Sodium Butyrate Induces Mitophagy and Apoptosis of Bovine Skeletal Muscle Satellite Cells through the Mammalian Target of Rapamycin Signaling Pathway.

Sodium butyrate (NaB) is one of the short-chain fatty acids and is notably produced in large amounts from dietary fiber in the gut. Recent evidence suggests that NaB induces cell proliferation and apoptosis. Skeletal muscle is rich in plenty of mitochondrial. However, it is unclear how NaB acts on host muscle cells and whether it is involved in mitochondria-related functions in myocytes. The present study aimed to investigate the role of NaB treatment on the proliferation, apoptosis, and mitophagy of bovine skeletal muscle satellite cells (BSCs). The results showed that NaB inhibited proliferation, promoted apoptosis of BSCs, and promoted mitophagy in a time- and dose-dependent manner in BSCs. In addition, 1 mM NaB increased the mitochondrial ROS level, decreased the mitochondrial membrane potential (MMP), increased the number of autophagic vesicles in mitochondria, and increased the mitochondrial DNA (mtDNA) and ATP level. The effects of the mTOR pathway on BSCs were investigated. The results showed that 1 mM NaB inhibited the mRNA and protein expression of mTOR and genes AKT1, FOXO1, and EIF4EBP1 in the mTOR signaling pathway. In contrast, the addition of PP242, an inhibitor of the mTOR signaling pathway also inhibited mRNA and protein expression levels of mTOR, AKT1, FOXO1, and EIF4EBP1 and promoted mitophagy and apoptosis, which were consistent with the effect of NaB treatment. NaB might promote mitophagy and apoptosis in BSCs by inhibiting the mTOR signaling pathway. Our results would expand the knowledge of sodium butyrate on bovine skeletal muscle cell state and mitochondrial function.

Multi-transcriptomics reveals RLMF axis-mediated signaling molecules associated with bovine feed efficiency.

The regulatory axis plays a vital role in interpreting the information exchange and interactions among mammal organs. In this study on feed efficiency, it was hypothesized that a rumen-liver-muscle-fat (RLMF) regulatory axis exists and scrutinized the flow of energy along the RLMF axis employing consensus network analysis from a spatial transcriptomic standpoint. Based on enrichment analysis and protein-protein interaction analysis of the consensus network and tissue-specific genes, it was discovered that carbohydrate metabolism, energy metabolism, immune and inflammatory responses were likely to be the biological processes that contribute most to feed efficiency variation on the RLMF regulatory axis. In addition, clusters of genes related to the electron respiratory chain, including ND (2,3,4,4L,5,6), NDUF (A13, A7, S6, B3, B6), COX (1,3), CYTB, UQCR11, ATP (6,8), clusters of genes related to fatty acid metabolism including APO (A1, A2, A4, B, C3), ALB, FG (A, G), as well as clusters of the ribosomal-related gene including RPL (8,18A,18,15,13, P1), the RPS (23,27A,3A,4X), and the PSM (A1-A7, B6, C1, C3, D2-D4, D8 D9, E1) could be the primary effector genes responsible for feed efficiency variation. The findings demonstrate that high feed efficiency cattle, through the synergistic action of the regulatory axis RLMF, may improve the efficiency of biological processes (carbohydrate metabolism, protein ubiquitination, and energy metabolism). Meanwhile, high feed efficiency cattle might enhance the ability to respond to immunity and inflammation, allowing nutrients to be efficiently distributed across these organs associated with digestion and absorption, energy-producing, and energy-storing organs. Elucidating the distribution of nutrients on the RLMF regulatory axis could facilitate an understanding of feed efficiency variation and achieve the study on its molecular regulation.

Blood transcriptome reveals immune and metabolic-related genes involved in growth of pasteurized colostrum-fed calves.

