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 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.

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.

Validation of an automatic method for reconstruction, delineation, and measurement of levator hiatus in clinical practice.

OBJECTIVES: To evaluate the concordance between an automatic software program and manual evaluation in reconstructing, delineating, and measuring the levator hiatus (LH) on maximal Valsalva maneuver. METHODS: This was a retrospective study analyzing archived raw ultrasound imaging data of 100 patients underwent transperineal ultrasound (TPUS) examination. Each data were assessed by the automatic Smart Pelvic System software program and manual evaluation. The Dice similarity index (DSI), mean absolute distance (MAD), and Hausdorff distance (HDD) were calculated to quantify delineation accuracy of LH. Agreement between automatic and manual measurement of levator hiatus area was assessed by intraclass correlation coefficient (ICC) and Bland-Altman method. RESULTS: The satisfaction rate of automatic reconstruction was 94%. Six images were recognized as unsatisfactory reconstructed images for some gas in the rectum and anal canal. Compared with satisfactory reconstructed images, DSI of unsatisfactory reconstructed images was lower, MAD and HDD were larger (p = 0.001, p = 0.001, p = 0.006, respectively). The ICC was up to 0.987 in 94 satisfactory reconstructed images. CONCLUSIONS: The Smart Pelvic System software program had good performance in reconstruction, delineation, and measurement of LH on maximal Valsalva maneuver in clinical practice, despite misidentification of the border of posterior aspect of LH due to the influence of gas in the rectum.

Identification of Candidate Genes and Functional Pathways Associated with Body Size Traits in Chinese Holstein Cattle Based on GWAS Analysis.

Body size is one of the most economically important traits of dairy cattle, as it is significantly associated with cow longevity, production, health, fertility, and environmental adaptation. The identification and application of genetic variants using a novel genetic approach, such as genome-wide association studies (GWASs), may give more insights into the genetic architecture of complex traits. The identification of genes, single nucleotide polymorphisms (SNPs), and pathways associated with the body size traits may offer a contribution to genomic selection and long-term planning for selection in dairy cows. In this study, we performed GWAS analysis to identify the genetic markers and genes associated with four body size traits (body height, body depth, chest width, and angularity) in 1000 Chinese Holstein cows. We performed SNPs genotyping in 1000 individuals, based on the GeneSeek Genomic Profiler Bovine 100 K. In total, we identified 11 significant SNPs in association with body size traits at the threshold of Bonferroni correction (5.90 × 10-7) using the fixed and random model circulating probability unification (FarmCPU) model. Several genes within 200 kb distances (upstream or downstream) of the significant SNPs were identified as candidate genes, including MYH15, KHDRBS3, AIP, DCC, SQOR, and UBAP1L. Moreover, genes within 200 kb of the identified SNPs were significantly enriched (p ≤ 0.05) in 25 Gene Ontology terms and five Kyoto Encyclopedia of Genes and Genomes pathways. We anticipate that these results provide a foundation for understanding the genetic architecture of body size traits. They will also contribute to breeding programs and genomic selection work on Chinese Holstein cattle.

CircRNA-1967 participates in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression.

Circular RNAs (circRNAs), a novel class of non-coding RNAs, can interact with miRNAs through a sequence-driven sponge mechanism, thereby regulating the expression of their downstream target genes. CircRNA-1967 was found in secondary hair follicles (SHFs) of cashmere goats, but its functions are not clear. Here, we showed that both circRNA-1967 and its host gene BNC2 had significantly higher expression in SHF bulge at anagen than those at telogen of cashmere goats. Also, circRNA-1967 participates in the differentiation of SHF stem cells (SHF-SCs) into hair follicle lineage in cashmere goats. RNA pull-down assay verified that circRNA-1967 interacts with miR-93-3p. We also indicated that circRNA-1967 promoted LEF1 expression in SHF-SCs of cashmere goats. By dual-luciferase reporter analysis, we found that circRNA-1967 up-regulated LEF1 expression through the miR-93-3p-mediated pathway. The results from this study demonstrated that circRNA-1967 participated in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression. Our founding might constitute a novel pathway for revealing the potential mechanism of the differentiation of SHF-SCs into hair follicle lineage in cashmere goats. Also, these results provided a valuable basis for further enhancing the intrinsic regeneration of cashmere goat SHFs with the formation and growth of cashmere fibers.

