Combining Transcriptomics and Proteomics to Screen Candidate Genes Related to Bovine Birth Weight.

The placenta is a vital organ in bovine reproduction, crucial for blood supply, nutrient transport, and embryonic development. It plays an essential role in the intrauterine growth of calves. However, the molecular mechanisms governing placental function in calves remain inadequately understood. METHODS: We established transcriptome and proteome databases for low-birth-weight (LB) and high-birth-weight (HB) calf placentae, identifying key genes and proteins associated with birth weight through bioinformatics analyses that included functional enrichment and protein-protein interactions (PPIs). Both mRNA and protein levels were validated. RESULTS: A total of 1494 differentially expressed genes (DEGs) and 294 differentially expressed proteins (DEPs) were identified when comparing the LB group to the HB group. Furthermore, we identified 53 genes and proteins exhibiting significant co-expression across both transcriptomic and proteomic datasets; among these, 40 were co-upregulated, 8 co-downregulated, while 5 displayed upregulation at the protein level despite downregulation at the mRNA level. Functional enrichment analyses (GO and KEGG) and protein-protein interaction (PPI) analysis indicate that, at the transcriptional level, the primary factor contributing to differences in calf birth weight is that the placenta of the high-birth-weight (HB) group provides more nutrients to the fetus, characterized by enhanced nutrient transport (SLC2A1 and SLC2A11), energy metabolism (ACSL1, MICALL2, PAG2, COL14A1, and ELOVL5), and lipid synthesis (ELOVL5 and ELOVL7). In contrast, the placenta of the low-birth-weight (LB) group prioritizes cell proliferation (PAK1 and ITGA3) and angiogenesis. At the protein level, while the placentae from the HB group exhibit efficient energy production and lipid synthesis, they also demonstrate reduced immunity to various diseases such as systemic lupus erythematosus and bacterial dysentery. Conversely, the LB group placentae excel in regulating critical biological processes, including cell migration, proliferation, differentiation, apoptosis, and signal transduction; they also display higher disease immunity markers (COL6A1, TNC CD36, CD81, Igh-1a, and IGHG) compared to those of the HB group placentae. Co-expression analysis further suggests that increases in calf birth weight can be attributed to both high-efficiency energy production and lipid synthesis within the HB group placentae (ELOVL5, ELOVL7, and ACSL1), alongside cholesterol biosynthesis and metabolic pathways involving CYP11A1 and CYP17A1. CONCLUSION: We propose that ELOVL5, ELOVL7, ACSL1, CYP11A1, and CYP17A1 serve as potential protein biomarkers for regulating calf birth weight through the modulation of the fatty acid metabolism, lipid synthesis, and cholesterol levels.

Genome-Wide Identification and Characterization of Long Non-Coding RNAs in Longissimus dorsi Skeletal Muscle of Shandong Black Cattle and Luxi Cattle.

There is an increasing understanding of the possible regulatory role of long non-coding RNAs (LncRNA). Studies on livestock have mainly focused on the regulation of cell differentiation, fat synthesis, and embryonic development. However, there has been little study of skeletal muscle of domestic animals and the potential role of lncRNA. In this study, the transcriptome numbers of longissimus muscle of different beef cattle (Shandong black catle and Luxi catlle) were used to construct muscle related lncRNAs-miRNA-mRNA interaction network through bioinformatics analysis. This is helpful to clarify the molecular mechanism of bovine muscle development, and can be used to promote animal husbandry and improve animal husbandry production. According to the screening criteria of |FC|≧2 and q < 0.05, a total of 1,415 transcripts (of which 480 were LncRNAs) were differentially expressed (q < 0.05) in the different breeds. Further, we found that the most differentially expressed LncRNAs were found on chromosome 9, in which the differentially expressed LncRNAs targeted 1,164 protein coding genes (MYORG, Wnt4, PAK1, ADCY7,etc) (upstream and downstream<50 Kb). In addition, Pearson's correlation coefficients of co-expression levels indicated a potential trans regulatory relationship between the differentially expressed LncRNAs and 43844 mRNAs (r > 0.9). The identified co-expressed mRNAs (MYORG, Dll1, EFNB2, SOX6, MYOCD, and MYLK3) are related to the formation of muscle structure, and enriched in muscle system process, strained muscle cell differentiation, muscle cell development, striated muscle tissue development, calcium signaling, and AMPK signaling. Additionally, we also found that some LncRNAs (LOC112444238, LOC101903367, LOC104975788, LOC112441863, LOC112449549, and LOC101907194) may interact with miRNAs related to cattle muscle growth and development. Based on this, we constructed a LncRNAs-miRNA-mRNA interaction network as the putative basis for biological regulation in cattle skeletal muscle. Interestingly, a candidate differential LncRNA (LOC104975788) and a protein-coding gene (Pax7) contain miR-133a binding sites and binding was confirmed by luciferase reporter assay. LOC104975788 may combined miR-133a competitively with Pax7, thus relieving the inhibitory effect of miR-133a on Pax7 to regulate skeletal muscle development. These results will provide the theoretical basis for further study of LncRNA regulation and activity in different cattle breeds.

A Study of the Regulatory Mechanism of the CB1/PPARγ2/PLIN1/HSL Pathway for Fat Metabolism in Cattle.