The quality of colostrum is a key factor contributing to healthy calf growth, and pasteurization of colostrum can effectively reduce the counts of pathogenic microorganisms present in the colostrum. Physiological changes in calves fed with pasteurized colostrum have been well characterized, but little is known about the underlying molecular mechanisms. In this study, key genes and functional pathways through which pasteurized colostrum affects calf growth were identified through whole blood RNA sequencing. Our results showed that calves in the pasteurized group (n = 16) had higher body height and daily weight gain than those in the unpasteurized group (n = 16) in all months tested. Importantly, significant differences in body height were observed at 3 and 4 months of age (p < 0.05), and in daily weight gain at 2, 3, and 6 months of age (p < 0.05) between the two groups. Based on whole blood transcriptome data from 6-months old calves, 630 differentially expressed genes (DEGs), of which 235 were upregulated and 395 downregulated, were identified in the pasteurized compared to the unpasteurized colostrum groups. Most of the DEGs have functions in the immune response (e.g., CCL3, CXCL3, and IL1A) and metabolism (e.g., PTX3 and EXTL1). Protein-protein interaction analyses of DEGs revealed three key subnetworks and fifteen core genes, including UBA52 and RPS28, that have roles in protein synthesis, oxidative phosphorylation, and inflammatory responses. Twelve co-expression modules were identified through weighted gene co-expression network analysis. Among them, 17 genes in the two modules that significantly associated with pasteurization were mainly involved in the tricarboxylic acid cycle, NF-kappa B signaling, and NOD-like receptor signaling pathways. Finally, DEGs that underwent alternative splicing in calves fed pasteurized colostrum have roles in the immune response (SLCO4A1, AKR1C4, and MED13L), indicative of potential roles in immune regulation. Results from multiple analytical methods used suggest that differences in calf growth between the pasteurized and unpasteurized groups may be due to differential immune activity. Our data provide new insights into the impact of pasteurization on calf immune and metabolic-related pathways through its effects on gene expression.

Identification and characterization of hypothalamic circular RNAs associated with bovine residual feed intake.

Residual feed intake (RFI) is crucial economic indicator used for calculating the feed efficiency of growing beef cattle. circRNA plays an important biological role in gene transcriptional regulation, but little is known about its potential functional regulation underlying RFI phenotypic variation. As the core center of regulation of animal feeding, the hypothalamus is closely associated with RFI. Therefore, the present study aimed to identify the key genes and functional pathways contributing to variance in cattle RFI phenotypes using RNA sequencing from hypothalamic tissue samples, in order to gain insight into the potential regulatory role of circRNAs in bovine RFI phenotypic variation. Differentially expressed genes were detected by RNA sequencing for beef cattle in the high and low RFI groups, followed by GO, KEGG enrichment, and circRNA-miRNA co-expression network analysis. A total of 257 circRNAs were differentially expressed between the two groups, with 128 significantly upregulated and 129 significantly downregulated genes in H group compared to L group. Among them, 9 unique circRNAs were present in group L and 4 unique circRNAs were present in group H. GO and KEGG enrichment analysis of the source genes of the differentially expressed circRNAs revealed that they were mainly involved in metabolic processes, such as cellular metabolic processes, cellular macromolecular metabolic processes, and regulatory pathways related to nutrient metabolism, including protein and amino acid metabolism, as well as vitamin metabolism and pancreatic secretion associated with the animal feeding behavior. The circRNAs detected in this study were mostly novel, and have not been investigated directly to be associated with the RFI phenotype. Interestingly, most miRNAs of differentially expressed circRNAs predicted based on the circRNA-miRNA co-expression network analysis by using top 50 differentially expressed circRNAs and 13 unique circRNAs, have been reported to be related to animal RFIs, implying that circRNAs in bovine hypothalamic tissue may regulate phenotypic variation in RFI through miRNAs. The study results illustrate the complex biological functions of the hypothalamus in regulating feed efficiency and showing the potential role of circRNAs in the feeding behavior regulation of livestock, which would contributing to expanding the understanding of circRNA.

Integrative Analysis of miRNA-mRNA in Ovarian Granulosa Cells Treated with Kisspeptin in Tan Sheep.

