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The current study reports the identification of previously undiscovered single-nucleotide polymorphisms (SNPs) in the bovine AGPAT3 gene and further investigates their associations with milk production traits. Our results demonstrate that the major allele C of the SNP g.12264 C > T is positively correlated with test-day milk yield, protein percentage and 305-day milk yield. Importantly, in silico analysis showed that the C/T transition at this locus gives rise to two new transcription factor binding sites (TFBS), E2F1 and Nkx3-2. Polymorphism g.18658 G > A was the only SNP associated with milk urea nitrogen (MUN) with the G allele related to an increase in milk urea nitrogen as well as fat percentage. The GG genotype of SNP g.28731 A > G was associated with the highest fat and protein percentage and lowest 305-day milk yield and somatic cell score (SCS). The association between AGPAT3 locus and milk production traits could be utilized in marker-assisted selection for the genetic improvement of milk production traits and, probably in conjunction with other traits, for selection to improve fitness of dairy cattle.

Milk contains a number of beneficial fatty acids including short and medium chain and unsaturated conjugated and nonconjugated fatty acids. In this study, microRNA sequencing of mammary tissue collected in early-, peak-, mid-, and late-lactation periods was performed to determine the miRNA expression profiles. miR-16a was one of the differentially expressed miRNA and was selected for in-depth functional studies pertaining to fatty acid metabolism. The mimic of miR-16a impaired fat metabolism [triacylglycerol (TAG) and cholesterol] while knock-down of miR-16a promoted fat metabolism in vitro in bovine mammary epithelial cells (BMECs). In addition, the in vitro work with BMECs also revealed that miR-16a had a negative effect on the cellular concentration of cis 9-C18:1, total C18:1, C20:1, and C22:1 and long-chain polyunsaturated fatty acids. Therefore, these data suggesting a negative effect on fatty acid metabolism extend the discovery of the key role of miR-16a in mediating adipocyte differentiation. Through a combination of bioinformatics analysis, target gene 3' UTR luciferase reporter assays, and western blotting, we identified large tumor suppressor kinase 1 ( LATS1 ) as a target of miR-16a. Transfection of siRNA- LATS1 into BMECs led to increases in TAG, cholesterol, and cellular fatty acid concentrations, suggesting a positive role of LATS1 in mammary cell fatty acid metabolism. In summary, data suggest that miR-16a regulates biological processes associated with intracellular TAG, cholesterol, and unsaturated fatty acid synthesis through LATS1 . These data provide a theoretical and experimental framework for further clarifying the regulation of lipid metabolism in mammary cells of dairy cows.

Background: As cattle represent one of the most important livestock species for meat production, control of muscle development in regards to quality is an important research focus., Objectives: In this study, the phenotypic quality traits and its associations with DNA methylation levels of the longissimus muscle in two cattle breeds were studied., Methods: The pH value, water loss rate, fat and protein and fatty acid content were measured in three beef cattle breeds of longissimus mucle; The longissimus mucle was analyzed by MethylRAD-seq and RNA-seq. The differentially methylated and differentially expressed related genes were subjected to BSP., Results: Methylation status of longissimus mucle was analyzed by MethylRAD-seq. Compared with Simmental, there were 39 differentially methylated and expressed genes in muscle of Yunling cattle, and 123 differentially methylated and expressed genes in Wenshan muscle. A combined analysis of MethylRAD-seq and RNA-seq results revealed differential methylation and expression level of 18 genes between Simmental and Wenshan cattle, and 14 genes between Simmental and Yunling cattle. In addition, 28 genes were differentially methylated between Wenshan and Yunling cattle. Results of promoter methylation analysis of ACAD11, FADS6 and FASN showed that the overall degree of DNA methylation of FADS6 and FASN was negatively correlated with their expression levels. Methylation level of FASN in Simmental was greater than Yunling and Wenshan. The degree of methylation at the FADS6 CpG4 site was significantly higher in Simmental than that in Yunling. The levels of methylation at the CpG7 locus of the Simmental and Yunling breeds were greater than Wenshan cattle. A negative correlation was detected between the methylation levels and the expression of FASN CpG1, CpG2, CpG3, CpG5, CpG7, and CpG10., Conclusion: The functional and molecular regulatory mechanism of the genes related to meat quality can be revealed systematically from aspects of the genetic and epigenetic regulation. These studies will help to further explore the molecular mechanisms and phenotypic differences that regulate growth and quality of different breeds of cattle.

