Identification and characterization of long non-coding RNAs in mammary gland tissues of Chinese Holstein cows.

LncRNAs (Long non-coding RNA) is an RNA molecule with a length of more than 200 bp. LncRNAs can directly act on mRNA, thus affecting the expression of downstream target genes and proteins, and widely participate in many important physiological and pathological regulation processes of the body. In this study, RNA-Seq was performed to detect lncRNAs from mammary gland tissues of three Chinese Holstein cows, including three cows at 7 d before calving and the same three cows at 30 d postpartum (early lactation stage). A total of 1,905 novel lncRNAs were detected, 57.3% of the predicted lncRNAs are ≥ 500 bp and 612 lncRNAs are intronic lncRNAs. The exon number of lncRNAs ranged from 2 to 10. A total of 96 lncRNAs were significantly differentially expressed between two stages, of which 47 were upregulated and 49 were downregulated. Pathway analysis found that target genes were mainly concentrated on the ECM-receptor interaction, Jak-STAT signaling pathway, PI3K-Akt signaling pathway, and TGF-beta signaling pathway. This study revealed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows at non-lactation and early lactation periods, and provided a basis for studying the functions of lncRNAs in Holstein cows during different lactation periods.

The mammary gland of dairy cows is the main place of milk synthesis and secretion, and plays a vital role in the process of milk production. LncRNAs (Long non-coding RNAs) are a class of non-coding RNAs with a length greater than 200 bp and do not encode protein, which can regulate gene expression at the transcriptional, post-transcriptional and chromatin levels, with biological functions such as regulating cell proliferation, differentiation, and apoptosis. Relevant studies in humans and model animals have shown that lncRNAs participate in mammalian mammary gland development and lactation, but there are few studies on lncRNAs regulation of mammary gland development and lactation in dairy cows. Therefore, this study aims to reveal the potential role of lncRNAs in the mammary gland of dairy cows through screening, identification, and functional research of differentially expressed lncRNAs at different periods of mammary gland development (pregnancy and early lactation period). It provides a reference for the follow-up study on the regulatory mechanism of dairy cows' mammary gland health.

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.

Potential Role of Lauric Acid in Milk Fat Synthesis in Chinese Holstein Cows Based on Integrated Analysis of Ruminal Microbiome and Metabolome.

The composition and metabolic profile of the ruminal microbiome have an impact on milk composition. To unravel the ruminal microbiome and metabolome affecting milk fat synthesis in dairy cows, 16S rRNA and internal transcribed spacer (ITS) gene sequencing, as well as ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods were used to investigate the significant differences in ruminal bacterial and fungal communities as well as metabolome among Chinese Holstein cows with contrasting milk fat contents under the same diet (H-MF 5.82 ± 0.41% vs. L-MF 3.60 ± 0.12%). Another objective was to culture bovine mammary epithelial cells (BMECs) to assess the effect of metabolites on lipid metabolism. Results showed that the acetate-to-propionate ratio and xylanase activity in ruminal fluid were both higher in H-MF. Microbiome sequencing identified 10 types of bacteria and four types of fungi differently abundant at the genus level. Metabolomics analysis indicated 11 different ruminal metabolites between the two groups, the majority of which were lipids and organic acids. Among these, lauric acid (LA) was enriched in fatty acid biosynthesis with its concentration in milk fat of H-MF cows being greater (217 vs. 156 mg per 100 g milk), thus, it was selected for an in vitro study with BMECs. Exogenous LA led to a marked increase in intracellular triglyceride (TG) content and lipid droplet formation, and it upregulated the mRNA abundance of fatty acid uptake and activation (CD36 and ACSL1), TG synthesis (DGAT1, DGAT2 and GPAM), and transcriptional regulation (SREBP1) genes. Taken together, the greater relative abundance of xylan-fermenting bacteria and fungi, and lower abundance of bacteria suppressing short-chain fatty acid-producing bacteria or participating in fatty acid hydrogenation altered lipids and organic acids in the rumen of dairy cows. In BMECs, LA altered the expression of genes involved in lipid metabolism in mammary cells, ultimately promoting milk fat synthesis. Thus, it appears that this fatty acid plays a key role in milk fat synthesis.

