Transcriptional regulation of bovine elongation of very long chain fatty acids protein 6 in lipid metabolism and adipocyte proliferation.

The elongation of very long chain fatty acids protein 6 (ELOVL6) gene encodes a key enzyme that plays a role in lipogenesis through the catalytic elongation of both saturated and monounsaturated fatty acids. Previous studies have described the high expression of bovine ELOVL6 in adipose tissues. However, transcriptional regulation and the functional role of ELOVL6 in lipid metabolism and adipocyte proliferation remain unexplored. Here, a 1.5 kb fragment of the 5'-untranslated region promoter region of ELOVL6 was amplified from the genomic DNA of Qinchuan cattle and sequenced. The core promoter region was identified through unidirectional 5'-end deletion of the promoter plasmid vector. In silico analysis predicted important transcription factors that were then validated through site-directed mutation and small interfering RNA interference with an electrophoretic mobility shift assay. We found that the binding of KLF6 and PU.1 transcription factors occurred in the region -168/+69. Both perform a vital regulatory function in the transcription of bovine ELOVL6. Overexpression of ELOVL6 significantly upregulated the expression of peroxisome proliferator activated receptor γ (PPARγ), but inhibited the expression of fatty acid-binding protein 4 (FABP4), while silencing of ELOVL6 negatively regulated the messenger RNA expression level of PPARγ, FABP4, ACSL, and FATP1. In addition, ELOVL6 promotes adipocyte proliferation by regulating the cell-cycle genes' expression. Taken together, these findings provide useful information about the transcriptional regulation and functional mechanisms of bovine ELOVL6 in lipid metabolism and adipocyte proliferation in Qinchuan cattle.

Coordinate regulation by transcription factors and DNA methylation in the core promoter region of SIRT6 in bovine adipocytes.

Sirtuin6 (SIRT6) is an ADP-ribosyltransferase and NAD+-dependent deacylase of acetyl groups and long-chain fatty acyl groups, and has been shown as a regulator of insulin secretion, glucose metabolism, lipid metabolism, and cancer. In this study, we determined that the bovine SIRT6 showed higher levels of mRNA expression in the testis, longissimus thoracis, and subcutaneous fat tissue. To elucidate the molecular regulation mechanism of bovine SIRT6 expression, we obtained a 2-kb fragment containing the 5'-regulatory region, and the functional proximal minimal promoter of bovine SIRT6 was identified in the -472/-73 bp region. The CCAAT enhancer binding protein beta (CEBPß), paired box 6 (PAX6), Kruppel-like factor 2 (KLF2), myb proto-oncogene protein (CMYB), nuclear respiratory factor 1 (NRF1), and E2F transcription factor 1 (E2F1) binding sites, as transcriptional activators or repressors in the core promoter region of SIRT6, were determined by electrophoretic mobility shift assay (EMSA) experiments and luciferase reporter assays. In addition, the results from methylation assay and luciferase report assay showed that the bovine SIRT6 promoter activity was coordinately regulated by methylation and NRF1 or E2F1 during bovine adipocyte differentiation. Taken together, this study illuminated the underlying mechanism of methylation and transcription regulation of SIRT6 expression in bovine adipocytes.

Current situation and future prospects for beef production in China - A review.

The beef industry is an important part of livestock and meat production in China. China ranks third in the world for beef production. With the rapid development of the Chinese economy, beef consumption has grown rapidly, and beef consumption has been increasing with rising per capita gross domestic production. However, the domestic beef industry in China has not been able to keep pace with growth in consumption, making China a net importer of beef from other countries. Moreover, the volume of production has increased little despite rising demand. The slowing of growth in beef production in recent years has led to a sharp rise in beef prices. Domestic beef production and consumption is restricted by a shortage of beef cattle inventory. The Chinese beef industry is facing many technical problems including transformation of traditional practices, feeding and management systems, and genetic improvement of cattle breeds. The long-term, sustainable development of the Chinese beef industry is an important issue for China.

Bovine miR-146a regulates inflammatory cytokines of bovine mammary epithelial cells via targeting the TRAF6 gene.

