Molecular regulatory mechanism of key LncRNAs in subclinical mastitic cows with folic acid supplementation.

BACKGROUND: Folic acid is a water-soluble B vitamin (B9), which is closely related to the body's immune and other metabolic pathways. The folic acid synthesized by rumen microbes has been unable to meet the needs of high-yielding dairy cows. The incidence rate of subclinical mastitis in dairy herds worldwide ranged between 25%~65% with no obvious symptoms, but it significantly causes a decrease in lactation and milk quality. Therefore, this study aims at exploring the effects of folic acid supplementation on the expression profile of lncRNAs, exploring the molecular mechanism by which lncRNAs regulate immunity in subclinical mastitic dairy cows. RESULTS: The analysis identified a total of 4384 lncRNA transcripts. Subsequently, differentially expressed lncRNAs in the comparison of two groups (SF vs. SC, HF vs. HC) were identified to be 84 and 55 respectively. Furthermore, the weighted gene co-expression network analysis (WGCNA) and the KEGG enrichment analysis result showed that folic acid supplementation affects inflammation and immune response-related pathways. The two groups have few pathways in common. One important lncRNA MSTRG.11108.1 and its target genes (ICAM1, CCL3, CCL4, etc.) were involved in immune-related pathways. Finally, through integrated analysis of lncRNAs with GWAS data and animal QTL database, we found that differential lncRNA and its target genes could be significantly enriched in SNPs and QTLs related to somatic cell count (SCC) and mastitis, such as MSTRG.11108.1 and its target gene ICAM1, CXCL3, GRO1. CONCLUSIONS: For subclinical mastitic cows, folic acid supplementation can significantly affect the expression of immune-related pathway genes such as ICAM1 by regulating lncRNAs MSTRG.11108.1, thereby affecting related immune phenotypes. Our findings laid a ground foundation for theoretical and practical application for feeding folic acid supplementation in subclinical mastitic cows.

Transcriptome sequencing analysis for the identification of stable lncRNAs associated with bovine Staphylococcus aureus mastitis.

BACKGROUND: Staphylococcus aureus (S. aureus) mastitis is one of the most difficult diseases to treat in lactating dairy cows worldwide. S. aureus with different lineages leads to different host immune responses. Long non-coding RNAs (lncRNAs) are reported to be widely involved in the progress of inflammation. However, no research has identified stable lncRNAs among different S. aureus strain infections. In addition, folic acid (FA) can effectively reduce inflammation, and whether the inflammatory response caused by S. aureus can be reduced by FA remains to be explored. METHODS: lncRNA transcripts were identified from Holstein mammary gland tissues infected with different concentrations of S. aureus (in vivo) and mammary alveolar cells (Mac-T cells, in vitro) challenged with different S. aureus strains. Differentially expressed (DE) lncRNAs were evaluated, and stable DE lncRNAs were identified in vivo and in vitro. On the basis of the gene sequence conservation and function conservation across species, key lncRNAs with the function of potentially immune regulation were retained for further analysis. The function of FA on inflammation induced by S. aureus challenge was also investigated. Then, the association analysis between these keys lncRNA transcripts and hematological parameters (HPs) was carried out. Lastly, the knockdown and overexpression of the important lncRNA were performed to validate the gene function on the regulation of cell immune response. RESULTS: Linear regression analysis showed a significant correlation between the expression levels of lncRNA shared by mammary tissue and Mac-T cells (P < 0.001, R2 = 0.3517). lncRNAs PRANCR and TNK2-AS1 could be regarded as stable markers associated with bovine S. aureus mastitis. Several HPs could be influenced by SNPs around lncRNAs PRANCR and TNK2-AS1. The results of gene function validation showed PRANCR regulates the mRNA expression of SELPLG and ITGB2 within the S. aureus infection pathway and the Mac-T cells apoptosis. In addition, FA regulated the expression change of DE lncRNA involved in toxin metabolism and inflammation to fight against S. aureus infection. CONCLUSIONS: The remarkable association between SNPs around these two lncRNAs and partial HP indicates the potentially important role of PRANCR and TNK2-AS1 in immune regulation. Stable DE lncRNAs PRANCR and TNK2-AS1 can be regarded as potential targets for the prevention of bovine S. aureus mastitis. FA supplementation can reduce the negative effect of S. aureus challenge by regulating the expression of lncRNAs.