Identification and Classification of Long Non-Coding RNAs in the Mammary Gland of the Holstein Cow.

The mammary glands, responsible for milk secretion, are regulated at a local level by various hormones, growth factors, non-coding RNAs, and other elements. Recent research has discovered the presence of lncRNAs in these glands, with suggestions that they may be essential for the maintenance and function of mammary glands. Besides directly controlling the gene and protein expression, lncRNAs are believed to play a significant part in numerous physiological and pathological processes. This study focused on examining the mammary gland tissues of Chinese Holstein cows, to identify and categorize long non-coding RNAs (lncRNAs). The research intended to distinguish lncRNAs in the mammary tissues of Holstein cows and contrast them between lactation and non-lactation periods. In this study, mammary gland tissues were sampled from three Holstein cows in early lactation (n = 3, 30 days postpartum) and non-lactation (n = 3, 315 days postpartum) on a large dairy farm in Jiangsu province. Mammary tissue samples were collected during early lactation and again during non-lactation. In total, we detected 1905 lncRNAs, with 57.3% being 500 bp and 612 intronic lncRNAs. The exon count for lncRNAs varied from 2 to 10. It was observed that 96 lncRNA expressions markedly differed between the two stages, with 83 genes being upregulated and 53 downregulated. Enrichment analysis results revealed that Gene Ontology (GO) analysis was primarily abundant in cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that target genes were predominantly abundant in metabolic pathways, fatty acid biosynthesis, the immune system, and glycosphingolipid biosynthesis. This study analyzed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows during both lactation and non-lactation stages, forming a foundation for further investigation into the functional roles of lncRNAs in Holstein cows throughout lactation.

The emerging regulatory mechanisms and biological function of circular RNAs in skeletal muscle development.

Circular RNAs (circRNAs) are novel noncoding RNAs that assume a covalently closed-loop structure. Because of technical limitations in research, circRNAs were long considered to be byproducts of the RNA splicing process. Recently, emerging evidence has indicated that circRNAs can regulate gene expression by sponging microRNAs (miRNAs) or proteins, functioning as protein scaffolds, regulating transcription and splicing, and acting as templates for translation, thereby extending the functional complexity and diversity of eukaryotic transcriptomes. Remarkably, an increasing number of studies have revealed that circRNAs are stable, evolutionarily conserved, and are often expressed in a tissue- or developmental stage-specific patterns, especially abundant in muscle tissue. circRNAs are emerging as powerful regulators in diverse cellular processes and diseases, particularly in skeletal muscle myogenesis. Here, we describe circRNAs discovery, classification, and regulatory mechanisms, highlight the current understanding of circRNAs in regulating skeletal muscle development, and tell the story of how circRNAs, once thought to be "splicing noise", have become "genetic treasures".

Polymorphisms of the IL-17A Gene Influence Milk Production Traits and Somatic Cell Score in Chinese Holstein Cows.

The cow's milk production characteristics are a significant economic indicator in the livestock industry. Serum cytokines such as interleukin-17 (IL-17) may be potential indicators for bovine mastitis concerning the milk somatic cell count (SCC) and somatic cell score (SCS). The current study aims to find previously undiscovered single nucleotide polymorphisms in the bovine (IL-17A) gene and further investigates their associations with milk production traits in Chinese Holstein cows. Twenty Chinese Holstein cows were randomly chosen from six farms in Jiangsu Province, China. The DNA was extracted from selected samples of bloods for PCR amplification Sequence analyses were used to find SNPs in the bovine (IL-17A) gene. The discovered five SNPs are g-1578A>G, g-1835G>A, and g-398T>A in the 5'UTR; g3164T>C and g3409G>C in the exon region. The genotyping of Holstein cows (n = 992) was performed based on Sequenom Mass ARRAY and SNP data. The connection between SNPs, milk production variables, and the somatic cell score was investigated using the least-squares method. Based on the results, SNP g-398T>A had a significant linkage disequilibrium with g3164T>C. SNPs were found to have significant (p < 0.05) correlations with the test-day milk yield. In conclusion, IL-17A affects cow's milk production traits significantly.

