This study investigated the single nucleotide polymorphisms (SNPs) of Fatty acid desaturase 2 (FADS2) gene and further explored their genetic effects on conventionally collected milk production traits in Chinese Holstein cows using 18,264 test-day records of 841 cows. One missense mutation c. 908 C > T (SNP site in the complementary DNA sequence), which caused an amino acid change from alanine to valine (294Ala > Val), and two 3’ untranslated region (UTR) SNPs, c.1571 G > A and c.2776 A > G were finally identified. The SNP c.908 C > T was significantly associated with test-day milk yield, fat percentage and 305-day milk, fat and protein yield. In particular, the T allele of the SNP c.908 C > T showed a significant association with decreased somatic cell score (SCS) in the investigated population. Significant relationship between the SNP c.1571 G > A and 305-day milk yield showed that genotype GG was linked to the highest milk yield. Substituting the allele G for A at the c.2776 A > G locus resulted in a decrease of protein percentage. Our results demonstrated that FADS2 was an interesting candidate for selection to increase milk production and improve resistance against mastitis.

This study determined the associations of FADS2 c.1571G>A with milk FAs content and revealed that cows with the GG genotype had improved levels of delta-6 desaturase substrates (linoleic acid, C18:2n-6; p < 0.001) and decreased levels of desaturase products (gamma-linolenic acid, C18:3n-6; p < 0.001), indicating a reduction in FADS2 expression or delta-6 desaturase activity caused by this polymorphism. Computer alignment demonstrated that c.1571G>A occurred within a potential miR-744 binding site. When the c.1571G allele was present, the luciferase activity of reporter constructs was significantly suppressed by miR-744, while no such effect was observed with the A allele. Overexpression of miR-744 in bovine mammary epithelial cells (with the 1571GG genotype) downregulated FADS2 expression at both mRNA and protein levels. In contrast, inhibition of endogenous miR-744 with a specific inhibitor dramatically upregulated FADS2 expression. Taken together, these lines of evidence indicated that the c.1571A minor allele abolished the ability of miR-744 to bind FADS2, with a consequent increase in FADS2 expression levels and synthesis of omega-6 LC-PUFAs.

Context: The regulation of milk lipids is important for the evaluation of dairy cows’ performance. Lipids are produced and secreted by mammary gland under the regulation of steroid hormones, growth factors and microRNAs (miRNAs). MicroRNAs have been verified to be involved in numerous biological processes. Previous studies have shown that miR-141 is expressed at higher levels in dairy cows at peak lactation than in those at early lactation. However, the roles of miR-141 in bovine mammary epithelial cells (BMECs) and the mechanisms how it affects lipid metabolism are as yet unknown. Aims: The aims of this study were to clarify (i) the molecular mechanisms of miR-141 in milk lipid metabolism, and (ii) how miR-141 affects milk lipid metabolism in BMECs. Methods: Triglycerides were observed in BMECs using triglyceride analysis after overexpression or inhibition of miR-141; selected potential candidate genes that are targeted by miR-141 using TargetScan. The regulatory relationship among miR-141, SIRT1 gene and lipid metabolism-related genes (SREBF1, FASN and PPARγ) by using the dual luciferase assay, quantitative real-time PCR and western blotting. Key results: Through overexpression or inhibition of miR-141 expression, we found that miR-141 promoted lipid metabolism in BMECs and an increase in triglycerides was observed in these cells. Further, miR-141 targets the 3′UTR of SIRT1 mRNA, and negative regulates the expression of SIRT1 gene in BMECs. Also, the expression levels of SREBF1, FASN and PPARγ, which are related to milk lipid metabolism, were also altered after overexpression miR-141. Conclusions: Our results have revealed that miR-141 could promote milk lipid metabolism in BMECs by means of negative regulates SIRT1 gene and positive effects lipid metabolism-related genes (SREBF1, FASN and PPARγ) in BMECs. Implications: Our research indicates that miR-141 could be considered a marker in cattle breeding to obtain high quality dairy products. It would be useful to study the function of miRNAs in milk lipid metabolism and synthesis. In the long term these findings might be helpful in developing practical means to improve the quality of ruminant milk.

Long non-coding RNAs (LncRNAs) are generally longer than 200 bp in length and play an important regulatory role in the growth and development of skeletal muscle. In the previous work, the non-coding RNAs with abundant expression in bovine tissues were screened out. After quantitative real-time PCR (qPCR), 33 lncRNAs with differential expression in various bovine tissues were identified. Differential expression analysis base on tissue expression profiles of 33 lncRNAs, a long non-coding RNA LncRNA13, which may have effects on bovine muscle development, was found. The expression levels in embryo muscle and adult cattle muscle were significantly different (p < 0.01), so it is speculated that it may have a certain impact on the development of cattle muscle. It was named LncRNA 5.8S rRNA-OT1, and its overexpression vector pcDNA3.1-LncRNA 5.8S rRNA-OT1 was cloned and constructed. The purpose of this study is to further explore its impact on the proliferation and differentiation of bovine muscle cells and accumulate data to lay a foundation for further exploration of the function of LncRNA 5.8S rRNA-OT1 and add basic data for the study of the regulatory mechanism of lncRNA.

Long noncoding RNAs have been identified as important regulators of gene expression and animal development. The expression of natural antisense transcripts (NATs) transcribed in the opposite direction to protein-coding genes is usually positively correlated with the expression of homologous sense genes and is the key factor for expression. Here, we identified a conserved noncoding antisense transcript, CFL1-AS1, that plays an important role in muscle growth and development. CFL1-AS1 overexpression and knockout vectors were constructed and transfected into 293T and C2C12 cells. CFL1-AS1 positively regulated CFL1 gene expression, and the expression of CFL2 was also downregulated when CFL1-AS1 was knocked down. CFL1-AS1 promoted cell proliferation, inhibited apoptosis and participated in autophagy. This study expands the research on NATs in cattle and lays a foundation for the study of the biological function of bovine CFL1 and its natural antisense chain transcript CFL1-AS1 in bovine skeletal muscle development. The discovery of this NAT can provide a reference for subsequent genetic breeding and data on the characteristics and functional mechanisms of NATs.

Bovine mastitis causes significant economic losses in the dairy industry. Emerging evidence highlights the critical role of long non-coding RNAs (lncRNAs) in inflammation-associated epigenetic regulation through competing endogenous RNA (ceRNA) networks. In this study, we established a bovine mastitis model in three healthy primiparous Holstein cows by intramammary infection with S. aureus. Infected and control mammary tissue samples were then collected for transcriptomic profiling, which identified 2005 differentially expressed lncRNAs. Among them, BMNCR was significantly upregulated in S. aureus-infected mammary tissues and S. aureus-stimulated BMECs. We evaluated the coding potential of BMNCR and confirmed its non-coding nature. Functional studies in BMECs demonstrated that knockdown of BMNCR suppressed proliferation, promoted apoptosis, and altered the expression of inflammatory factors, including IL-2, IL-6, IL-8, and IL-12. Mechanistically, BMNCR acted as a sponge for bta-miR-145, thereby leading to the derepression of ANO6. Silencing ANO6 partly recapitulated the effects of BMNCR knockdown, impairing proliferation and increasing IL-8 expression. Collectively, these findings suggest that the BMNCR/miR-145/ANO6 axis is involved in the regulation of inflammatory responses and epithelial homeostasis during bovine mastitis, with BMNCR functioning as a protective regulator in this process.