It has been reported previously that bovine miR-146a (bta-miR-146a) is significantly differentially expressed in mammary glands infected with mastitis, compared with healthy udders. This suggests that bta-miR-146a plays an important role in the regulation of mammary inflammation. However, the specifics of this function have yet to be elucidated. Bovine mammary epithelial cells (bMEC) represent the first line of defense against pathogens and have important roles in initiating and regulating inflammatory responses and innate immunity during infection. In this study, a double luciferase reporter assay was used to confirm that bta-miR-146a directly targets the 3' UTR of the tumor-necrosis factor receptor-associated factor 6 (TRAF6) gene. To elucidate the role of bta-miR-146a in innate immune responses, either a mimic or inhibitor of bta-miR-146a was transfected into bMEC stimulated with lipopolysaccharide, which activates the innate immune response through the toll-like receptor (TLR) 4/nuclear factor (NF)-κB signaling pathway. Forty-eight hours posttransfection, quantitative real-time PCR and Western blots were used to detect the expressions of the related genes and proteins, respectively. An ELISA was used to measure the quantity of inflammatory factors in culture supernatants. The results showed that bta-miR-146a significantly inhibits both mRNA and protein expression levels of bovine TRAF6, and ultimately suppresses downstream expression of NF-κB mRNA and protein. As a result, production of NF-κB-dependent inflammatory mediators such as tumor necrosis factor α, IL-6, and IL-8 are suppressed following lipopolysaccharide stimulation of bMEC. Thus, we concluded that bta-miR-146a acts as a negative feedback regulator of bovine inflammation and innate immunity through downregulation of the TLR4/TRAF6/NF-κB pathway. This study presents a potential regulatory mechanism of bta-miR-146a on immune responses in bovine mammary infection and may provide a potential therapeutic target for mastitis.

Expression patterns of miR-146a and miR-146b in mastitis infected dairy cattle.

This study reports a significant up-regulation of bta-miR-146a and bta-miR-146b expression levels in bovine mammary tissues infected with subclinical, clinical and experimental mastitis. Potential target genes are involved in multiple immunological pathways. These results suggest a regulatory function of both miRNAs for the bovine inflammatory response in mammary tissue.

Expression of the SIRT2 gene and its relationship with body size traits in Qinchuan cattle (Bos taurus).

Silent information regulator 2 (SIRT2) is a member of the sirtuin family of class III NAD (nicotinamide adenine dinucleotide)-dependent protein deacetylases and may regulate senescence, metabolism and apoptosis. The aims of this study were to investigate whether the SIRT2 gene could be used as a candidate gene in the breeding of Qinchuan cattle. Real-time polymerase chain reaction (RT-PCR) results showed that among all types of tissue that were analyzed, the highest mRNA expression levels of the gene were found in subcutaneous fat. DNA sequencing of 468 individual Qinchuan cattle identified two novel, single nucleotide polymorphisms (g.19501 C > T and g.19518 C > T) in the 3' untranslated region (3'UTR) of the SIRT2 gene. The frequencies of SNP g.19501 C > T and g.19518 C > T were in Hardy-Weinberg disequilibrium in all the samples (chi-square test, χ2 < χ0.052). An association analysis showed that the two loci were significantly correlated with some body size traits and the H2H2 (-CT-CT-) diplotypes performed better than other combinations. These results indicated that the variations in the SIRT2 gene and their corresponding genotypes may be considered as molecular markers for economic traits in cattle breeding.

Molecular Characterization of Bovine SMO Gene and Effects of Its Genetic Variations on Body Size Traits in Qinchuan Cattle (Bos taurus).