Emerging functions of circular RNA in the regulation of adipocyte metabolism and obesity.

As noncoding RNAs, circular RNAs (circRNAs) are covalently enclosed endogenous biomolecules in eukaryotes that have tissue specificity and cell specificity. circRNAs were once considered a rare splicing byproduct. With the development of high-throughput sequencing, it has been confirmed that they are expressed in thousands of mammalian genes. To date, only a few circRNA functions and regulatory mechanisms have been verified. Adipose is the main tissue for body energy storage and energy supply. Adipocyte metabolism is a physiological process involving a series of genes and affects biological activities in the body, such as energy metabolism, immunity, and signal transmission. When adipocyte formation is dysregulated, it will cause a series of diseases, such as atherosclerosis, obesity, fatty liver, and diabetes. In recent years, many noncoding RNAs involved in adipocyte metabolism have been revealed. This review provides a comprehensive overview of the basic structure and biosynthetic mechanism of circRNAs, and further discusses the circRNAs related to adipocyte formation in adipose tissue and liver. Our review will provide a reference for further elucidating the genetic regulation mechanism of circRNAs involved in adipocyte metabolism.

Epigenetic Regulation Mechanisms of the Cofilin-1 Gene in the Development and Differentiation of Bovine Primary Myoblasts.

As the quality of beef products has received increasing attention, it is essential to explore the underlying transcriptional and epigenetic mechanisms of meat traits. Our project uses Qinchuan cattle as the research subject. First, we examined the spatiotemporal expression pattern of the CFL1 gene in a panel of fetal bovine, calf, and adult cattle samples. Then, we performed DNA methylation experiments of CFL1 on myogenesis and muscle maturation using the BSP amplification and COBRA sequencing techniques and found that high DNA methylation levels showed low expression levels. Next, we performed an assay between bta-miR-182 and the CFL1 gene and demonstrated that miR-182 could promote bovine primary myoblast differentiation by negatively regulated the expression of CFL1. Finally, we constructed an adenovirus overexpression and interference vector and found that CFL1 could suppress the differentiation of bovine primary myoblasts. In summary, our experiment comprehensively analyzes the epigenetic regulation mechanisms of the CFL1 gene in the development and differentiation of bovine primary myoblasts. This has far-reaching significance for improving the meat production and meat quality of Qinchuan cattle. This can provide reliable data support and a theoretical research basis for the rapid and efficient breeding selection of local yellow cattle and the genetic improvement of meat quality.

Analysis of Non-Genetic Factors Affecting Wood's Model of Daily Milk Fat Percentage of Holstein Cattle.

This research paper aimed to explore the characteristics of Holstein cattle's milk fat percentage lactation curve and its influencing factors. The Wood model was used for fitting the lactation curve of 398,449 DHI test-day milk fat percentage records of Holstein cows from 2018 to 2020 in 12 dairy farms in Jiangsu province, and the influencing factors­including farm size, parity, calving season, calving interval, and 305-days milk production­on the parameters of the lactation curve were analyzed. The results showed that the non-genetic factors such as dairy farm size, calving season, parity, calving interval, and 305-days milk yield have a significant impact on milk fat percentage (p < 0.01); the average R2 of the daily milk fat percentage curve was 0.9699; the lowest milk fat percentage was 3.54%; the time to reach the lowest milk fat percentage was 126 days; and the persistence of milk fat percentage was 3.59%. All of these factors explored in this study fit at different levels above 0.96. The Wood model performed well in the fitting and analysis of the milk fat percentage curve of Holstein cattle in Jiangsu Province. This study provides a reference for improving the milk fat percentage of Holstein cattle.