Kisspeptin is a peptide hormone encoded by the kiss-1 gene that regulates animal reproduction. Our studies revealed that kisspeptin can regulate steroid hormone production and promote cell proliferation in ovarian granulosa cells of Tan sheep, but the mechanism has not yet been fully understood. We speculated that kisspeptin might promote steroid hormone production and cell proliferation by mediating the expression of specific miRNA and mRNA in granulosa cells. Accordingly, after granulosa cells were treated with kisspeptin, the RNA of cells was extracted to construct a cDNA library, and miRNA-mRNA sequencing was performed. Results showed that 1303 expressed genes and 605 expressed miRNAs were identified. Furthermore, eight differentially expressed miRNAs were found, and their target genes were significantly enriched in progesterone synthesis/metabolism, hormone biosynthesis, ovulation cycle, and steroid metabolism regulation. Meanwhile, mRNA was significantly enriched in steroid biosynthesis, IL-17 signaling pathway, and GnRH signaling pathway. Integrative analysis of miRNA-mRNA revealed that the significantly different oar-let-7b targets eight genes, of which EGR1 (early growth response-1) might play a significant role in regulating the function of granulosa cells, and miR-10a regulates lipid metabolism and steroid hormone synthesis by targeting HNRNPD. Additionally, PPI analysis revealed genes that are not miRNA targets but crucial to other biological processes in granulosa cells, implying that kisspeptin may also indirectly regulate granulosa cell function by these pathways. The findings of this work may help understand the molecular mechanism of kisspeptin regulating steroid hormone secretion, cell proliferation, and other physiological functions in ovarian granulosa cells of Tan sheep.

miR-377 Inhibits Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells by Targeting FHL2.

Non-coding RNAs, especially microRNAs (miRNAs), play an important role in skeletal muscle growth and development. miR-377 regulates many basic biological processes and plays a key role in tumor cell proliferation, migration and apoptosis. Nevertheless, the function of miR-377 during skeletal muscle development and how it regulates skeletal muscle satellite cells (SMSCs) remains unclear. In the present study, we proposed to elucidate the regulatory mechanism of miR-377 in the proliferation and differentiation of bovine primary SMSCs. Our results showed that miR-377 can significantly inhibit the proliferation of SMSCs. In addition, we found that miR-377 can reduce myotube formation and restrain skeletal myogenic differentiation. Moreover, the results obtained from the biosynthesis and dual luciferase experiments showed that FHL2 was the target gene of miR-377. We further probed the function of FHL2 in muscle development and found that FHL2 silencing significantly suppressed the proliferation and differentiation of SMSCS, which is contrary to the role of miR-377. Furthermore, FHL2 interacts with Dishevelled-2 (Dvl2) to enable Wnt/ß-catenin signaling pathway, consequently regulating skeletal muscle development. miR-377 negatively regulates the Wnt/ß-catenin signaling pathway by targeting FHL2-mediated Dvl2. Overall, these findings demonstrated that miR-377 regulates the bovine SMSCs proliferation and differentiation by targeting FHL2 and attenuating the Wnt/ß-catenin signaling pathway.

Genomic Selection for Milk Production Traits in Xinjiang Brown Cattle.

One-step genomic selection is a method for improving the reliability of the breeding value estimation. This study aimed to compare the reliability of pedigree-based best linear unbiased prediction (PBLUP) and single-step genomic best linear unbiased prediction (ssGBLUP), single-trait and multitrait models, and the restricted maximum likelihood (REML) and Bayesian methods. Data were collected from the production performance records of 2207 Xinjiang Brown cattle in Xinjiang from 1983 to 2018. A cross test was designed to calculate the genetic parameters and reliability of the breeding value of 305 daily milk yield (305 dMY), milk fat yield (MFY), milk protein yield (MPY), and somatic cell score (SCS) of Xinjiang Brown cattle. The heritability of 305 dMY, MFY, MPY, and SCS estimated using the REML and Bayesian multitrait models was approximately 0.39 (0.02), 0.40 (0.03), 0.49 (0.02), and 0.07 (0.02), respectively. The heritability and estimated breeding value (EBV) and the reliability of milk production traits of these cattle calculated based on PBLUP and ssGBLUP using the multitrait model REML and Bayesian methods were higher than those of the single-trait model REML method; the ssGBLUP method was significantly better than the PBLUP method. The reliability of the estimated breeding value can be improved from 0.9% to 3.6%, and the reliability of the genomic estimated breeding value (GEBV) for the genotyped population can reach 83%. Therefore, the genetic evaluation of the multitrait model is better than that of the single-trait model. Thus, genomic selection can be applied to small population varieties such as Xinjiang Brown cattle, in improving the reliability of the genomic estimated breeding value.

