Bovine mastitis has become increasingly important issues for farmers and consumers, leading to large economic losses in the dairy industry worldwide. Because treatment of mastitis is difficult and costly, improved mastitis resistance through selective breeding would be advantageous. The toll-like receptor 4 (TLR4) is an important player in recognising pathogens and activating immune responses. However, its roles in mastitis occurrence and the underlying molecular mechanisms are unclear. In this study, a single nucleotide polymorphism, rs8193069 (T → C) in TLR4 gene was detected in a Holstein cow resource population in southern China. Association analysis with 5-year production traits, haematology, and biochemistry parameters revealed that individuals with genotype CC had significantly lower somatic cell counts (SCC), lower fat percentage, but higher 305-day milk (p < 0.05) and total milk yield (p < 0.01). Both genotypes CC and CT had lower lymphocyte counts (#LYMPH) (p < 0.01) and basophil counts (#BASO) (p < 0.05) than TT. Genotype CC had a less level of triglyceride (p < 0.01) and creatine kinase (p < 0.05) than CT. Further analysis based on the production data revealed significant positive correlations between SCC and #LYMPH. Analysis of TLR4 protein structure and properties suggested that the missense mutation on the 674th amino acid from Thr to Ile reduced the flexibility and hydrophilicity of TIR domain, implying a weakened binding ability of TLR4 to its adaptors. In conclusion, allele C of rs8193069 was the major allele in Holstein cows that indicated a greater genetic potential to mastitis resistance and milk yields, probably via the LPS-TLR4 inflammatory signalling. This study offers a marker to improve mastitis resistance in the dairy cow population in southern China.

The embryonic loss during early stage of gestation is one of the major causes of infertility for domestic ruminants, causing huge economic losses to pasture. Maternal recognition of pregnancy and implantation are the crucial process for determining the successful establishment and development of pregnancy in cattle. The research on molecular mechanisms of pregnancy recognition will facilitate illustrating the complex process of pregnancy establishment and help to improve pregnancy outcomes. In this study, we performed transcriptomic analysis of primary bovine endometrial epithelial cells (BEND) with or without IFNT and hormones intervention through RNA sequencing. We eventually identified 608 differentially expressed genes (DEGs) including 409 up-regulated genes and 199 down-regulated genes in IFNT and hormones-treated group compared with control group. Gene Ontology (GO) enrichment analysis demonstrated that the majority of DEGs were implicated in immune system process, response to external stimulus, response to cytokine, regulation of response to stress. Results from KEGG analysis showed a significant enrichment of NOD-like receptor signaling pathway, antigen processing and presentation, necroptosis, oxidative phosphorylation, RIG-I-like receptor signaling pathway. Additionally, a set of promising candidate genes, including (USP18, STAT1, PSMB8, IFIH1, MX2, IFI44, DHX58, CASP8, DRAM1, CXCR4), were characterized by constructing an integrated interaction network. Specifically, the mRNA expression of HOXA11, PTGS1 and PTGS2 were remarkably suppressed by silencing DRAM1 under IFNT and hormone administration, thus speculating that DRAM1 might play a crucial role in early pregnancy by regulating endometrial function. The results of this study depicted a relatively comprehensive transcriptional profiles of BEND in response to IFNT and hormones, which contributes to a better understanding of gene interaction network and underlying regulatory mechanisms in endometrium of ruminants during early pregnancy.

Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three weeks after insemination. During early pregnancy, the expression of interferon-stimulating genes (ISGs) in peripheral blood leukocytes (PBL) is significantly increased. However, due to different strains, detection sample types, detection methods, threshold value, etc. the specific effectiveness of early pregnancy diagnosis using ISGs is worth further study. The purpose of this study was to test interferon-stimulated protein 15 ( ISG15 ), 2'–5'-oligoadenylate synthetase 1 ( OAS1 ) and radical S-adenosyl methionine domain containing 2 ( RSAD2 ) for early pregnancy diagnosis in dairy cows. The expression of ISG15 , OAS1 , and RSAD2 in PBL of pregnant and non-pregnant heifers on days 0, 14, 18, 21 and 28 after artificial insemination (AI) was detected by fluorescence quantitative polymerase chain reaction (PCR). The sensitivity and specificity of the pregnancy diagnosis was analyzed using expression of these three genes separately or in combination by receiver operating characteristic curve. The combination with the highest accuracy used probe primers and duplex fluorescence quantitative PCR. The single quantitative PCR results showed that expression of ISG15 , OAS1 and RSAD2 on day 18 after AI was significantly higher in pregnant than in non-pregnant cows. When these three genes were used separately, or in combination, for early pregnancy diagnosis, the sensitivity for the RSAD2 gene was 100%, and the combination of ISG15 with RSAD2 was 94.7%. The duplex quantitative PCR showed that, although the sensitivity of ISG15 alone was 100%, its specificity was only 88.2% (cut-off value 1.402). The sensitivity of RSAD2 alone was 89.5%, and the specificity was 88.2%; however, when the two genes were used in combination, the sensitivity, specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR. These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using ISG15 and RSAD2 was established. Simultaneous detection of expression of ISG15 and RSAD2 by duplex quantitative PCR can effectively improve the diagnostic accuracy for dairy cows.