New Insight into Muscle-Type Cofilin (CFL2) as an Essential Mediator in Promoting Myogenic Differentiation in Cattle.

Meat quality and meat composition are not separated from the influences of animal genetic improvement systems; the growth and development of skeletal muscle are the primary factors in agricultural meat production and meat quality. Though the muscle-type cofilin (CFL2) gene has a crucial influence on skeletal muscle fibers and other related functions, the epigenetic modification mechanism of the CFL2 gene regulating meat quality remains elusive. After exploring the spatiotemporal expression data of CFL2 gene in a group of samples from fetal bovine, calf, and adult cattle, we found that the level of CFL2 gene in muscle tissues increased obviously with cattle age, whereas DNA methylation levels of CFL2 gene in muscle tissues decreased significantly along with cattle age by BSP and COBRA, although DNA methylation levels and mRNA expression levels basically showed an opposite trend. In cell experiments, we found that bta-miR-183 could suppress primary bovine myoblast differentiation by negatively regulated CFL2. In addition, we packaged recombinant adenovirus vectors for CFL2 gene knockout and overexpression and found that the CFL2 gene could promote the differentiation of primary bovine myoblasts by regulating marker genes MYOD, MYOG and MYH3. Therefore, CFL2 is an essential mediator for promoting myogenic differentiation by regulating myogenic marker genes in cattle myoblasts.

Genome-Wide Association Study for Udder Conformation Traits in Chinese Holstein Cattle.

Udder conformation traits are one of the most economic traits in dairy cows, greatly affecting animal health, milk production, and producer profitability in the dairy industry. Genetic analysis of udder structure and scores have been developed in Holstein cattle. In our research, we conducted a genome-wide association study for five udder traits, including anterior udder attachment (AUA), central suspensory ligament (CSL), posterior udder attachment height (PUAH), posterior udder attachment width (PUAW), and udder depth (UD), in which the fixed and random model circulating probability unification (FarmCPU) model was applied for the association analysis. The heritability and the standard errors of these five udder traits ranged from 0.04 ± 0.00 to 0.49 ± 0.03. Phenotype data were measured from 1000 Holstein cows, and the GeneSeek Genomic Profiler (GGP) Bovine 100 K SNP chip was used to analyze genotypic data in Holstein cattle. For GWAS analysis, 984 individual cows and 84,407 single-nucleotide polymorphisms (SNPs) remained after quality control; a total of 18 SNPs were found at the GW significant threshold (p < 5.90 × 10−7). Many candidate genes were identified within 200kb upstream or downstream of the significant SNPs, which include MGST1, MGST2, MTUS1, PRKN, STXBP6, GRID2, E2F8, CDH11, FOXP1, SLF1, TMEM117, SBF2, GC, ADGRB3, and GCLC. Pathway analysis revealed that 58 Gene Ontology (GO) terms and 18 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched with adjusted p values, and these GO terms and the KEGG pathway analysis were associated with biological information, metabolism, hormonal growth, and development processes. These results could give valuable biological information for the genetic architecture of udder conformation traits in dairy Holstein cattle.

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.

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.

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.

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.

Menin regulates lipid deposition in mouse hepatocytes via interacting with transcription factor FoxO1.

Non-alcoholic fatty liver disease (NAFLD) is rapidly being recognized as the leading cause of chronic liver disease worldwide. Men1, encoding protein of menin, is a key causative gene of multiple endocrine neoplasia type 1 syndrome including pancreatic tumor. It is known that insulin that secretes by endocrine tissue pancreatic islets plays a critical role in hepatic metabolism. Mouse model of hemizygous deletion of Men1 was shown to have severe hepatic metabolism disorders. However, the molecular function of menin on lipid deposition in hepatocytes needs to be further studied. Transcriptome sequencing does show that expression suppression of Men1 in mouse hepatocytes widely affect signaling pathways involved in hepatic metabolism, such as fatty acid metabolism, insulin response, glucose metabolism and inflammation. Further molecular studies indicates that menin overexpression inhibits expressions of the fat synthesis genes Srebp-1c, Fas, and Acc1, the fat differentiation genes Pparγ1 and Pparγ2, and the fat transport gene Cd36, thereby inhibiting the fat accumulation in hepatocytes. The biological process of menin regulating hepatic lipid metabolism was accomplished by interacting with the transcription factor FoxO1, which is also found to be critical for lipid metabolism. Moreover, menin responds to insulin in hepatocytes and mediates its regulatory effect on hepatic metabolism. Our findings suggest that menin is a crucial mediation factor in regulating the hepatic fat deposition, suggesting it could be a potential important therapeutic target for NAFLD.