Effects of Seasonal Heat Stress during Late Gestation on Growth Performance, Metabolic and Immuno-Endocrine Parameters of Calves.

Heat stress during late gestation could affect subsequent lactation performance, resulting in damage to the immune function, health, and growth performance of calves. This study aimed to compare the effects of 33 days of summer stress (Summer group, 70.15 < THI < 74.28) with 33 days of winter during late gestation (Winter group, 57.55 < THI < 67.25) on the growth, hormones, oxidative stress, and immune function of calves. Calves (Summer, n = 28; Winter, n = 23) were separated from cows immediately after birth and fed with 2 L colostrum within 2 h and 8−10 h after birth, respectively, and weaned at 60 days of age. Bodyweight (BW) was measured at birth and weaning. Withers height (WH), body length, and chest girth were measured at birth, 30 days, and 60 days of age. The health of calves ranging in age from 1 to 7 days was recorded. Plasma interferon-γ (IFN-γ), superoxide dismutase (SOD), adrenocorticotropin (ACTH), gonadotropin-releasing hormone (GnRH), IgG, cortisol, heat shock protein 70 (Hsp70), growth hormone (GH), insulin, lipid peroxide (LPO), and tumor necrosis factor-α (TNF-α) levels were measured in calves at 0 (before colostrum feeding), 3, 7, 14, 28, and 56 days of age. The pregnancy period of the Summer group was shortened by 1.44 days. The Winter and Summer groups had the same birth weight. One week after birth, the incidence of diarrhea was 57.14% and 21.74% in Summer and Winter groups, respectively. Compared with the Winter group, TNF-α in the Summer group increased significantly before colostrum feeding. ACTH and LPO decreased significantly at 3 days of age, ACTH and TNF-α decreased significantly at 7 days of age, Hsp70 increased significantly, ACTH was significantly reduced at 14 days of age, and Hsp70 increased dramatically at 7 days of age. SOD and TNF-α increased statistically at 28 days of age, LPO decreased significantly, and IFN-γ decreased significantly at 56 days of age, while IgG and GH increased significantly. We conclude that maternal heat stress during late gestation can damage the oxidative stress and immune plasma indexes of offspring before weaning.

Identification and Characterization of Circular RNAs in Mammary Tissue from Holstein Cows at Early Lactation and Non-Lactation.

In this study, circular RNAs (circRNAs) from Holstein cow mammary tissues were identified and compared between early lactation and non-lactation. After analysis, 10,684 circRNAs were identified, ranging from 48 to 99,406 bp, and the average size was 882 bp. The circRNAs were mainly distributed on chromosomes 1 to 11, and 89.89% of the circRNAs belonged to sense-overlapping circRNA. The exons contained with circRNAs ranged from 1 to 47 and were concentrated from 1 to 5. Compared with the non-lactating cows, 87 circRNAs were significantly differentially expressed in the peak lactation cows. There were 68 upregulated circRNAs and 19 downregulated circRNAs. Enrichment analysis of circRNAs showed that GO analysis mainly focused on immune response, triglyceride transport, T cell receptor signaling pathway, etc. Pathway analysis mainly focused on cytokine-cytokine receptor interaction, T helper 17 cell differentiation, fatty acid biosynthesis, the JAK-STAT signaling pathway, etc. Specific primers were designed for two proximal ends of the circRNA junction sites to allow for PCR validation of four randomly selected circRNAs and carry out circRNA-miRNA interaction research. This study revealed the expression profile and characteristics of circRNAs in mammary tissue from Holstein cows at early lactation and non-lactation, thus providing rich information for the study of circRNA functions and mechanisms, as well as potential candidate miRNA genes for studying lactation in Holstein cows.

Genome-wide recombination map construction from single sperm sequencing in cattle.

