Sperm motility is the main index used to assess the quality of bull semen. It may also be used to evaluate the fertility potential of bulls. Protein-coding mRNA and long noncoding RNA (lncRNA) ...participate in the regulation of spermatogenesis. Here, we employed strand-specific RNA sequencing to profile the semen transcriptome (mRNA and lncRNA) of six paired full-sibling Holstein bulls with divergent sperm motility and to determine the functions of mRNA and lncRNA in sperm motility. Among 20,875 protein-encoding genes detected in semen, 19 were differentially expressed between the high sperm motility group (H: H1, H2, and H3) and low sperm motility group (L: L1, L2, and L3). Of the 11,561 lncRNAs identified in sperm, 2,517 were differentially expressed between the H and L groups. We found that TCONS_00041733 lncRNA targets the node gene EFNA1 (ephrin A1), involved in male reproductive physiology. Our study provides a global mRNA and lncRNA transcriptome of bull semen, as well as novel insights into the regulation of neighboring protein coding by lncRNAs and the influence of mRNAs on sperm motility.
Current knowledge about the evolutionary history of donkeys is still incomplete due to the lack of archeological and whole-genome diversity data. To fill this gap, we have de novo assembled a ...chromosome-level reference genome of one male Dezhou donkey and analyzed the genomes of 126 domestic donkeys and seven wild asses. Population genomics analyses indicate that donkeys were domesticated in Africa and conclusively show reduced levels of Y chromosome variability and discordant paternal and maternal histories, possibly reflecting the consequences of reproductive management. We also investigate the genetic basis of coat color. While wild asses show diluted gray pigmentation (Dun phenotype), domestic donkeys display non-diluted black or chestnut coat colors (non-Dun) that were probably established during domestication. Here, we show that the non-Dun phenotype is caused by a 1 bp deletion downstream of the TBX3 gene, which decreases the expression of this gene and its inhibitory effect on pigment deposition.
Neutrophils are the first effectors of inflammatory response triggered by mastitis infection, and are important defense cells against pathogenic Escherichia coli (E. coli). DNA methylation, as a ...critical epigenetic mechanism for regulating gene function, is involved in bovine mastitis.
In this study, we sequenced the blood neutrophils of healthy and E. coli-infected mastitic half-sib cows for the overall DNA methylation levels using transcriptome sequencing and reduced representation bisulfite sequencing. The methylation levels in the mastitis cows (MCs) were decreased compared with healthy cows (HCs). A total of 494 differentially methylated regions were identified, among which 61 were up-methylated and 433 were down-methylated (MCs vs. HCs). The expression levels of 1094 differentially expressed genes were up-regulated, and 245 genes were down-regulated. Twenty-nine genes were found in methylation and transcription data, among which seven genes' promoter methylation levels were negatively correlated with expression levels, and 11 genes were differentially methylated in the exon regions. The bisulfite sequencing PCR and quantitative real-time PCR validation results demonstrated that the promoter methylation of CITED2 and SLC40A1 genes affected differential expression. The methylation of LGR4 exon 5 regulated its own alternative splicing. The promoter methylation of bta-miR-15a has an indirect effect on the expression of its target gene CD163. The CITED2, SLC40A1, and LGR4 genes can be used as candidates for E. coli-induced mastitis resistance.