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.

Lycium barbarum polysaccharides alleviate LPS-induced inflammatory responses through PPARγ/MAPK/NF-κB pathway in bovine mammary epithelial cells.

As the main component of the Gram-negative bacterial cell wall, lipopolysaccharide (LPS) is well documented as an inducer of inflammation in bovine mammary cells. Lycium barbarum (goji) polysaccharides (LBP) have been used in nonruminants as prebiotics to improve growth performance, immune ability, and antioxidant capacity. We aimed to investigate the underlying effects of LBPs on proinflammatory responses in LPS-stimulated primary bovine mammary epithelial cells (bMECs). Cells were isolated from mammary tissue of three lactating Holstein cows without clinical disease (30.26 ± 3.1 kg/d of milk yield; 175 ± 6 DIM). For the pre-experimental treatment, bMECs were precultured with serum-free medium for 12 h. Treatments were as follows: pretreatment with culture medium devoid of LPS or LBP for 30 h (CON); CON for 24 h followed by challenge with 2 µg/mL LPS for 6 h (LPS); pretreatment with 100 or 300 µg/mL LBP for 24 h followed by LPS challenge (2 µg/mL) for 6 h (LBP(100)+LPS; LBP(300)+LPS). To further determine if the effect of LBP on immuneregulation is peroxisome proliferator-activated receptor-γ (PPARγ) activation dependent, an inhibitor of PPARγ, GW9662, at a concentration of 1 µM was used. Cells treated with LBP at 100, 300, and 500 µg/mL had upregulated protein abundance of PPARγ, while PGC1α had a higher expression only at 300 µg/mL of LBP treatment. Compared with CON, cells pretreated with LBP at 100 and 300 µg/mL had greater protein abundance of SCD1 and SREBP1. 5-Ethynyl-2'-deoxyuridine (EdU) staining and cell wound healing assays showed that the negative effect of LPS alone on cell proliferation was reversed by pretreatment with LBP at both 100 and 300 µg/mL. Upregulation of gene and protein abundance of proinflammatory factors and cytokines (COX-2, NLRP3, TNF-α, IL-1ß, and IL-6) induced by LPS stimulation were alleviated by LBP pretreatment at 300 µg/mL (more than 2-fold decrease). Compared with LPS challenge alone, phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38, JNK, and ERK) pathways was downregulated following LBP treatment. Additionally, inhibition of PPARγ by GW9662 weakened the protective effect of LBP on LPS-induced protein abundance of phosphorylated p65, COX-2, IL-1ß, and TNF-α. These results indicated that the protective effect of LBP on LPS-induced bMECs inflammatory responses is PPARγ activation-dependent. As such, this knowledge might help design strategies for intervening against the detrimental effects of bovine mastitis. INTERPRETIVE SUMMARY: Current research examined Lycium barbarum polysaccharides (LBP) for combating LPS-induced inflammatory responses in primary bovine mammary epithelial cells. We uncovered a preventive role of LBP in reducing detrimental effects induced by LPS including inhibition of NF-κB and MAPK along with peroxisome proliferator-activated receptor-γ (PPARγ) activation. The decrease in cell proliferation due to LPS was curtailed by pretreatment with LBP. Moreover, the effect of LBP on regulation of inflammatory responses in bovine mammary epithelial cell was PPARγ dependent. Collectively, data suggest that LBP reverses LPS-induced inflammatory response via MAPK/NF-κB signaling in a PPARγ-activation-dependent manner. Thus, the study provides new insights into therapeutic strategies for combating mastitis using LBP and highlighted the link between PPARγ and regulation of mammary cell inflammation.

Genome-Wide Association Study Candidate Genes on Mammary System-Related Teat-Shape Conformation Traits in Chinese Holstein Cattle.