Identifying the key genes and functional enrichment pathways associated with feed efficiency in cattle.

Residual feed intake (RFI) is a measurement of feed efficiency, and is inversely correlated with feed efficiency. The differentially expressed genes (DEGs) associated with RFI vary substantially among studies, posing great challenges in finding the RFI-related marker genes. This study attempted to resolve this issue by integrating and comparing the multiple transcriptome sequencing data associated with RFI in the cattle liver, using differential, functional enrichment, protein-protein interaction (PPI) network, weighted co-expression network (WGCNA), and gene set enrichment analyses (GSEA) to identify the candidate genes and functional enrichment pathways that are closely associated with RFI. Four candidate genes namely SHC1, GPX4, ACADL, and IGF1 were identified and validated as the marker genes for RFI. Four functional enrichment pathways, namely the fatty acid metabolism, sugar metabolism, energy metabolism, and protein ubiquitination were also found to be closely related to RFI. This study identified several genes and signaling pathways with shared characteristics, which will provide new insights into the molecular mechanisms related to the regulation of feed efficiency, and provide basis for molecular markers related to feed efficiency in beef cattle.

HandyCNV: Standardized Summary, Annotation, Comparison, and Visualization of Copy Number Variant, Copy Number Variation Region, and Runs of Homozygosity.

Detection of CNVs (copy number variants) and ROH (runs of homozygosity) from SNP (single nucleotide polymorphism) genotyping data is often required in genomic studies. The post-analysis of CNV and ROH generally involves many steps, potentially across multiple computing platforms, which requires the researchers to be familiar with many different tools. In order to get around this problem and improve research efficiency, we present an R package that integrates the summarization, annotation, map conversion, comparison and visualization functions involved in studies of CNV and ROH. This one-stop post-analysis system is standardized, comprehensive, reproducible, timesaving, and user-friendly for researchers in humans and most diploid livestock species.

Characterization and Duodenal Transcriptome Analysis of Chinese Beef Cattle With Divergent Feed Efficiency Using RNA-Seq.

Residual feed intake (RFI) is an important measure of feed efficiency for agricultural animals. Factors associated with cattle RFI include physiology, dietary factors, and the environment. However, a precise genetic mechanism underlying cattle RFI variations in duodenal tissue is currently unavailable. The present study aimed to identify the key genes and functional pathways contributing to variance in cattle RFI phenotypes using RNA sequencing (RNA-seq). Six bulls with extremely high or low RFIs were selected for detecting differentially expressed genes (DEGs) by RNA-seq, followed by conducting GO, KEGG enrichment, protein-protein interaction (PPI), and co-expression network (WGCNA, n = 10) analysis. A total of 380 differentially expressed genes was obtained from high and low RFI groups, including genes related to energy metabolism (ALDOA, HADHB, INPPL1), mitochondrial function (NDUFS1, RFN4, CUL1), and feed intake behavior (CCK). Two key sub-networks and 26 key genes were detected using GO analysis of DEGs and PPI analysis, such as TPM1 and TPM2, which are involved in mitochondrial pathways and protein synthesis. Through WGCNA, a gene network was built, and genes were sorted into 27 modules, among which the blue (r = 0.72, p = 0.03) and salmon modules (r = -0.87, p = 0.002) were most closely related with RFI. DEGs and genes from the main sub-networks and closely related modules were largely involved in metabolism; oxidative phosphorylation; glucagon, ribosome, and N-glycan biosynthesis, and the MAPK and PI3K-Akt signaling pathways. Through WGCNA, five key genes, including FN1 and TPM2, associated with the biological regulation of oxidative processes and skeletal muscle development were identified. Taken together, our data suggest that the duodenum has specific biological functions in regulating feed intake. Our findings provide broad-scale perspectives for identifying potential pathways and key genes involved in the regulation of feed efficiency in beef cattle.