BACKGROUND: Meiotic recombination is one of the important phenomena contributing to gamete genome diversity. However, except for human and a few model organisms, it is not well studied in livestock, including cattle. RESULTS: To investigate their distributions in the cattle sperm genome, we sequenced 143 single sperms from two Holstein bulls. We mapped meiotic recombination events at high resolution based on phased heterozygous single nucleotide polymorphism (SNP). In the absence of evolutionary selection pressure in fertilization and survival, recombination events in sperm are enriched near distal chromosomal ends, revealing that such a pattern is intrinsic to the molecular mechanism of meiosis. Furthermore, we further validated these findings in single sperms with results derived from sequencing its family trio of diploid genomes and our previous studies of recombination in cattle. CONCLUSIONS: To our knowledge, this is the first large-scale single sperm whole-genome sequencing effort in livestock, which provided useful information for future studies of recombination, genome instability, and male infertility.

The polymorphism of bovine Cofilin-1 gene sequence variants and association analysis with growth traits in Qinchuan cattle.

In our study, four single nucleotide polymorphisms (SNPs) were identified in exon 2 of cofilin-1 (CFL1) gene in 488 Chinese Qinchuan (QC) cattle, which included two missense mutations T 2084G and G 2107C, two synonymous mutations T 2052C and T 2169C. Further, we evaluated haplotype frequency and linkage disequilibrium (LD) coefficient of four SNPs. At SNP T 2052C, G 2107C and T 2169C, the QC cattle population belonged to intermediate genetic diversity (0.25 < PIC-value < 0.5), whereas SNP T-2084G belonged to low polymorphism (PIC-value < 0.25). Haplotype analysis showed that 6 different haplotypes (frequency > 0.03). LD analysis showed that SNP G 2107C and T 2169C, SNP G 2107C and T 2084G were high LD, respectively (r2 > 0.33). Association analysis indicated that SNP T 2052C was significantly associated with body length, chest breadth, chest depth and body mass in the QC population (p < 0.01 or p < 0.05). SNP G 2107C was significantly associated with rump length (p < 0.05). SNP T 2169C was significantly associated with chest breadth and chest depth (p < .01 or p < .05). The results of our study suggest that the CFL1 gene may be a strong candidate gene that affects growth traits in the QC cattle breeding program.

Directed Expression of Tracheal Antimicrobial Peptide as a Treatment for Bovine-Associated Staphylococcus Aureus-Induced Mastitis in Mice.

Bovine mastitis is perplexing the dairy industry since the initiation of intensive dairy farming, which has caused a reduction in the productivity of cows and an escalation in costs. The use of antibiotics causes a series of problems, especially the formation of bacterial antimicrobial resistance. However, there are limited antibiotic-free therapeutic strategies that can effectively relieve bacterial infection of bovine mammary glands. Hence, in this study, we constructed a mammary gland tissue-specific expression vector carrying the antimicrobial peptide of bovine-derived tracheal antimicrobial peptide (TAP) and evaluated it in both primary bovine mammary epithelial cells (pBMECs) and mice. The results showed that the vector driven by the ß-lactoglobulin gene (BLG) promoter could efficiently direct the expression of TAP in pBMECs and the mammary gland tissue of mice. In addition, significant antibacterial effects were observed in both in vitro and in vivo experiments when introducing this vector to bovine-associated Staphylococcus aureus-treated pBMECs and mice, respectively. This study demonstrated that the mammary gland tissue-specific expression vector could be used to introduce antimicrobial peptide both in in vitro and in vivo and will provide a new therapeutic strategy in the treatment of bovine mastitis.

Metformin Inhibits Lipoteichoic Acid-Induced Oxidative Stress and Inflammation Through AMPK/NRF2/NF-κB Signaling Pathway in Bovine Mammary Epithelial Cells.

