Comprehensive Analysis of Transcriptome and Metabolome Reveals Regulatory Mechanism of Intramuscular Fat Content in Beef Cattle.

The intramuscular fat (IMF) content of beef determined the meat quality, and the market value of beef varies with different breeds. To provide some new approaches for improving meat quality and cattle breed improvement, 24-month-old Qinchuan cattle (Q, n = 6), Nanyang cattle (N, n = 6), and Japanese black cattle (J, n = 6) were selected. IMF content of the J group (16.92 ± 1.08%) is remarkably higher than that of indigenous Chinese cattle (Q, 13.38 ± 1.08%, and N, 12.35 ± 1.22%). Monounsaturated fatty acids and polyunsaturated fatty acids in the J group are higher than the Q and creatine, lysine, and glutamine are the three most abundant amino acids in beef, which contribute to the flavor formation. Similarly, IMF content-related genes were enriched in four vital KEGG pathways, including fatty acid metabolism, biosynthesis of unsaturated fatty acids, fatty acid elongation, and insulin resistance. Moreover, weighted genes coexpression network analysis (WGCNA) revealed that ITGB1 is the critical gene associated with the IMF content. This study compares transcriptome and metabolome of local and high-IMF cattle breeds, providing data for native cattle breeding and improvement of beef quality.

Genome-Wide Association Study Reveals Novel Loci Associated with Body Conformation Traits in Qinchuan Cattle.

A genome-wide association study (GWAS) is an effective tool for identifying the dominant genes of complex economic traits in livestock by statistical analysis of genotype data and measured phenotype data. In this study, we rigorously measured 14 body conformation traits in 254 Qinchuan cattle, comprising body weight (BW), body height (BOH), back height (BAH), buttock height (BUH), chest depth (CD), chest width (CW), hip cross height (HCH), body length (BL), hip width (HW), rump length (RL), pin bone width (PBW), chest girth (CG), abdomen circumference (AG), and calf circumference (CC). After quality control, 281,889 SNPs were generated for GWAS with different traits. A total of 250 suggestive SNPs (p < 3.54 × 10-6) were screened and 37 candidate genes were annotated. Furthermore, we performed a linkage disequilibrium analysis of SNP loci and considered published studies, identifying the eight genes (ADAMTS17, ALDH1A3, CHSY1, MAGEL2, MEF2A, SYNM, CNTNAP5, and CTNNA3) most likely to be involved in growth traits. This study provides new insights into the regulatory mechanisms of bovine body size development, which can be very useful in the development of management and breeding strategies.

MAPK family genes' influences on myogenesis in cattle: Genome-wide analysis and identification.

The mitogen-activated protein kinase (MAPK) family is highly conserved in mammals, and is involved in a variety of physiological phenomena like regeneration, development, cell proliferation, and differentiation. In this study, 13 MAPK genes were identified in cattle and their corresponding protein properties were characterized using genome-wide identification and analysis. Phylogenetic analysis showed that the 13 BtMAPKs were cluster grouped into eight major evolutionary branches, which were segmented into three large subfamilies: ERK, p38 and JNK MAPK. BtMAPKs from the same subfamily had similar protein motif compositions, but considerably different exon-intron patterns. The heatmap analysis of transcriptome sequencing data showed that the expression of BtMAPKs was tissue-specific, with BtMAPK6 and BtMAPK12 highly expressed in muscle tissues. Furthermore, knockdown of BtMAPK6 and BtMAPK12 revealed that BtMAPK6 had no effect on myogenic cell proliferation, but negatively affected the differentiation of myogenic cells. In contrast, BtMAPK12 improved both the cell proliferation and differentiation. Taken together, these results provide novel insights into the functions of MAPK families in cattle, which could serve as a basis for further studies on the specific mechanisms of the genes in myogenesis.

Long Non-Coding RNA BNIP3 Inhibited the Proliferation of Bovine Intramuscular Preadipocytes via Cell Cycle.

The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5' RACE and 3' RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5'RACE, and 464 bp of 3'RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2'- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality.

Identification of the hub genes related to adipose tissue metabolism of bovine.

Due to the demand for high-quality animal protein, there has been consistent interest in how to obtain more high-quality beef. As well-known, the adipose content of beef has a close connection with the taste and quality of beef, and cattle with different energy or protein diet have corresponding effects on the lipid metabolism of beef. Thus, we performed weighted gene co-expression network analysis (WGCNA) with subcutaneous adipose genes from Norwegian red heifers fed different diets to identify hub genes regulating bovine lipid metabolism. For this purpose, the RNA sequencing data of subcutaneous adipose tissue of 12-month-old Norwegian red heifers (n = 48) with different energy or protein levels were selected from the GEO database, and 7,630 genes with the largest variation were selected for WGCNA analysis. Then, three modules were selected as hub genes candidate modules according to the correlation between modules and phenotypes, including pink, magenta and grey60 modules. GO and KEGG enrichment analysis showed that genes were related to metabolism, and participated in Rap, MAPK, AMPK, VEGF signaling pathways, and so forth. Combined gene interaction network analysis using Cytoscape software, eight hub genes of lipid metabolism were identified, including TIA1, LOC516108, SNAPC4, CPSF2, ZNF574, CLASRP, MED15 and U2AF2. Further, the expression levels of hub genes in the cattle tissue were also measured to verify the results, and we found hub genes in higher expression in muscle and adipose tissue in adult cattle. In summary, we predicted the key genes of lipid metabolism in the subcutaneous adipose tissue that were affected by the intake of various energy diets to find the hub genes that coordinate lipid metabolism, which provide a theoretical basis for regulating beef quality.

Nutrient digestibility, rumen microbial protein synthesis, and growth performance in sheep consuming rations containing sea buckthorn pomace.

This experiment was conducted to investigate nutrient digestibility, rumen microbial protein synthesis, and growth performance when different proportions of sea buckthorn pomace (SBP) were included in the diet of sheep. A total of forty1/2 Dorper × 1/2 thin-tailed Han ram lambs (BW = 22.2 ± 0.92 kg, age =120 ± 11 d; mean ± SD) were selected and divided into four groups in a randomized design and were randomly allocated to one of four treatment diets. Diets were formulated isonitrogenously and contained different levels of SBP: 1) 0% SBP (control), 2) 7.8% of DM SBP (8SBP), 3) 16.0% of DM SBP (16SBP), and 4) 23.5% of DM SBP (24SBP). A portion of corn and forages were replaced with SBP. DMI and ADG increased linearly (P = 0.001), but feed efficiency was not affected (P ≥ 0.460) by increasing SBP inclusion rate. As the SBP inclusion increased, OM, NDF, and ADF digestibility decreased linearly (P ≤ 0.005) and that CP increased linearly (P = 0.012). Response to inclusion level of SBP was quadratic (P = 0.003) for the estimated microbial CP yield with the greatest at intermediate SBP levels. For intestinally absorbable dietary protein, quadratic (P = 0.029) effects were observed among treatments. The metabolizable protein (MP) supplies were linearly (P < 0.0001) improved with increasing SBP inclusion rate. The results indicated that SBP can be incorporated in the ration of ram lambs and improve MP supply and ADG. However, high content of it in the diet was adverse for nutrient digestibility. The optimal proportion was 16.0% under the condition of this experiment.