Smoothened (Smo)-mediated Hedgehog (Hh) signaling pathway governs the patterning, morphogenesis and growth of many different regions within animal body plans. This study evaluated the effects of genetic variations of the bovine SMO gene on economically important body size traits in Chinese Qinchuan cattle. Altogether, eight single nucleotide polymorphisms (SNPs: 1-8) were identified and genotyped via direct sequencing covering most of the coding region and 3'UTR of the bovine SMO gene. Both the p.698Ser.>Ser. synonymous mutation resulted from SNP1 and the p.700Ser.>Pro. non-synonymous mutation caused by SNP2 mapped to the intracellular C-terminal tail of bovine Smo protein; the other six SNPs were non-coding variants located in the 3'UTR. The linkage disequilibrium was analyzed, and five haplotypes were discovered in 520 Qinchuan cattle. Association analyses showed that SNP2, SNP3/5, SNP4 and SNP6/7 were significantly associated with some body size traits (p < 0.05) except SNP1/8 (p > 0.05). Meanwhile, cattle with wild-type combined haplotype Hap1/Hap1 had significantly (p < 0.05) greater body length than those with Hap2/Hap2. Our results indicate that variations in the SMO gene could affect body size traits of Qinchuan cattle, and the wild-type haplotype Hap1 together with the wild-type alleles of these detected SNPs in the SMO gene could be used to breed cattle with superior body size traits. Therefore, our results could be helpful for marker-assisted selection in beef cattle breeding programs.

HSP90 expression correlation with the freezing resistance of bull sperm.

To date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing-thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing-thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen-thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P < 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing-thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm.

Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells.

The accuracy of sixteen commonly used internal reference genes was assessed in skeletal muscle-derived satellite cells of Qinchuan cattle at different stages of proliferation and induction of differentiation to determine the most suitable ones. Quantitative real-time PCR and three commonly used algorithmic programs, GeNorm, NormFinder and BestKeeper, were used to evaluate the stability of expression of the candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K , RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) in skeletal muscle-derived satellite cells at 0, 12, 24, 36 and 48 h of growth and after differentiation for 0, 2, 4, 6 and 8 days. The expression of two satellite cell marker genes, CCNA2 and MYF5, was used for validation analysis. The results of the software analyses showed that GAPDH and RPS15A were the most stable reference gene combinations during in vitro proliferation of bovine skeletal muscle-derived satellite cells, RPS15A and RPS9 were the most stable reference gene combinations during in vitro induction of differentiation of the cells, and PPIA was the least stable reference gene during proliferation and differentiation and was not recommended. This study lays the foundation for the selection of reference genes for qRT-PCR during the proliferation and induction of differentiation of bovine skeletal muscle-derived satellite cells.

Analysis of stability of reference genes for qPCR in bovine preadipocytes during proliferation and differentiation in vitro.

The stability of internal reference genes is crucial to the reliability of gene expression results using real-time fluorescence quantitative PCR (qRT-PCR). Inappropriate reference genes may lead to inaccurate results or even wrong conclusions. This study aims to identify stable reference genes for analyzing the expression of proliferation-related and differentiation-inducing genes in bovine primary preadipocytes (BPPs) in vitro. In this study, the stability of 16 candidate internal reference genes (GAPDH, ACTB, PPIA, LRP10, HPRT1, YWHAZ, B2M, TBP, EIF3K, RPS9, UXT, 18S rRNA, RPLP0, MARVELD, EMD and RPS15A) for qRT-PCR at proliferation and differentiation stages of BPPs was investigated by three different algorithms (geNorm, NormFinder and BestKeeper). The expression of two marker genes, PCNA and LPL, was used to determine the validity of the candidate reference genes (RGs) at the proliferation and differentiation stages, respectively. The results showed that GAPDH and RPS15A were the most stable RGs in the proliferation of bovine primary preadipocyte, while PPIA was the least stable internal reference gene. RPLP0 and EIF3K were the most stable RGs in the differentiation induction of bovine primary preadipocyte, while GAPDH was the least stable internal reference gene. This study of RGs laid the foundation for subsequent research into the mechanism of proliferation and differentiation of BPPs in vitro using qRT-PCR.

Three novel SNPs in the coding region of PPARγ gene and their associations with meat quality traits in cattle.