This study explored the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-induced mastitis, thereby helping explain the function of DNA methylation in the pathogenesis of mastitis and provided new target genes and epigenetic markers for mastitis resistance breeding in dairy cattle.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Complement component 3 (C3) is the key molecule of the three pathways of complement activation (alternative, classical, and lectin pathways), which are involved in phagocytosis, inflammation, and ...immunoregulation processes to destroy infectious microorganisms. In this study, three novel single-nucleotide polymorphisms (SNPs) (g.-1293C>G located in the 5'-flanking region, g.56T>C in exon I, and g.7017C>T in exon XII) of the C3 gene were detected using created restriction site polymerase chain reaction, restriction fragment length polymorphism, and DNA sequencing in 952 cattle from three Chinese breeds. The genotypes and haplotypes were analyzed to investigate the polymorphisms and their possible implications, with particular investigative focus on their associations with serum C3 level, complement hemolytic activity (CH50 and ACH50), and milk production traits. The g.56T>C SNP in exon I affected the serum ACH50 (P<0.01) and the milk somatic cell score (SCS) (PT SNP in exon XII significantly affected the serum ACH50 values (P0.05). The C3 expression levels were significantly increased in lung and mammary tissues (P<0.05), while significantly decreased in heart, spleen, liver, and kidney tissues in mastitis cows compared with those in healthy animals (P<0.01), respectively. Bacterial counts of serum antibacterial activities were also completed to verify the effect of SNPs on resistance to mastitis pathogens. Genetically resistant cows (CCC/GCC) had serum with noticeably higher antibacterial activity against S. aureus and E. coli in vitro than the genetically susceptible CCC/GTT cows (P<0.05). Results from this study imply that the C3 gene plays a role in resistance to bacterial infection and that it can be used as a molecular marker for complement activity and traits related to milk production.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation and gene alternative splicing drive spermatogenesis. In screening DNA methylation markers and transcripts related to sperm motility, semen from three pairs of full-sibling Holstein ...bulls with high and low motility was subjected to reduced representation bisulphite sequencing. A total of 948 DMRs were found in 874 genes (gDMRs). Approximately 89% of gDMR-related genes harboured alternative splicing events, including SMAD2, KIF17, and PBRM1. One DMR in exon 29 of PBRM1 with the highest 5mC ratio was found, and hypermethylation in this region was related to bull sperm motility. Furthermore, alternative splicing events at exon 29 of PBRM1 were found in bull testis, including PBRM1-complete, PBRM1-SV1 (exon 28 deletion), and PBRM1-SV2 (exons 28-29 deletion). PBRM1-SV2 exhibited significantly higher expression in adult bull testes than in newborn bull testes. In addition, PBRM1 was localized to the redundant nuclear membrane of bull sperm, which might be related to sperm motility caused by sperm tail breakage. Therefore, the hypermethylation of exon 29 may be associated with the production of PBRM1-SV2 in spermatogenesis. These findings indicated that DNA methylation alteration at specific loci could regulate gene splicing and expression and synergistically alter sperm structure and motility.
Copy number variations (CNVs) have been demonstrated as crucial substrates for evolution, adaptation and breed formation. Chinese indigenous cattle breeds exhibit a broad geographical distribution ...and diverse environmental adaptability. Here, we analyzed the population structure and adaptation to high altitude of Chinese indigenous cattle based on genome-wide CNVs derived from the high-density BovineHD SNP array. We successfully detected the genome-wide CNVs of 318 individuals from 24 Chinese indigenous cattle breeds and 37 yaks as outgroups. A total of 5,818 autosomal CNV regions (683 bp-4,477,860 bp in size), covering ~14.34% of the bovine genome (UMD3.1), were identified, showing abundant CNV resources. Neighbor-joining clustering, principal component analysis (PCA), and population admixture analysis based on these CNVs support that most Chinese cattle breeds are hybrids of
(hereinafter to be referred as
) and
(
). The distribution patterns of the CNVs could to some extent be related to the geographical backgrounds of the habitat of the breeds, and admixture among cattle breeds from different districts. We analyzed the selective signatures of CNVs positively involved in high-altitude adaptation using pairwise Fst analysis within breeds with a strong
background (taurine-type breeds) and within
×
hybrids, respectively. CNV-overlapping genes with strong selection signatures (at top 0.5% of Fst value), including
(Fst = 0.490),
(Fst = 0.440), and
(Fst = 0.420) within taurine-type breeds, and
(Fst = 0.233),
(Fst = 0.222), and
(Fst=0.154) within hybrids, were potentially involved in the adaptation to hypoxia. Thus, we provide a new profile of population structure from the CNV aspects of Chinese indigenous cattle and new insights into high-altitude adaptation in cattle.