Investigation of genetic markers for intramuscular fat in the hybrid Wagyu cattle with bulked segregant analysis.

ulked Segregant Analysis (BSA) is a rapid strategy for identifying genetic markers in specific regions of the phenotypical population and it has been widely used for QTLs mapping in smaller mixed F2 and F3 populations. We applied a modified BSA method to assessed genome-wide homozygous and heterozygous linkage patterns in the Chinese Wagyu Beef Cattle F2/F3 mixed population. Two overlapped regions from F2 and F3 populations on autosomes were found with high-density heterozygote alleles between high and low intramuscular fat groups. Regions from 24.8 M ~ 29.6 M of chromosome 23 were identified as most significantly correlated to the intramuscular fat in our samples. We also identified other 4 potential loci on chromosomes 5, 9, 15, and 21 correlated with Intramuscular fat. This study provided a novel low-cost method for QTLs mapping and identify molecular markers of phenotypical changes in a small mixed population.

Discovering Copy Number Variation in Dual-Purpose XinJiang Brown Cattle.

Copy number variants (CNVs), which are a class of structural variant, can be important in relating genomic variation to phenotype. The primary aims of this study were to discover the common CNV regions (CNVRs) in the dual-purpose XinJiang-Brown cattle population and to detect differences between CNVs inferred using the ARS-UCD 1.2 (ARS) or the UMD 3.1 (UMD) genome assemblies based on the 150K SNP (Single Nucleotide Polymorphisms) Chip. PennCNV and CNVPartition methods were applied to calculate the deviation of the standardized signal intensity of SNPs markers to detect CNV status. Following the discovery of CNVs, we used the R package HandyCNV to generate and visualize CNVRs, compare CNVs and CNVRs between genome assemblies, and identify consensus genes using annotation resources. We identified 38 consensus CNVRs using the ARS assembly with 1.95% whole genome coverage, and 33 consensus CNVRs using the UMD assembly with 1.46% whole genome coverage using PennCNV and CNVPartition. We identified 37 genes that intersected 13 common CNVs (>5% frequency), these included functionally interesting genes such as GBP4 for which an increased copy number has been negatively associated with cattle stature, and the BoLA gene family which has been linked to the immune response and adaption of cattle. The ARS map file of the GGP Bovine 150K Bead Chip maps the genomic position of more SNPs with increased accuracy compared to the UMD map file. Comparison of the CNVRs identified between the two reference assemblies suggests the newly released ARS reference assembly is better for CNV detection. In spite of this, different CNV detection methods can complement each other to generate a larger number of CNVRs than using a single approach and can highlight more genes of interest.

Genome-wide association study of milk and reproductive traits in dual-purpose Xinjiang Brown cattle.