Mastitis is a common disease for dairy cows that exerts tremendously detrimental impacts on the productivity of cows and economic viability of pasture. Dihydromyricetin (DMY) is a flavonoid monomeric compound that possesses anti-inflammatory and antioxidant activity. This study aimed at dissecting the effects of DMY on the lactation performance, blood parameters, gut microbiota, and metabolite profiles of dairy cows with subclinical mastitis (SM). The results showed that dietary supplementation with DMY resulted in a reduction in milk somatic cell count, an increase in serum T-AOC and CAT activity, as well as a decrease in serum MDA content. DMY significantly enhanced the prevalence of Coprococcus and Roseburia and reduced the proportion of Cyanobacteria, Proteobacteria, and Dehalobacterium. The amino acid degradation, antibiotic resistance, and O-antigen building blocks biosynthesis (E. coli) capacity of gut microbes were notably diminished by DMY supplementation in cows with SM. Moreover, fecal and plasma metabolomic analysis revealed that DMY intervention reduced the abundance of pro-inflammatory metabolites including arachidonic acid analogues, ω-6 PUFA, and structural components of bacteria. Nevertheless, the levels of anti-inflammatory and antioxidant metabolites involving secondary bile acids, antioxidant vitamins, specific amino acid analogues, etc. were elevated by DMY administration. Overall, DMY might ameliorate SM via enhancing antioxidant capacity and improving the structure of the hindgut microbial community and metabolite profiles in dairy cows. These findings underscore the potential of DMY as a valuable dietary supplement for the improvement of mammary inflammatory diseases in dairy cows.

High-yielding dairy cows suffer from a high metabolic load and oxidative stress, which lead to systemic inflammation and metabolic disorders, increasing the susceptibility of these cows to various production diseases. Dihydromyricetin (DMY) has demonstrated potent antioxidant and anti-inflammatory physiological functions; however, research into its application in ruminants remains limited. This study investigated whether DMY supplementation is associated with the maintenance of metabolic homeostasis through the regulation of gut microbiota and metabolite profiles. A total of 14 mid-lactation Holstein dairy cows were randomly divided into two groups (n = 7 per group) and supplemented with DMY at 0 or 0.05% in their basal diet for 60 consecutive days. The effects of DMY on the blood biochemical indicators and the antioxidant capacity of the dairy cows were then determined. Alterations to the gut microbiome and the fecal and plasma metabolome were analyzed through 16S rDNA sequencing and untargeted metabolomics. The results showed that DMY significantly improved the activity of serum glutathione peroxidase (GSH-Px) and presented a trend of increasing the total antioxidant capacity (T-AOC). The abundance of multiple fiber-degrading and beneficial commensal bacteria in the gut, including Fibrobacter_succinogenes, Ruminococcus_albus, and Turicibacter, was significantly elevated by the DMY intervention, accompanied by the upregulation of 8,11,14-eicosatrienoic acid, myricetin, dihydro-3-coumaric acid, PGE1, L-leucine, nicotinuric acid, pantothenic acid, and pyruvate in the feces and plasma. Moreover, DMY supplementation notably reduced the abundance of potential pathogenic microbes, such as Chloroflexi, Deltaproteobacteria, RFP12, and Succinivibrio, and downregulated the levels of 12-hydroxydodecanoic acid, 12,13-DHOME (12,13-dihydroxy-9Z-octadecenoic acid), 16-hydroxyhexadecanoic acid, niacin, and glycerol 3-phosphate. These differential metabolites were principally enriched in the mTOR signaling pathway; pantothenate, nicotinate, and thiamine metabolism; glutathione metabolism; and glycolysis/gluconeogenesis. In summary, dietary supplementation with DMY increased the abundance of intestinal fiber-degrading bacteria and multiple metabolites with known anti-inflammatory and antioxidant properties in the feces and plasma, and was associated with alterations in metabolic pathways involving B-vitamins, amino acids, and glutathione. This suggests a potential role for DMY in supporting metabolic homeostasis in dairy cows.

Genome-wide association studies (GWAS) have been a successful tool for identifying quantitative trait loci (QTL) for economically important traits in dairy cows. However, the availability of QTLs linked to phenotypic traits is limited in the literature. In this study, we used GWAS, haplotype association, and fine-mapping analyses to identify candidate variants associated with milk production, health, and reproductive traits in 380 Chinese Holstein cattle from Southern China using whole-genome sequence data. GWAS identified 91 genome-wide significant signals that were annotated to 63 genes associated with milk production, health, and reproductive traits in dairy cattle. Haplotype association analysis further revealed that eight GWAS signals within three QTLs were associated with milk production and health traits of cows. Fine-mapping analysis revealed that 3 GWAS signals (6_92530313_G_A, 10_17185230_G_A, and 10_17209112_T_G) were the potential causal variants. Several candidate genes, including ANKS1B, IL17RD, CNOT6L, AOC1, and TLE3, have been confirmed to be associated with milk production, health, and reproductive traits in dairy cows. These findings significantly contribute to unraveling the genetic basis of economically important traits in Holstein cattle.