The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that regulates adipogenesis and many other biological processes. In the present study, we carried out PCR-SSCP and DNA sequencing analyses to examine SNPs in coding region of the PPARγ gene. A total of 660 individuals from five Chinese cattle breeds were genotyped. We identified three SNPs and their associations with meat quality traits were analyzed in 108 Qinchuan cattle. Two missense mutations and one synonymous mutation were found: 200 A>G (genotypes AA, AB and BB) resulting in D7G change, the silent substitution 42895 C>T (genotypes JJ and JI) and 72472 G>T (genotypes CC, DC and DD) producing Q448H change, respectively. The frequencies of PPARγ-A allele were 0.86, 0.83, 0.80, 0.72 and 0.87 for Qinchuan, Nanyang, Jiaxian, Luxi and Xianan populations, respectively. The frequencies of PPARγ-J allele varied from 0.87 to 0.96 in the five populations. In the 72472 G>T locus, the frequencies of PPARγ-C allele were higher than PPARγ-D allele in the five populations, and ranged from 0.58 to 0.82. Least squares analysis revealed that in 42895 C>T locus, there was a significant effect on tenderness in 18-20 months Qinchuan cattle (P<0.01), and in the 72472 G>T locus, animals with the genotype DC had lower mean values than these with genotype CC (P<0.01) for back fat thickness in 18-20 months, and animals with the genotype DD had lower mean values than these with genotypes CC and DC (P<0.01) for water holding capacity in 21-24 months (P<0.01). The SNPs we have identified may contribute to establishing a more efficient selection program for improving of genetic characteristics in indigenous Chinese cattle.

The advantages of low-density lipoproteins in the cryopreservation of bull semen.

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen-thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P<0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P>0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P<0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.

Expression Profiling of microRNA From Peripheral Blood of Dairy Cows in Response to Staphylococcus aureus-Infected Mastitis.

As the main pathogen causing dairy cow mastitis, Staphylococcus aureus can cause subclinical mastitis, which is difficult to be diagnosed. It seriously affects milk quality and the economic benefits of the dairy industry. Therefore, it is very necessary to find biomarkers for early diagnosis of S. aureus-infected mastitis in peripheral blood of dairy cows. In this study, S. aureus was used to infect the mammary gland tissues of dairy cows, and a mastitis model was successfully constructed. The RNAseq technology was used to determine the expression profiles of microRNA (miRNA) from peripheral blood of dairy cows infected with S. aureus at 0, 1, 3, 5, and 7 days. A total of 288 differentially expressed miRNAs (DIE-miRNAs) were found, of which 108 were known miRNAs and 180 were novel predicted miRNAs. Bioinformatics analysis results showed that the above DIE-miRNAs might be involved in 10 immune system-related signaling pathways (i.e., chemokine signaling pathway, leukocyte transendothelial migration, natural killer cell-mediated cytotoxicity, toll-like receptor signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Wnt signaling pathway, cell adhesion molecules, cytokine-cytokine receptor interaction, and ECM-receptor interaction), thus regulating the process of S. aureus mastitis. It was also found that the expression variation of up-regulated expression of miR-320a, miR-19a, and miR-19b as well as down-regulated expression of miR-143, miR-205, and miR-24 reached a significant level on the 5th and 7th day of infection, suggesting that they might play an important biological role in mastitis and provide a direction for the research and development of molecular therapy technology for mastitis. However, at different times after S. aureus infection, miR-1301 was significantly up-regulated in peripheral blood. miR-2284r was significantly down-regulated, suggesting that these two miRNAs might be the new blood biomarkers for S. aureus-infected dairy cow mastitis. The above results laid a new foundation for the research and development of molecular diagnosis and biological therapy technology for S. aureus-infected mastitis in dairy cow.

A novel polymorphism of GDF5 gene and its association with body measurement traits in Bos taurus and Bos indicus breeds.

Body measurement traits, influenced by genes and environmental factors, play numerous important roles in the value assessment of productivity and economy. Growth differentiate factor 5 (GDF5), involved in the development and maintenance of bone and cartilage, is an important candidate gene for body measurement traits selection through marker-assisted selection (MAS). In this study, based on the PCR-RFLP technology, we discovered and evaluated the potential association of the single nucleotide polymorphism (SNP) (T586C in exon 1) of the bovine GDF5 gene with body measurement traits in 985 Bos taurus breed, 42 Bos indicus breed and 76 Bos indicus x Bos taurus individuals. As the SNP marker, there were the significant effects on the Body length (BL) in the Bos taurus (BT) and Bos indicus x Bos taurus (BMY) populations (P < 0.05). In BT population, animals with the genotype TT had lower mean values for BL and Hip width (HW) than these with the TC and CC genotype (P < 0.01). In BMY population, animals with the genotype TC had lower mean values for BL than these with the genotype CC (P < 0.05). These results suggest that the SNP of the GDF5 gene could be a very useful genetic marker for body measurement traits in the bovine reproduction and breeding.