The objective of this research was to explore the effect of metformin on the lipoteichoic acid (LTA)-induced mastitis model using isolated primary bovine mammary epithelial cells (PBMECs). The PBMECs were exposed to either 3 mM metformin for 12 h as a metformin group (MET) or 100 µg/mL LTA for 6 h as LTA group (LTA). Cells pretreated with 3 mM metformin for 12 h followed by washing and 100 µg/mL LTA exposure for 6 h served as the MET + LTA group. Phosphate-buffered saline was added to cells as the control group. PBMECs pretreated with different metformin doses were analyzed by a flow cytometry (annexin V-fluorescein isothiocyanate assay) to detect the cell apoptotic rate. We performed quantitative reverse transcriptase-polymerase chain reaction and Western blot analysis to evaluate the inflammatory and oxidative responses to metformin and LTA by measuring cellular cytotoxicity, mRNA expression, and protein expression. Immunofluorescence was used to evaluate nuclear localization. The results showed that the gene expression of COX2, IL-1ß, and IL-6 significantly increased in the cells challenged with LTA doses compared to control cells. In inflammatory PBMECs, metformin attenuated LTA-induced expression of inflammatory genes nuclear factor κB (NF-κB) p65, tumor necrosis factor α, cyclooxygenase 2, and interleukin 1ß, as well as the nuclear localization and phosphorylation of NF-κBp65 protein, but increased the transcription of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-targeted antioxidative genes heme oxygenase-1 (HO-1) and Gpx1, as well as the nuclear localization of HO-1 protein. Importantly, metformin-induced activation of Nrf2 is AMP-activated protein kinase (AMPK)-dependent; as metformin-pretreated PBMECs activated AMPK signaling via the upregulation of phosphorylated AMPK levels, cell pretreatment with metformin also reversed the translocation of Nrf2 that was LTA inhibited. This convergence between AMPK and Nrf2 pathways is essential for the anti-inflammatory effect of metformin in LTA-stimulated PBMECs. Altogether, our results indicate that metformin exerts anti-inflammation and oxidative stress through regulation of AMPK/Nrf2/NF-κB signaling pathway, which highlights the role of AMPK as a potential therapeutic strategy for treatment of bovine mastitis.

Abundance of solute carrier family 27 member 6 (SLC27A6) in the bovine mammary gland alters fatty acid metabolism.

Milk fatty acid (FA) composition is associated with the nutritional value of milk and is known to vary with the stage of lactation. Although biochemical aspects controlling FA metabolism in the bovine mammary gland are well-established, less is known about the underlying molecular mechanisms. Thus, to address some of these shortcomings, the present study sought to evaluate milk FA composition and mammary transcriptome profiles at different stages of lactation. Compared with 90 d of lactation, at 315 d of lactation, there was an increase in the concentrations of C18:2, polyunsaturated fatty acids (PUFA), and short-chain fatty acids (SCFA), and a decrease in C16:0 and long-chain fatty acids (LCFA) in milk. To further identify candidate genes and pathways responsible for these phenotypic differences, the transcriptome of bovine mammary tissue at 90 d (peak) and 315 d (late) of lactation was profiled using RNA-seq. A total of 827 differentially expressed genes were identified. Bioinformatic analysis revealed that the major differentially modulated lipid metabolic pathways were the PPAR signaling pathway, alpha-linolenic acid metabolism and linoleic acid metabolism. Compared with peak lactation, the mammary tissue at late lactation had lower abundance of genes related to FA transport and activation (CD36, SLC27A6, ACSM1, FABP3 and FABP4). Thus, to further explore the role of FA transport into mammary cells, we knocked down fatty acid transport protein 6 (solute carrier family 27 member 6, SLC27A6) in the bovine mammary epithelial cells (BMECs) using siRNA. The knockdown of SLC27A6 dramatically downregulated the mRNA abundance of genes associated with FA activation (ACSL4), oxidation (CPT1A) and transport (CD36), while the abundance of genes associated with transcription regulation (PPARG), diacylglycerol acyltransferase 1 (DGAT1), FA binding (FABP3), and desaturation (FADS2) was upregulated. In addition, SLC27A6 silenced the intracellular content of triglyceride (TG) and the percentage of C18:1cis9 and C20:4cis5,8,11,14 was greater, whereas that of C16:0 and C18:0 was lower. Overall, in vivo results indicated that LCFA transport into mammary cells during late lactation partly explains the difference in the FA profiles. In vitro analyses underscored how FA transport via SLC27A6 could dictate in part the intracellular utilization of FA for TG synthesis versus oxidation. The data provide strong support for a central role of SLC27A6 in the regulation of FA metabolism in BMECs.

