria from 2014 to 2021 and included records of approximately 54,000 Fleckvieh cows. Analyses were performed in two datasets, including test-day records from 5 to 150 or 5 to 305 days in milk. Prediction models were applied to obtain MIR- and SCS-based phenotypes (MIRprob, MIRSCSprob, LF). To estimate heritabilities and genetic correlations bivariate linear animal models were applied for all traits. A lactation model was used for CM, defined as a binary trait, and a test-day model for all other continuous traits. In addition to the random animal genetic effect, the fixed effects year-season of calving and parity-age at calving and the random permanent environmental effect were considered in all models. For CM the random herd-year effect, for continuous traits the random herd-test day effect and the covariate days in milk (linear and quadratic) were additionally fitted. The obtained genetic parameters were similar in both datasets. The heritability found for CM was expectedly low (h2 = 0.02). For SCS and MIRSCSprob, heritability estimates ranged from 0.23 to 0.25, and for MIRprob and LF from 0.15 to 0.17. CM was highly correlated with SCS and MIRSCSprob (ra = 0.85 to 0.88). Genetic correlations of CM were moderate with MIRprob (ra = 0.26 and 0.37) during 150 and 305 days in milk, respectively and low with LF (h2 = 0.10 and 0.11). However, basic selection index calculations indicate that the added value of the new MIR-predicted phenotypes is limited for genetic evaluation of udder health.

Transcriptomic Changes and Regulatory Networks Associated with Resistance to Mastitis in Xinjiang Brown Cattle.

Xinjiang brown cattle are highly resistant to disease and tolerant of roughage feeding. The identification of genes regulating mastitis resistance in Xinjiang brown cattle is a novel means of genetic improvement. In this study, the blood levels of IL-1ß, IL-6, IL-10, TNF-α, and TGF-ß in Xinjiang brown cattle with high and low somatic cell counts (SCCs) were investigated, showing that cytokine levels were higher in cattle with high SCCs. The peripheral blood transcriptomic profiles of healthy and mastitis-affected cattle were constructed by RNA-seq. Differential expression analysis identified 1632 differentially expressed mRNAs (DE-mRNAs), 1757 differentially expressed lncRNAs (DE-lncRNAs), and 23 differentially expressed circRNAs (DE-circRNAs), which were found to be enriched in key pathways such as PI3K/Akt, focal adhesion, and ECM-receptor interactions. Finally, ceRNA interaction networks were constructed using the differentially expressed genes and ceRNAs. It was found that keynote genes or mRNAs were also enriched in pathways such as PI3K-Akt, cholinergic synapses, cell adhesion molecules, ion binding, cytokine receptor activity, and peptide receptor activity, suggesting that the key genes and ncRNAs in the network may play an important role in the regulation of bovine mastitis.

Identification of candidate genes associated with milk production and mastitis based on transcriptome-wide association study.

Genetic research for the assessment of mastitis and milk production traits simultaneously has a long history. The main issue that arises in this context is the known existence of a positive correlation between the risk of mastitis and lactation performance due to selection. The transcriptome-wide association study (TWAS) approach endeavors to combine the expression quantitative trait loci and genome-wide association study summary statistics to decode complex traits or diseases. Accordingly, we used the farmgtex project results as a complete bovine database for mastitis and milk production. The results of colocalization and TWAS approaches were used for the detection of functional associated candidate genes with milk production and mastitis traits on multiple tissue-based transcriptome records. Also, we used the david database for gene ontology to identify significant terms and associated genes. For the identification of interaction networks, the genemania and string databases were used. Also, the available z-scores in TWAS results were used for the calculation of the correlation between tissues. Therefore, the present results confirm that LYNX1, DGAT1, C14H8orf33, and LY6E were identified as significant genes associated with milk production in eight, six, five, and five tissues, respectively. Also, FBXL6 was detected as a significant gene associated with mastitis trait. CLN3 and ZNF34 genes emerged via both the colocalization and TWAS approaches as significant genes for milk production trait. It is expected that TWAS and colocalization can improve our perception of the potential health status control mechanism in high-yielding dairy cows.