The pituitary gland is a key endocrine organ responsible for growth and development. To get closer to understanding important molecular mechanisms at work in the bovine pituitary and identifying the core genes controlling growth, in the present study we have analyzed the transcriptome profiling of the pituitary glands of two cattle breeds (Wenshan and Simmental cattle) with extreme growth differences using high-throughput RNA sequencing. Our study revealed that the overall gene expression levels are quite similar between the two breeds. A total of 18,065 annotated genes were identified, which accounted for 85% of the annotated genes in cattle. The five most abundant hormone genes (GH, PRL, POMC, CGA, and LHB) were relatively stable in both breeds, indicating their pivotal roles in maintaining the basic functions of the pituitary. In addition, 105 genes were identified as differentially expressed between Wenshan and Simmental pituitary glands, including 83 known and 22 novel genes. Functional association analyses of the differentially expressed genes between the breeds revealed 60 enriched gene ontology terms and 3 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Interestingly, the most enriched KEGG pathway, cell adhesion molecules, can modulate pituitary hormone secretion by cell-cell contact. Our findings demonstrated that SYTL2, SLC38A4, and NCAM2 are new candidates for crucial functions in the secretory pathways of the pituitary gland. These results will further understanding of the important molecular mechanisms at work in the bovine pituitary., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The research reported in this Research Communication aimed to describe the influence of Toll-like receptor 4 gene polymorphisms on milk production traits in Chinese Holstein cows. Toll-like receptor 4 (TLR4) is an important member of the toll-like receptor gene family that is widely found in various organisms. Since TLR4 can identify molecular patterns from various pathogenic microorganisms and induce natural and acquired immunity, it plays an important role in disease resistance in dairy cows. Two single nucleotide polymorphisms (SNPs) of TLR4 (c.-226 G > C and c.2021 C > T) that were previously found to be associated with health traits were genotyped using Sequenom MassARRAY (Sequenom Inc., San Diego, CA) for Chinese Holstein cows (n = 866). The associations between SNPs or their haplotypes and milk production traits and somatic cell count were analyzed by the generalized linear model procedure of Statistics Analysis System software (SAS). The c.-226 G > C and c.2021 C > T showed low linkage disequilibrium (r2 = 0·192). There was no association between these two SNPs and SCC, but significant effects were found for SNP c.-226 G > C on test-day milk yield, fat content, protein content, and total solid and milk urea nitrogen (P T and the SNP haplotypes on test-day milk yield, fat content, protein content, lactose content and total solids (P C was located within several potential transcription factor binding sites, including transcription factor AP-2. The polymorphisms c.-226 G > C and c.2021 C > T had significant effects on the milk production for Chinese Holstein, and these SNP could be used for molecular marker-assisted selection of milk production.

Lactoferrin is an iron-binding protein found in cow's milk that plays an important role in preventing mastitis caused by intramammary infection. In this study, 20 Chinese Holstein cows were selected randomly for PCR amplification and sequencing of the bovine lactoferrin gene promoter region and used for SNP discovery in the region between nucleotide positions -461 to -132. Three SNPs (-270T>C, -190G>A and -156A>G) were identified in bovine lactoferrin, then Chinese Holstein cows (n=866) were genotyped using Sequenom MassARRAY (Sequenom Inc., San Diego, CA) based on the previous SNP information in this study, and the associations between SNPs or haplotype and milk somatic cell score (SCS) and production traits were analyzed by the least squares method in the GLM procedure of SAS. SNPs -270T>C and -156A>G showed close linkage disequilibrium (r(2)=0.76). The SNP -190G>A showed a significant association with SCS, and individuals with genotype GG had higher SCS than genotypes AG and AA. Associations were found between the SNPs -270T>C and -190G>A with SCS and the milk composition. The software MatInspector revealed that these SNPs were located within several potential transcription factor binding sites, including NF-κB p50, KLF7 and SP1, and may alter gene expression, but further investigation will be required to elucidate the biological and practical relevance of these SNPs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The single nucleotide polymorphisms (SNPs) in the 5' upstream of bovine IL8 gene were investigated in 810 Chinese Holstein cows from 35 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The Real-time PCR and Western blot were used to detect the mRNA and protein levels of genotype Chinese Holstein dairy cows. The results showed that one SNP -105G>A was detected, designating three genotypes (GG, GA and AA) with respective frequencies of 0.38, 0.46, and 0.16. The significant association of the SNP -105G>A with somatic cell score (SCS) was identified. Genotype GG had a significantly lower SCS than genotype GA or AA (P < 0.01), and the relative mRNA expression and protein level of GG was found to be the highest. These results suggest that the genotype GG may be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein dairy cows.

To explore the relation of Interleukin-8 (IL8) gene polymorphism with immunity against mastitis in dairy cow, the polymorphism of IL8 gene was investigated in 610 Chinese Holstein cow from 30 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, and milk yield, milk fat percentage, milk protein percentage, 305 day corrected milk yield, 305 day milk fat yield, 305 day milk protein yield and somatic cell score (SCS) were measured and analyzed, and the mRNA levels of IL8 genotypes in blood were detected by real-time PCR. The results showed that three genotypes, KK, KA and AA were detected with frequencies of 0.187, 0.451, and 0.362, respectively. The gene frequencies of K and A were 0.412 and 0.588, respectively. The significant association of the IL8 mutations with milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield, SCS, and significant association with milk protein percentage were identified, while their association with milk fat percentage were not significant. KK had higher milk yield, 305 day milk protein yield, 305 day corrected milk yield and 305 day milk fat yield than AA or KA, and the least square mean of SCS of KK was significantly lower than that of AA or KA. AA had significant lower milk protein yield than KK or KA. The relative IL8 mRNA level of KK in blood was the highest. These findings demonstrated that IL8 genotype significantly correlated with mastitis resistance and the locus could be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein.