Polymorphisms of the ACSL1 Gene Influence Milk Production Traits and Somatic Cell Score in Chinese Holstein Cows.

Improving the quality of milk is a challenge for zootechnicians and dairy farms across the globe. Long-chain acyl-CoA synthetase 1 (ACSL1) is a significant member of the long-chain acyl-CoA synthetase gene family. It is widely found in various organisms and influences the lactation performance of cows, including fat percentage, milk protein percentage etc. Our study was aimed to investigate the genetic effects of single nucleotide polymorphisms (SNPs) in ACSL1 on milk production traits. Twenty Chinese Holstein cows were randomly selected to extract DNA from their blood samples for PCR amplification and sequencing to identify SNPs of the bovine ACSL1 gene, and six SNPs (5'UTR-g.20523C>G, g.35446C>T, g.35651G>A, g.35827C>T, g.35941G>A and g.51472C>T) were discovered. Then, Holstein cow genotyping (n = 992) was performed by Sequenom MassARRAY based on former SNP information. Associations between SNPs and milk production traits and somatic cell score (SCS) were analyzed by the least-squares method. The results showed that SNP g.35827C>T was in high linkage disequilibrium with g.35941G>A. Significant associations were found between SNPs and test-day milk yield (TDMY), fat content (FC), protein content (PC) and SCS (p < 0.05). Among these SNPs, SNP 5'UTR-g.20523C>G showed an extremely significant effect on PC and SCS (p < 0.01). The SNP g.35446C>T showed a statistically significant effect on FC, PC, and SCS (p < 0.01), and also TDMY (p < 0.05). The SNP g.35651G>A had a statistically significant effect on PC (p < 0.01). The SNP g.35827C>T showed a highly significant effect on TDMY, FC, and SCS (p < 0.01) and significantly influenced PC (p < 0.05). Lastly, SNP g.51472C>T was significantly associated with TDMY, FC, and SCS (p < 0.05). In summary, the pleiotropic effects of bovine ACSL1 for milk production traits were found in this paper, but further investigation will be required on the intrinsic correlation to provide a theoretical basis for the research on molecular genetics of milk quality traits of Holstein cows.

Genome-Wide DNA Methylation Analysis of Mammary Gland Tissues From Chinese Holstein Cows With Staphylococcus aureus Induced Mastitis.

Staphylococcus aureus intramammary infection is one of the most common causes of chronic mastitis in dairy cows, whose development may be associated with epigenetic changes in the expression of important host defense genes. This study aimed to construct a genome-wide DNA methylation profile of the mammary gland of Chinese Holstein cows (n = 3) following experimentally induced S. aureus mastitis, and to explore the potential gene regulatory mechanisms affected by DNA methylation during S. aureus mastitis. DNA was extracted from S. aureus-positive (n = 3) and S. aureus-negative (n = 3) mammary gland quarters and subjected to methylation-dependent restriction-site associated DNA sequencing (Methyl-RAD Seq). Results showed that CmCGG/CmCWGG DNA methylation sites were unevenly distributed and concentrated on chromosomes 5, 11, and 19, and within intergenic regions and intron regions of genes. Compared with healthy control quarters, 9,181 significantly differentially methylated (DM) CmCGG sites and 1,790 DM CmCWGG sites were found in the S. aureus-positive quarters (P < 0.05, |log2FC| > 1). Furthermore, 363 CmCGG differently methylated genes (DMGs) and 301 CmCWGG DMGs (adjusted P < 0.05, |log2FC| > 1) were identified. Gene ontology and KEGG enrichment analysis indicated that CmCGG DMGs are involved in immune response pathways, while the CmCWGG DMGs were mainly enriched in gene ontology terms related to metabolism. The mRNAs of 526 differentially methylated CmCGG genes and 124 differentially methylated CmCWGG genes were also significantly differentially expressed (RNA-Seq data) in the same samples, herein denoted differentially methylated and expressed genes (DMEGs) (P < 0.05). Functional enrichment analysis of DMEGs revealed roles related to biological processes, especially the regulation of immune response to diseases. CmCGG DMEGs like IL6R, TNF, BTK, IL1R2, and TNFSF8 enriched in several immune-related GO terms and pathways indicated their important roles in host immune response and their potential as candidate genes for S. aureus mastitis. These results suggest potential regulatory roles for DNA methylation in bovine mammary gland processes during S. aureus mastitis and serves as a reference for future epigenetic regulation and mechanistic studies.