BACKGROUND: Dual-purpose cattle are more adaptive to environmental challenges than single-purpose dairy or beef cattle. Balance among milk, reproductive, and mastitis resistance traits in breeding programs is therefore more critical for dual-purpose cattle to increase net income and maintain well-being. With dual-purpose Xinjiang Brown cattle adapted to the Xinjiang Region in northwestern China, we conducted genome-wide association studies (GWAS) to dissect the genetic architecture related to milk, reproductive, and mastitis resistance traits. Phenotypic data were collected for 2410 individuals measured during 1995-2017. By adding another 445 ancestors, a total of 2855 related individuals were used to derive estimated breeding values for all individuals, including the 2410 individuals with phenotypes. Among phenotyped individuals, we genotyped 403 cows with the Illumina 150 K Bovine BeadChip. RESULTS: GWAS were conducted with the FarmCPU (Fixed and random model circulating probability unification) method. We identified 12 markers significantly associated with six of the 10 traits under the threshold of 5% after a Bonferroni multiple test correction. Seven of these SNPs were in QTL regions previously identified to be associated with related traits. One identified SNP, BovineHD1600006691, was significantly associated with both age at first service and age at first calving. This SNP directly overlapped a QTL previously reported to be associated with calving ease. Within 160 Kb upstream and downstream of each significant SNP identified, we speculated candidate genes based on functionality. Four of the SNPs were located within four candidate genes, including CDH2, which is linked to milk fat percentage, and GABRG2, which is associated with milk protein yield. CONCLUSIONS: These findings are beneficial not only for breeding through marker-assisted selection, but also for genome editing underlying the related traits to enhance the overall performance of dual-purpose cattle.

The study of blood transcriptome profiles in Holstein cows with miscarriage during peri-implantation.

OBJECTIVE: In this study, the transcriptome profile of cow experiencing miscarriage during peri-implantation was investigated. METHODS: Total transcriptomes were checked by RNA sequencing, and the analyzed by bioinformatics methods, the differentially expressed genes (DEGs) were analysed with hierarchical clustering and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. RESULTS: The results suggested that serum progesterone levels were significantly decreased in cows that miscarried as compared to the pregnant cows at 18, 21, 33, 39, and 51 days after artificial insemination. The RNA sequencing results suggested that 32, 176, 5, 10, and 2 DEGs were identified in the pregnant cows and miscarried cows at 18, 21, 33, 39, and 51 d after artificial insemination. And 15, 101, 1, 2, and 2 DEGs were upregulated, and 17, 74, 4, and 8 DEGs were downregulated in the cows in the pregnant and miscarriage groups, respectively at 18, 21, 33, and 39, but no gene was downregulated at 51 d after artificial insemination. These DEGs were distributed to 13, 20, 3, 6, and 20 pathways, and some pathway essential for pregnancy, such as cell adhesion molecules, tumor necrosis factor signaling pathway and PI3K-Akt signaling pathway. CONCLUSION: This analysis has identified several genes and related pathways crucial for pregnancy and miscarriage in cows, as well as these genes supply molecular markers to predict the miscarriage in cows.

Effects of soya oligosaccharides and soya oligopeptides on lipid metabolism in hyperlipidaemic rats.

In the present study, we aimed to examine the effects of soya oligosaccharides (SOS) and soya oligopeptides (SOP) on blood lipid levels, release of vasoactive substances, antioxidant activity and faecal bile acid (FBA) excretion in rats fed a high-fat diet (HFD). Male Sprague-Dawley rats were evenly divided into five groups according to diets as follows: regular diet (control), HFD, HFD enriched with 2 % of SOS (SOS), HFD enriched with 3 % of SOP (SOP) and HFD enriched with 2 % SOS and 3 % SOP (SOSP). The results showed that SOS and SOP significantly reduced plasma total cholesterol, LDL-cholesterol and TAG, whereas HDL-cholesterol concentration was significantly increased. Furthermore, SOS and SOP reduced plasma apoB, apoE and the apoB:apoAI ratio, whereas apoAI was significantly increased. Moreover, SOS and SOP also reduced plasma thromboxane A2 (TXA2) and the TXA2:prostacyclin (PGI2) ratio, whereas plasma PGI2 and nitric oxide were significantly increased. In addition, SOS and SOP significantly reduced serum and liver malondialdehyde concentrations and increased FBA excretion. However, we did not observe obvious influences of SOS and SOP on superoxide dismutase activities in the liver of HFD-fed rats. The combination of 2 % SOS and 3 % SOP showed a more marked effect than SOS or SOP alone in improving the lipid profile, release of vasoactive substances and increasing FBA excretion (P < 0.05). In summary, SOS and SOP might help prevent atherosclerosis through improving abnormal blood lipid levels, regulating vasoactive substances and protecting against oxidative stress.