DNA methylation haplotype block signatures responding to Staphylococcus aureus subclinical mastitis and association with production and health traits.

BACKGROUND: DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis. RESULTS: Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield). CONCLUSIONS: In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures.

Milk exosomal microRNA profiling identified miR-375 and miR-199-5p for regulation of immune response during subclinical mastitis of crossbred cattle.

BACKGROUND: The dairy industry has experienced significant economic losses as a result of mastitis, an inflammatory disease of cows, including both subclinical and clinical cases. Milk exosome microRNAs have gained attention due to their stable and selective wrapping nature, offering potential for the prognosis and diagnosis of bovine mastitis, the most common pathological condition of the mammary gland. METHODS AND RESULTS:  In the present investigation, the microRNA profile of milk exosomes was explored using high-throughput small RNA sequencing data in sub-clinical mastitic and healthy crossbred Vrindavani cattle. In both groups, 349 microRNAs were identified, with 238 (68.19%) microRNAs co-expressed; however, 35 and 76 distinct microRNAs were found in subclinical mastitic and healthy cattle, respectively. Differential expression analysis revealed 11 microRNAs upregulated, and 18 microRNAs were downregulated in sub-clinical mastitic cattle. The functional annotation of the target genes of differentially expressed known and novel microRNAs including bta-miR-375, bta-miR-199-5p and bta-miR-12030 reveals their involvement in the regulation of immune response and inflammatory mechanisms and could be involved in development of mastitis. CONCLUSIONS: The analysis of milk exosomal miRNAs cargos hold great promise as an approach to study the underlying molecular mechanisms associated with mastitis in high milk producing dairy cattle. Concurrently, the significantly downregulated miR-375 may upregulate key target genes, including CTLA4, IHH, IRF1, and IL7R. These genes are negative regulators of immune response pathways, which could be associated with impaired inflammatory mechanisms in mammary cells. According to the findings, bta-miR-375 could be a promising biomarker for the development of mastitis in dairy cattle.

LncRNA CA12-AS1 targets miR-133a to promote LPS-induced inflammatory response in bovine mammary epithelial cells.

Bovine mastitis seriously affects milk production and quality and causes huge economic losses in the dairy industry. Recent studies have shown that long non-coding RNAs (lncRNAs) may regulate bovine mastitis. In this study, the expression of lncRNA CA12-AS1 was significantly upregulated in LPS-induced bovine mammary epithelial cells (bMECs) but negatively correlated with the expression of miR-133a, suggesting that it may be related to the inflammatory response in bMECs. Dual luciferase reporter gene assay revealed that miR-133a is a downstream target gene of lncRNA CA12-AS1. Furthermore, lncRNA CA12-AS1 silencing negatively regulated the expression of miR-133a inhibited the secretion of inflammatory factors (IL-6, IL-8 and IL-1ß) and decreased the mRNA expression levels of nuclear factor kappa B (NF-κB) (p65/p50) and apoptosis-related genes (BAX, caspase3 and caspase9). LncRNA CA12-AS1 silencing also promoted the mRNA expression levels of the Tight junction (TJ) signaling pathway-related genes (Claudin-1, Occludin and ZO-1), apoptotic gene BCL2, proliferation-related genes (CDK2, CDK4 and PCNA) and the viability of bMECs. However, overexpression of lncRNA CA12-AS1 reversed the above effects. These results revealed that lncRNA CA12-AS1 is a pro-inflammatory regulator, and its silencing can alleviate bovine mastitis by targeting miR-133a, providing a novel strategy for molecular therapy of cow mastitis.

Genetic parameters for novel mastitis traits defined by combining test-day somatic cell score and differential somatic cell count in the first lactation of Japanese Holsteins.