Comparative analyses of copy number variations between Bos taurus and Bos indicus.

BACKGROUND: Bos taurus and Bos indicus are two main sub-species of cattle. However, the differential copy number variations (CNVs) between them are not yet well studied. RESULTS: Based on the new high-quality cattle reference genome ARS-UCD1.2, we identified 13,234 non-redundant CNV regions (CNVRs) from 73 animals of 10 cattle breeds (4 Bos taurus and 6 Bos indicus), by integrating three detection strategies. While 6990 CNVRs (52.82%) were shared by Bos taurus and Bos indicus, large CNV differences were discovered between them and these differences could be used to successfully separate animals into two subspecies. We found that 2212 and 538 genes uniquely overlapped with either indicine-specific CNVRs and or taurine-specific CNVRs, respectively. Based on FST, we detected 16 candidate lineage-differential CNV segments (top 0.1%) under selection, which overlapped with eight genes (CTNNA1, ENSBTAG00000004415, PKN2, BMPER, PDE1C, DNAJC18, MUSK, and PLCXD3). Moreover, we obtained 1.74 Mbp indicine-specific sequences, which could only be mapped on the Bos indicus reference genome UOA_Brahman_1. We found these sequences and their associated genes were related to heat resistance, lipid and ATP metabolic process, and muscle development under selection. We further analyzed and validated the top significant lineage-differential CNV. This CNV overlapped genes related to muscle cell differentiation, which might be generated from a retropseudogene of CTH but was deleted along Bos indicus lineage. CONCLUSIONS: This study presents a genome wide CNV comparison between Bos taurus and Bos indicus. It supplied essential genome diversity information for understanding of adaptation and phenotype differences between the Bos taurus and Bos indicus populations.

miR-497 regulates fatty acid synthesis via LATS2 in bovine mammary epithelial cells.

Both mRNA and miRNA play an important role in the regulation of mammary fatty acid metabolism and milk fat synthesis. Although studies have shown a strong transcriptional control of fatty acid metabolism, less is known about the regulatory mechanisms of milk fat synthesis as a function of miRNA-mRNA interactions. In this study, we carried out transcriptome sequencing using mammary tissues from the early lactation period, peak lactation, mid-lactation and late lactation in dairy cows and identified key genes regulating milk fatty acid metabolism. A total of 32 differentially co-expressed gene were screened out. Large tumor suppressor kinase 2 (LATS2) was chosen for further study using luciferase reporter assays, qRT-PCR and western blotting. The aim was to demonstrate that miR-497 is an upstream regulator of LATS2, i.e. miR-497 and LATS2 are a potential miRNA/mRNA regulatory pair. The results indicated that miR-497 could inhibit the production of triglycerides (TAG) and unsaturated fatty acids in bovine mammary epithelial cells (BMECs). In contrast, LATS2 can promote the production of TAG and unsaturated fatty acids. "Rescue" experiments further verified the miR-497/LATS2 regulatory network. Overall, data underscored that the miR-497/LATS2 pathway exerts control on milk fat metabolism and provides a theoretical approach for improving milk quality via genetic means.