Analysis of Rumen Microbial Protein Abundance of Gayals based on Metaproteomics

Background: Presently, our understanding of the rumen of Gayals is still very shallow, which is recognized as the most effective and developed fiber degradation system in nature, with abundant microorganisms. Molecular biology technology is an effective means to study the microbial resources in the rumen. Methods: Rumen contents of 3 Gayals (Gayals, Bos frontalis; G) and 3 Yellow Cattle (Yunnan Yellow Cattle, Bos taurus; Y) were collected in this study. Rumen microbial proteins were extracted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), then to analyze the bioinformatics of protein abundance was performed through the bovine rumen transcriptome database (gene.uniGeneset.fasta). Result: The results were as follows: the differences in protein abundance of Gayals rumen bacteria in Firmicutes, Actinobacteria, Ruminococcus and Olsenella were significantly higher than Yellow cattle (P less than 0.05) and the difference in protein abundance of Chytridiomycota and Batrachochytrium in rumen fungus of Gayals was significantly less than that of Yellow cattle. Enrichment analysis by KEGG metabolism pathway of differentially expressed proteins in rumen microorganisms was performed, Gayals have higher abundance of β-glucosidase and 6-phosphate-β-glucosidase than Yellow Cattle.

Transcription expression of gene encoding cathelicidin CATHL4 in Vietnam indigenous yellow cattle

Cathelicidins, a family of host-defence peptides, are present in a diverse range of species, including fish, amphibians, birds, reptiles, and mammals. The evidence for the role of these cationic antimicrobial peptides in innate host defenses is convincing from the data of animal model and transgenic animal experiments as well as wildlife or domestic animals, indicating that the peptides protect against inflammatory and immune system after induction by bacterial infection. In this study, we present the assessment of transcription expression of CATHL4 gene which encodes indolicidin, a cathelicidin from indigenous yellow cattle of Vietnam. The research focused on RNA samples extracted from lung tissues and lymph node collected from diseased cattle which died of infectious respiratory symptoms and the healthy cattle which were slaughtered for food purposes. By quantitative real-time PCR, the relative expression of transcripts was determined and analyzed using the expression of YWHAZ gene as reference. The results showed that this gene was abundantly expressed at a higher level in tissues of the diseased cattle than in those of the healthy ones. In both infection and healthy states, the expression of CATHL4 in lymph nodes were higher than in lung tissues. This indicated that CATHL4 (indolicidin) may participate in functions against infectious pathogens.

Proteomic Analysis of Capacitated and Non-Capacitated Spermatozoa of Yanbian Yellow Cattle

Background: Sperm capacitation is a process which occurs prior to fertilization, and is essential for producing high-quality living embryos. The main purpose of this study was to explore the difference of proteomics between capacitated and non-capacitated sperm of Yanbian yellow cattle. Bioinformatic analyses of LC-MS/MS data included GO enrichment, KEGG pathway enrichment, and protein-protein interaction (PPI) analysis. Results: The results revealed 23 specific proteins in the capacitated group and 345 in the non-capacitated group. Compared with non-capacitated sperm, capacitated sperm exhibited 89 upregulated proteins and 509 downregulated proteins. Western blotting was used to confirm our proteomics data. The expression level of PSMD1 in the capacitated sperm group was significantly lower than that in the non-capacitated sperm group, and the expression level of HSPA5 was significantly higher than in the non-capacitated sperm group. Conclusions: Our results revealed that many proteins were differentially expressed between capacitated and non-capacitated sperm, particularly those involved in the proteasome signaling and protein transport signaling pathways. This work enhances our understanding of molecular processes involved in sperm viability in Yanbian yellow cattle, and provides a framework for future studies.

The effect of miR-6523a on growth hormone secretion in pituitary cells of Yanbian yellow cattle

Yanbian yellow cattle breeding is limited by its slow growth. We previously found that the miRNA miR-6523a is differentially expressed between Yanbian yellow cattle and Han Yan cattle, which differ in growth characteristics. In this study, we evaluated the effects of miR-6523a on growth hormone (GH) secretion in pituitary cells of Yanbian yellow cattle. Bioinformatics analyses using TargetScan and RNAhybrid, as well as dual luciferase reporter assays, showed that miR-6523a targets the 3′ untranslated region of somatostatin receptor 5 (SSTR5). We further found that the mRNA and protein expression levels of GH in pituitary cells were significantly higher in cells treated with miR-6523a mimic than in the control group (P = 0.0082 and P = 0.0069). The GH mRNA and protein expression levels were lower in cells treated with miR-6523a inhibitor than in the control group, but the difference was not significant (P = 0.064 and P = 0.089). SSTR5 mRNA and protein levels were inhibited by miR-6523a mimic compared with the control group (P = 0.0024 and P = 0.0028) and were elevated slightly by miR-6523a inhibitor (P = 0.093 and P = 0.091). These results prove that miR-6523a regulates GH secretion in pituitary cells by SSTR5. More broadly, these findings provide a basis for studies of the roles of miRNAs in animal growth and development.

miR-381 Targets KCTD15 to Regulate Bovine Preadipocyte Differentiation In Vitro

MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs ~21 to ~23 nucleotides in length and have become a popular research topic in recent years due to their regulation of gene expression and many physiological processes, including fat metabolism; however, the precise functional mechanisms underlying their regulation of fat metabolism are not fully understood. Here, we identified miR-381, which specifically targets the 3′ untranslated region (3′ UTR) of potassium channel tetramerization-domain-containing 15 (KCTD15) , and verified the mechanism regulating its expression and participation in adipogenesis. We used a dual luciferase-reporter assay and transfection-mediated miR-381 overexpression and inhibition in Yanbian yellow cattle preadipocytes to investigate the role of miR-381 in adipogenesis. The results showed that miR-381 directly targets the 3′ UTR of KCTD15 and downregulates its expression. Additionally, miR-381 overexpression using an miRNA mimic promoted triglyceride accumulation and upregulated adipogenic peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα) at both the protein and mRNA levels, whereas miR-381 inhibition produced the opposite effect. These results indicated that miR-381 regulates the differentiation of Yanbian yellow cattle preadipocytes by inhibiting KCTD15 expression, thereby highlighting the importance of miRNA-mediated regulation of adipogenesis. Furthermore, our findings suggested that miR-381 and its target gene(s) might represent new targets for investigating intramuscular fat deposits in cattle and treating human obesity.

Histological Analysis, Bioinformatics Profile, and Expression of Methylenetetrahydrofolate Reductase (MTHFR) in Bovine Testes

Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes, exerts a direct effect on spermatogenesis; however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, ≤2 years juvenile (y-c, n = 3; yak, n = 3) and ≥3 years adult (y-c, n = 3; yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in the number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher, but not statistically significant, in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.