In the dairy industry, mammary system traits are economically important for dairy animals, and it is important to explain their fundamental genetic architecture in Holstein cattle. Good and stable mammary system-related teat traits are essential for producer profitability in animal fitness and in the safety of dairy production. In this study, we conducted a genome-wide association study on three traits-anterior teat position (ATP), posterior teat position (PTP), and front teat length (FTL)-in which the FarmCPU method was used for association analyses. Phenotypic data were collected from 1000 Chinese Holstein cattle, and the GeneSeek Genomic Profiler Bovine 100K single-nucleotide polymorphisms (SNP) chip was used for cattle genotyping data. After the quality control process, 984 individual cattle and 84,406 SNPs remained for GWAS work analysis. Nine SNPs were detected significantly associated with mammary-system-related teat traits after a Bonferroni correction (p < 5.92 × 10-7), and genes within a region of 200 kb upstream or downstream of these SNPs were performed bioinformatics analysis. A total of 36 gene ontology (GO) terms and 3 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched (p < 0.05), and these terms and pathways are mainly related to metabolic processes, immune response, and cellular and amino acid catabolic processes. Eleven genes including MMS22L, E2F8, CSRP3, CDH11, PEX26, HAL, TAMM41, HIVEP3, SBF2, MYO16 and STXBP6 were selected as candidate genes that might play roles in the teat traits of cows. These results identify SNPs and candidate genes that give helpful biological information for the genetic architecture of these teat traits, thus contributing to the dairy production, health, and genetic selection of Chinese Holstein cattle.

circ01592 regulates unsaturated fatty acid metabolism through adsorbing miR-218 in bovine mammary epithelial cells.

The composition of fatty acids plays a key role in regulating milk flavor and quality. Therefore, to improve the quality of milk, it is particularly important to study the regulatory mechanism of fatty acid metabolism in dairy cows. In this study, the expression profiles at non-lactation, peak-lactation, mid-lactation and late-lactation were constructed by high-throughput sequencing. Considering non-lactation as the control group and the other points as the experimental groups, the differentially expressed genes were screened. ELOVL5 was significantly upregulated and was selected for subsequent analyses. Bioinformatics prediction, a dual-luciferase assay, qPCR analysis and western blot analysis were used for verification. The results showed that ELOVL5 was a downstream target gene of miR-218 that regulated milk fat metabolism. A dual-luciferase assay and expression level analysis showed that circ01592 can directly bind to miR-218 and that overexpression of circ01592 (pcDNA-circ01592) significantly reduced the expression of miR-218 and enhanced the expression of ELOVL5, the target gene of miR-218 in BMECs. A functional study of BMECs showed that circ01592 promoted the synthesis of TAG and increased the content of UFA. The function of miR-218 was opposite to that of circ01592. Overall, the data showed that circ01592 promoted TAG synthesis and fatty acid composition by binding miR-218, alleviating the inhibitory effect of miR-218 on ELOVL5 expression. These mechanisms provide a new research approach and theoretical basis for improving milk quality.

Progress on the Regulation of Ruminant Milk Fat by Noncoding RNAs and ceRNAs.

Milk fat is not only a key factor affecting the quality of fresh milk but also a major target trait forbreeding. The regulation of milk fat involves multiple genes, network regulation and signal transduction. To explore recent discoveries of pathway regulation, we reviewed the published literature with a focus on functional noncoding RNAs and epigenetic regulation in ruminants. Results indicate that miRNAs play key roles in the regulation of milk fat synthesis and catabolism in ruminants. Although few data are available, merging evidence indicates that lncRNAs and circRNAs act on milk fat related genes through indirect action with microRNAs or RNAs in the ceRNA network to elicit positive effects on transcription. Although precise regulatory mechanisms remain unclear, most studies have focused on the regulation of the function of target genes through functional noncoding RNAs. Data to help identify factors that can regulate their own expression and function or to determine whether self-regulation involves positive and/or negative feedback are needed. Despite the growing body of research on the role of functional noncoding RNA in the control of ruminant milk fat, most data are still not translatable for field applications. Overall, the understanding of mechanisms whereby miRNA, lncRNA, circRNA, and ceRNA regulate ruminant milk fat remains an exciting area of research.