[Genome array on differentially expressed genes of muscle tissue in intact male and castrated Qinchuan cattle].

Bovine genome array was used to construct gene expression profile to screen differentially expressed genes of the Longuissimus dorsi muscle (LDM) tissue between intact male and castrated Qinchuan cattle and investigate the molecular mechanism related to meat quality differences between the two groups. Significance Analysis of Microarray (SAM) methods was used to identify the differentially expressed genes. Go (gene ontology) and the pathway analyses were conducted on differentially expressed genes using a free web-based Molecular Annotation System 2.0 (MAS 2.0). About 11,000 probe sets represented about 10,000 genes were detected in LDM of 36 months old Qinchuan cattle. A total of 143 genes were identified to be differentially expressed genes. They were mainly involved in collagen fibril organization and synthesis, regulation of cell growth and development, ubiquitin-dependent protein catabolism, and striated muscle contraction etc. The enriched pathways mainly included ECM-receptor interaction, cell communication, and focal adhesion etc. Subsequently, real-time PCR was performed to validate eight differentially expressed genes screened out by the microarray approach and sufficient consistency was observed between the two methods. According to our study and published papers, the regulated pathways including ECM-receptor interaction, cell communication, focal adhesion, tight junction and genes including COL3A1, COL1A1, COL1A2, SPP1, FBN1, MMP2, ECM1, MYH3, MYH8, S100A4, ASPN, CFD etc were considered as the most important pathways and genes involved in meat quality differences between males and castrated Qinchuan cattle. Moreover, some genes had no annotation in GenBank were screened out, which were presumed to be unknown new genes. The roles that they may play in muscle metabolism need to be clarified in future.

[SNPs detection of IGF2 gene and its relationship with carcass and meat quality traits in Qinchuan cattle].

PCR-SSCP technology was used to analyze the correlation of polymorphisms IGF2 (Insulin-like growth factor) gene with carcass and meat quality traits in 186 Qinchuan cattle at the age of 24-month. C-->T mutation in 120 and A-->G mutation in 279 of IGF2 gene. Statistical analysis indicated that Qinchuan cattle with genotype BB and DD had significant differences in slaughter weight, carcass weight, carcass length, carcass chest depth and eye muscle area (P<0.05). The difference in thickness of back fat was significant (P<0.01). Significant differences in marbling, tenderness, pH24 of meat quality (P<0.05), but the differences in carcass depth and water holding capacity were not significant (P>0.05). Genotype AA, DD were predominant genotypes and A, D were predominant alleles. The population containing B, D allele had more excellent carcass and meat quality than others, especially in the capacity of fat accumulate.

[Genetic variation in RXRG gene and its relationship with twinning trait in cattle].

Retinoic X receptor-gamma (RXRG) gene was studied as a candidate gene for the twinning trait of bovine. A new SNP A1941G was detected by sequencing at 3'UTR. Different genotypes were determined in Luxi monotocous cows, Luxi twinning cows, Chinese Simmental cows, Angus cows and Simmental x Mongolia cows by restriction fragment length polymorphism (RFLP). The value of polymorphism information content indicated that this was a moderate polymorphism in Luxi monotocous cows and Luxi twinning cows. The chi(2) test indicated that the polymorphic locus in Luxi twinning cows did not fit Hardy-Weinberg equilibrium (Plt;0.05). The chi(2) test of associational analysis between genotypic distribution and twinning or monotocous trait in Luxi cows showed that the difference was very significant (Plt;0.01).

[Genetic diversity and origin of mitochondrial DNA D-loop region for some domestic goat breeds of China].