Fat metabolism is closely related to the economic characteristics of beef cattle. Therefore, regulating fat deposition and increasing intramuscular fat deposition are among the main goals of breeders. In this study, we aim to explore the regulatory role of CB1 gene on PPARγ2/PLIN1/HSL pathway in fat metabolism, and to further explore the differential expression of regulatory factors of this pathway in Shandong black cattle and Luxi cattle. In this study, CB1 overexpression stimulated lipid synthesis in adipocytes to some extent by increasing the levels of FASN and ACSL1. CB1 inhibitors reduce the lipid content in adipocytes and reduce the expression of GLUT1 and Insig1. In addition, overexpression of CB1 decreased the expression of PPARγ2 and led to an increase in PLIN1 expression and a decrease in HSL expression in adipocytes. We also found that the CB1/PPARγ2/PLIN1/HSL was differentially expressed in the different breeds of cattle and was involved in the regulation of fat metabolism, which affected the fatty acid content in the longissimus dorsi muscle of the two breeds. In short, CB1 participates in lipid metabolism by regulating HSL in the PPARγ2 and PLIN1 pathways, and improves lipid formation in adipocytes. In conclusion, CB1/PPARγ2/PLIN1/HSL pathway may be involved in the regulation of lipid metabolism.

Identification and Characterization of circRNA in Longissimus Dorsi of Different Breeds of Cattle.

Shandong black cattle is a new breed of cattle that is developed by applying modern biotechnology, such as somatic cloning, and conventional breeding methods to Luxi cattle. It is very important to study the function and regulatory mechanism of circRNAs in muscle differentiation among different breeds to improve meat quality and meat production performance and to provide new ideas for beef cattle meat quality improvements and new breed development. Therefore, the goal of this study was to sequence and identify circRNAs in muscle tissues of different breeds of cattle. We used RNA-seq to identify circRNAs in the muscles of two breeds of cattle (Shandong black and Luxi). We identified 14,640 circRNAs and found 655 differentially expressed circRNAs. We also analyzed the classification and characteristics of circRNAs in muscle tissue. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used on the parental genes of circRNAs. They were mainly involved in a variety of biological processes, such as muscle fiber development, smooth muscle cell proliferation, bone system morphogenesis, tight junctions and the MAPK, AMPK, and mTOR signaling pathways. In addition, we used miRanda to predict the interactions between 14 circRNAs and 11 miRNAs. Based on the above assays, we identified circRNAs (circ0001048, circ0001103, circ0001159, circ0003719, circ0003424, circ0003721, circ0003720, circ0001519, circ0001530, circ0005011, circ0014518, circ0000181, circ0000190, circ0010558) that may play important roles in the regulation of muscle growth and development. Using real-time quantitative PCR, 14 circRNAs were randomly selected to verify the real circRNAs. Luciferase reporter gene system was used to verify the binding site of miR-1 in circ0014518. Our results provide more information about circRNAs regulating muscle development in different breeds of cattle and lay a solid foundation for future experiments.

Different expression of lipid metabolism-related genes in Shandong black cattle and Luxi cattle based on transcriptome analysis.

To provide new ideas for improving meat quality and generating new breeds of cattle, the important candidate genes affecting fat deposition in two kinds of cattle were identified. Eighteen months Shandong black cattle (n = 3) and Luxi cattle (n = 3) were randomly assigned into two environmental. The longissimus dorsi muscles of Shandong Black Cattle and Luxi Cattle were collected and analyzed by fatty acid determination, high-throughput sequencing transcriptomics, qRT-PCR expression profile and western blot. The ratio of unsaturated fatty acids to saturated fatty acids was 1.37:1 and 1.24:1 in the muscle tissues of Shandong black cattle and Luxi cattle, respectively. The results of RNA-Seq analysis revealed 1320 DEGs between the longissimus dorsi of Shandong black cattle and Luxi cattle. A total of 867 genes were upregulated, and the other 453 genes were downregulated. With GO enrichment analysis, it was found that the identified DEGs were significantly enriched in regulation of the Wnt signaling pathway, negative regulation of the Wnt signaling pathway, cAMP metabolic process, fat cell differentiation and among other functions. We found that regulation of lipolysis in adipocytes was the significant enrichment pathway of upregulated genes and downregulated genes, PPAR signaling pathway and AMPK signaling pathway are highly representative pathways of lipid metabolism in Shandong black cattle. Network analysis showed that PPARGC1A, ADCY4, ANKRD6, COL1A1, FABP4, ADIPOQ, PLIN1, PLIN2, and LIPE genes were correlated with key loci genes in multiple metabolic pathways. Meanwhile we found that FABP4 and ADIPOQ had 7 common regulatory factors in different genes, which were PLIN1, PLIN2, PPARGC1A, RXRA, PCK1, LEPR, LEP. These genes were involved in regulation of lipolysis in adipocytes, adipocytokine signaling pathway, PPAR signaling pathway. FABP4 and ADIPOQ were selected as important candidate marker genes for fat deposition based on the results.

[Isolation and nuclear transfer of ES-like cells colonies derived from embryos being cloning of bovine somatic].

In this experiment, it was designed to carry out proliferous culture of bovine blastocysts(day 7) derived from embryos cloned through bovine somatic cell nuclear transfer, isolating and passaging of ES cells. The cells of blastocysts, which were planted on feeder layer, formed small colonies within 24 h. The nest-shape colonies occurred after culturing for 2-3 days. After the colonies in the same shape were isolated and passaged 4-5 times, many different size colonies with monolayer of multi-cells appeared. The colonies that had been passaged 4-5 times were planted into 4-wells multi-dishes without feeder layer. The colonies with monolayer of multi-cells appeared after 24 h, spread all over the bottom of the dishes, emerged epidermis-like cells that appeared reticulate after 4-7 days. These cells were used as donor cells to carry out nuclear transfer. The results showed that 80% (40/50) of the reconstructed embryos cleaved, 5% (2/40) and 2.5% (1/40) of them developed to the morulaes and blastocyst stage, respectively. It revealed that ES-like cells derived embryos constructed through somatic cell nuclear transfer have the developmental potentials.

Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.