In this study, we aimed to improve current udder health genetic evaluations by addressing the limitations of monthly sampled somatic cell score (SCS) for distinguishing cows with robust innate immunity from those susceptible to chronic infections. The objectives were to (1) establish novel somatic cell traits by integrating SCS and the differential somatic cell count (DSCC), which represents the combined proportion of polymorphonuclear leukocytes and lymphocytes in somatic cells and (2) estimate genetic parameters for the new traits, including their daily heritability and genetic correlations with milk production traits and SCS, using a random regression test-day model (RRTDM). We derived 3 traits, termed ML_SCS_DSCC, SCS_4_DSCC_65_binary, and ML_SCS_DSCC_binary, by using milk loss (ML) estimates at corresponding SCS and DSCC levels, thresholds established in previous studies, and a threshold established from milk loss estimates, respectively. Data consisted of test-day records collected during January 2021 through March 2022 from 265 herds in Hokkaido, Japan. From these records, we extracted records between 7 to 305 d in milk (DIM) in the first lactation to fit the RRTDM. The model included the random effect of herd-test-day, the fixed effect of year-month, fixed lactation curves nested with calving age groups, and random regressions with Legendre polynomials of order 3 for additive genetic and permanent environmental effects. The analysis was performed using Gibbs sampling with Gibbsf90+ software. The averages (ranges) of the daily heritability estimates over lactation were 0.086 (0.075-0.095) for SCS, 0.104 (0.073-0.127) for ML_SCS_DSCC, 0.137 (0.014-0.297) for SCS_4_DSCC_65_binary, and 0.138 (0.115-0.185) for ML_SCS_DSCC_binary; the heritability curve for SCS_4_DSCC_65_binary was erratic. Genetic correlations within the trait decreased as the DIM interval widened, especially for those integrating DSCC, indicating that these traits should be analyzed using RRTDM rather than repeatability models. The averages (ranges) of genetic correlations with milk yield over lactation were 0.01 (-0.22 to 0.28) for SCS, -0.05 (-0.40 to 0.13) for ML_SCS_DSCC, -0.08 (-0.17 to 0.09) for SCS_4_DSCC_65_binary, and -0.08 (-0.22 to 0.27) for ML_SCS_DSCC_binary. Compared with SCS, the newly defined traits exhibited slightly stronger negative genetic correlations with milk yield. Especially in late lactation stages, the genetic correlation between ML_SCS_DSCC and milk yield was significantly below zero, with a posterior median of -0.40. Furthermore, the new traits showed positive correlations with SCS, having estimates varying from 0.68 to 0.85 for ML_SCS_DSCC, 0.14 to 0.47 for SCS_4_DSCC_65_binary, and 0.61 to 0.66 for ML_SCS_DSCC_binary, depending on DIM. Considering that ML_SCS_DSCC and ML_SCS_DSCC_binary have relatively high heritability (compared with SCS) and favorable genetic correlations with milk production traits and SCS, their incorporation into breeding programs appears promising. Nevertheless, their genetic relationships with (sub)clinical mastitis require further investigation.

Methylome-wide analysis of milk somatic cells upon subclinical mastitis in dairy cattle.

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.

dCas9-guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by using Staphylococcus aureus.

Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.

Transcriptome sequencing analysis of bovine mammary epithelial cells induced by lipopolysaccharide.

Mastitis in cows is caused by the inflammation of the mammary glands due to an infection by external pathogenic bacteria. Mammary gland epithelial cells, which are in direct contact with the external environment, are responsible for the first line of defense of the mammary gland against pathogenic bacteria, playing an essential role in immune defense. To investigate the mechanism of bovine mammary epithelial cells in the inflammatory process, we treated the cells with LPS for 12 hours and analyzed the changes in mRNA by transcriptome sequencing. The results showed that compared to the control group, the LPS treatment group had 121 up-regulated genes and 18 down-regulated genes. GO and KEGG enrichment analysis revealed that these differential genes were mainly enriched in the IL-17 signaling pathway, Legionellosis, Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, and other signaling pathways. Furthermore, the expression of GRO1 and CXCL3 mRNAs increased significantly after LPS treatment. These findings provide new insights for the treatment of mastitis in cows in the future.