The complete sequences of mitochondrial DNA D-loop of 140 individuals in 10 Chinese goat (Capra hiruc) breeds were analyzed by DNA sequencing technology. The results showed that the length of mtDNA D-loop in Chinese goats was 1 211-1 213 bp. There were 84 different haplotypes and 171 polymorphic sites. The mean nucleotide diversity (Pi) and haplotype diversity (Hd) were 0.02063+/+0.00225 and 0.988+/-0.003, respectively, and the average number of nucleotide differences (k) was 24.896. The results showed an abundant genetic diversity in domestic goats of China. The NJ tree indicated that there were two main branches in Chinese domestic goats, thereinto, one branch was clustered with Capra aegagrus, and Capra falconeri was clustered alone, which indicated that Capra aegagrus had more contribution to Chinese domestic goat breeds.

Expression profiling of peripheral blood miRNA using RNAseq technology in dairy cows with Escherichia coli-induced mastitis.

E. coli is the main causative agent of mastitis in dairy cows, but the mechanism of molecular regulation underlying the occurrence and development of mastitis has not yet been fully elucidated. In this study, an E. coli-induced mastitis model was created and RNASeq technology was used to measure the miRNA expression profiles at different times post-infection (0, 1, 3, 5, 7 dpi), as well as to screen for differentially expressed miRNA. The results show detection of 2416 miRNAs, including 628 known miRNAs and 1788 newly discovered miRNAs. A total of 200 differentially expressed miRNAs were found at different time points. Bioinformatics analysis showed that these differentially expressed miRNAs may regulate the occurrence and development of mastitis in dairy cows through seven signal transduction pathways, namely cytokine-cytokine receptor interaction, MAPK signaling pathway, chemokine signaling pathway, leukocyte transendothelial migration, T cell receptor signaling pathway, Toll-like receptor signaling pathway, and cell adhesion molecules. In addition, bta-miR-200a, bta-miR-205, bta-miR-122, bta-miR-182 and the newly discovered conservative_15_7229 might be involved in immune process in late stage of E. coli-induced mastitis. The results of this study lay the foundation for molecular network analysis of mastitis and molecular breeding of dairy cows.

Comparison of microRNA Profiles between Bovine Mammary Glands Infected with Staphylococcus aureus and Escherichia coli.

MicroRNAs (miRNAs) play crucial roles in regulating innate and adaptive immunity in humans and animals. Infection with E. coli or S. aureus can cause inflammation of the mammary glands, which results in significant economic losses in dairy cattle. However, the regulatory mechanisms of miRNAs in response to E. coli or S. aureus infection in bovine mammary glands have not been thoroughly explored. To discover the differential expression of miRNA in bovine mammary gland challenged with E. coli or S. aureus, we performed miRNA sequencing on tissue samples. A total of 1838 miRNAs were identified, including 580 known-miRNAs (included in the miRbase database) and 1258 predicted novel miRNAs. The miRNA expression patterns indicated that, compared with control samples, 279 miRNAs and 305 miRNAs were differentially expressed miRNAs (DIE-miRNA) in S. aureus and E. coli infected tissues, respectively. Moreover, the results of comparison the DIE-miRNAs between the E. coli and S. aureus infected groups showed that 197 DIE-miRNAs are identical, 108 DIE-miRNAs are specific to the E. coli group, and 82 DIE-miRNAs are specific to the S. aureus group. Many DIE-miRNAs, such as bta-miR-144, bta-miR-451 and bta-miR-7863, might be the useful biomarkers of mastitis caused by E. coli and S. aureus. In addition, target genes of the DIE-miRNAs were predicted. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that these DIE-miRNAs are likely involved in many immune signaling pathways, including the Toll-like receptor signaling pathways, MAPK signaling pathway, cell adhesion molecules, TGF-ß signaling pathway, leukocyte trans endothelial migration, cytokine-cytokine receptor interaction, and chemokine signaling pathways. This study has provided supportive evidence that miRNAs may serve as diagnostic biomarkers of mastitis in dairy cows, and suggests potentially of effective strategies to combat mastitis.