Mastitis is one of the most prevalent diseases in dairy cattle; CXCR1 plays a key role in mastitis resistance via IL8 signaling pathway, with the CXCR1 SNPs showing a different degree of mastitis resistance. To investigate the situation of CXCR1 polymorphisms in Chinese Holstein cattle and determine the relationship between the CXCR1 SNPs and mastitis resistance, the CXCR1 SNPs in 610 Chinese Holstein cattle of 30 families were investigated using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The results showed that four SNPs, -1830A > G, -1768T > A, -344T > C, and 783C > A were detected at 5' upstream and coding region. The correlation analysis demonstrated that -1830AA, -1768TT, and -344TT correlated significantly with the lowest SCS for each site, respectively. Haplotype analysis revealed Haplo2 (ATTA) correlated significantly with the lowest SCS. These findings indicated a prospect genetic marker of mastitis resistance in dairy cattle., (Copyright © Taylor & Francis Group, LLC)

A total of 321 individuals from six cattle populations of four species in a bovine subfamily in China were studied using 12 pairs of microsatellite markers. The genetic diversities within and between populations were calculated. The phylogenetic trees were constructed by (delta mu)(2) and DA distances, and the divergence times between populations were estimated by (delta mu)(2). Altogether, 144 microsatellite alleles were detected including 24 private alleles and nine shared alleles. Chinese Holstein had the largest number of private alleles (10), whereas, Bohai black and Buffalo had the smallest number of private alleles (2). Chinese Holstein showed the highest genetic variability. Its observed number of alleles (Na), mean effective number of alleles (MNA), and mean heterozygosity (He) were 7.7500, 4.9722, and 0.7719, respectively, whereas, the Buffalo and Yak showed low genetic variability. In the phylogenetic trees, Luxi and Holstein grouped first, followed by Bohai and Minnan. Yak branched next and buffalo emerged as the most divergent population from other cattle populations. Luxi and Bohai were estimated to have diverged 0.039-0.105 million years ago (MYA), however, buffalo and Holstein diverged 0.501-1.337 MYA. The divergence time of Yak versus Minnan, Holstein and buffalo was 0.136-0.363, 0.273-0.729, and 0.326-0.600 MYA, respectively.

Levels of genetic differentiation, gene flow, and genetic structure of three indigenous cattle populations (Luxi, Bohai, and Minnan) and two reference cattle populations (Chinese Holstein and Qinhai yak) in China were estimated using the information from 12 microsatellites, and 141 microsatellite alleles were identified. The mean number of alleles per locus ranged from 2.9005 in yak to 4.9722 in Holstein. The observed heterozygosity ranged from 0.5325 (yak) to 0.7719 (Holstein); 29 private alleles were detected. The global heterozygote deficit across all populations amounted to 58.5% (p < 0.001). The overall significant (p < 0.001) deficit of heterozygotes because of inbreeding within breeds amounted to 43.2%. The five cattle populations were highly differentiated (F (st) = 26.9%, p < 0.001) at all loci. The heterozygote deficit within the population was highest in Luxi cattle and lowest in yak. The average number of effective migrants exchanged per generation was highest (1.149) between Luxi and Holstein, and lowest (0.509) between Luxi and yak. With the application of prior population information, cluster analysis achieved posterior probabilities from 91% to 98% of correctly assigning individuals to populations. Combining the information of cluster analysis, gene flow, and Structure analysis, the five cattle populations belong to three genetic clusters, a taurine (Luxi and Chinese Holstein), a zebu (Bohai and Minnan), and a yak cluster. This indicates that Bohai black is closer to Bos indicus than Luxi cattle. The evolution and development of three indigenous cattle populations are discussed.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Considerable evidence has demonstrated that the μ -calpain (CAPN1) gene and its inhibitor calpastatin (CAST) gene are major factors affecting meat quality. Marker-assisted selection (MAS) has been widely used to improve beef quality traits. Therefore, the objective of the present study was to investigate the single nucleotide polymorphisms (SNPs) of bovine CAPN1 and CAST genes using 367 animals representing the four main Chinese cattle breeds and to explore the effects of these SNPs on meat quality traits. Two SNPs within CAPN1 and one SNP in CAST were successfully identified in cattle. Genetic diversity analyses suggested that most SNPs in the four breeds exhibited a moderate genetic diversity. Moreover, associations between individual markers and meat quality traits were analyzed in Chinese Simmental cattle. The CAPN1 4558 A > G locus was found to be significantly associated with shear force value (SFV) and marbling score (BMS), and CAPN1 4684 C > T exerted a significant effect on SFV, while the CAST genotype was not significantly associated with any of the measured traits. SFV, commonly used to measure meat tenderness, represents an important quality trait as it contributes to the flavor of cooked meat. This work confirms the effect of CAPN1 on beef tenderness and lays an important foundation for future cattle breeding. [ABSTRACT FROM AUTHOR]