Folic acid supplementation regulates milk production variables, metabolic associated genes and pathways in perinatal Holsteins.

Perinatal period is the critical time in dairy cattle due to negative energy balance and high milk production stress. Being a key role in biosynthesis and methylation cycle, folic acid is considered essential for lactational and metabolic performance in dairy cattle. Thus, the current study was designed to evaluate the effect of folic acid supplementation on milk production phenotypic traits in periparturient cows. Transcriptomic screening was performed for milk production and metabolism-associated differentially expressed genes. The 123 cows having similar parity, weight and expected date of calving were randomly selected and divided into three groups; A (n = 41, folic acid 240 mg/500 kg cow/day), B (n = 40, FA 120 mg/500 kg cow/day) and C (Control, n = 42). Folic acid was supplemented for 21 days (14 days pre- and seven days post-calving), and three samples of blood lymphocytes were taken on day seven post-calving from each folic acid-treated and control group. In addition, the milk samples for each folic acid-treated group have been collected at 2nd, 3rd and 4th month of lactation. The increase in average milk yield noticed in group B were significantly (p-value < .05) higher than C and A. However, the data showed no noteworthy differences for milk fat and milk protein among the three groups. The transcriptomic analysis revealed that folic acid treatment regulated many key metabolic-related genes (DGAT2, ALOX5, LAP3, GPAT3, GGH, ALDOA, TKT) and pathways (glycolysis, folate biosynthesis, glutathione metabolism, etc.) in periparturient dairy cattle. It was concluded from the above findings that 120 mg/500 kg of folic acid quantity could be considered as a standard during the periparturient period to enhance the milk production performance of dairy cows. The transcriptomic profile revealed several metabolic and milk production-associated genes which could be a useful addition to the marker selection for the enhancement of metabolism and milk production of periparturient dairy cows.

Folic acid supplementation regulates key immunity-associated genes and pathways during the periparturient period in dairy cows.

OBJECTIVE: The current research was aimed to profile the transcriptomic picture of the peripheral blood lymphocytes (PBLs) associated with immunity in Chinese Holsteins supplemented orally with coated folic acid during the periparturient period. METHODS: The total of 123 perinatal cows were selected for this study and divided into three groups; group A (n = 41, 240mg/ 500 kg cow/day), group B (n = 40, 120mg/ 500 kg cow/day) and group C (n = 42, 0mg/cow/day) based on the quantity of folic acid fed. Three samples of PBLs were selected from each folic acid treated group (High, Low, and Control) and RNA sequencing method was carried out for transcriptomic analysis. RESULTS: The analysis revealed that a higher number of genes and pathways were regulated in response to high and low folic acid supplementation compared to the controls. We reported the novel pathways (TNF signaling, Antigen processing and presentation, Staphylococcus aureus infection and NF-kappa B signaling pathways) and the key genes (e.g. CXCL10, TNFRSFIA, CD4, BOLA-DQB, NFKBIA, and TNFSF13) having great importance in immunity and anti-inflammation in the periparturient cows in response to coated folic acid treatment. CONCLUSION: Collectively, our study profiled first-time transcriptomic analysis of bovine lymphocytes and compared the involved cytokines, genes, and pathways between High vs. Control and Low vs. Control. Our data suggest that the low folic acid supplementation (120 mg/500 kg) could be a good choice to boost appropriate immunity and anti-inflammation as well as might being applied to the health improvement of perinatal dairy cows.