Neutrophil cytosolic factor 4 (NCF4) is component of the nicotinamide dinucleotide phosphate oxidase complex, a key factor in biochemical pathways and innate immune responses. In this study, splice ...variants and functional single-nucleotide polymorphism (SNP) of NCF4 were identified to determine the variability and association of the gene with susceptibility to bovine mastitis characterized by inflammation. A novel splice variant, designated as NCF4-TV and characterized by the retention of a 48 bp sequence in intron 9, was detected in the mammary gland tissues of infected cows. The expression of the NCF4-reference main transcript in the mastitic mammary tissues was higher than that in normal tissues. A novel SNP, g.18174 A>G, was also found in the retained 48 bp region of intron 9. To determine whether NCF4-TV could be due to the g.18174 A>G mutation, we constructed two mini-gene expression vectors with the wild-type or mutant NCF4 g.18174 A>G fragment. The vectors were then transiently transfected into 293T cells, and alternative splicing of NCF4 was analyzed by reverse transcription-PCR and sequencing. Mini-gene splicing assay demonstrated that the aberrantly spliced NCF4-TV with 48 bp retained fragment in intron 9 could be due to g.18174 A>G, which was associated with milk somatic count score and increased risk of mastitis infection in cows. NCF4 expression was also regulated by alternative splicing. This study proposes that NCF4 splice variants generated by functional SNP are important risk factors for mastitis susceptibility in dairy cows.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The posttranscriptional gene regulation mediated by microRNA plays an important role in the development and function of male and female reproductive organs and germ cells in mammals, including ...cattle. In the present study, we identified novel and differentially expressed miRNAs in the testis and ovary in Holstein cattle by combining the Solexa sequencing with bioinformatics. In total 100 and 104 novel pre-miRNAs were identified in testicular and ovarian tissues, encoding 122 and 136 mature miRNAs, respectively. Of these, 6 miRNAs appear to be bovine-specific. A total of 246 known miRNAs were co-expressed in the testicular and ovarian tissues. Of the known miRNAs, twenty-one testis-specific and nine ovary-specific (1-23 reads) were found. Approximately 30.5% of the known bovine miRNAs in this study were found to have >2-fold differential expression within the two respective reproductive organ systems. The putative miRNA target genes of miRNAs were involved in pathways associated with reproductive physiology. Both known and novel tissue-specific miRNAs are expressed by Real-time quantitative PCR analysis in dairy cattle. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the bovine testicular and ovarian tissues, which provide important information on sex differences in miRNA expression. Diverse miRNAs may play an important regulatory role in the development of the reproductive organs in Holstein cattle.
Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP ...gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nd2Fe14B-type permanent magnets have been widely applied in various fields such as wind power, voice coil motors, and medical instruments. The large temperature dependence of coercivity, however, ...limits their further applications. We have systematically investigated the magnetic properties, thermal stabilities and coercivity mechanisms of the (Pr0.2Nd0.8)13Fe81-xB6Hfx (x=0, 0.5) nanocrystalline magnets fabricated by a spark plasma sintering (SPS) technique. The results indicate that the influence of Hf addition is significant on magnetic properties and thermal stabilities of the (PrNd)2Fe14B-type sintered magnets. It is shown that the sample with x = 0.5 at 300 K has much higher coercivity and remanent magnetization than those counterparts without Hf. The temperature coefficients of remanence (α) and coercivity (β) of the (Pr0.2Nd0.8)13Fe81-xB6Hfx magnets are improved significantly from -0.23 %/K, -0.57 %/K for the sample at x = 0 to -0.17 %/K, -0.49 %/K for the sample at x = 0.5 in the temperature range of 300-400 K. Furthermore, it is found out that the domain wall pinning mechanism is more likely responsible for enhancing the coercivity of the (Pr0.2Nd0.8)13Fe81-xB6Hfx magnets.
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