The spatio-temporal expression patterns of Circular RNA (circRNA) across organs and developmental stages are critical for its function and evolution analysis. However, they remain largely unclear in mammals. Here, we comprehensively analysed circRNAs in nine organs and three skeletal muscles of Guizhou miniature pig (S. scrofa), a widely used biomedical model animal. We identified 5,934 circRNAs and analysed their molecular properties, sequence conservation, spatio-temporal expression pattern, potential function, and interaction with miRNAs. S. scrofa circRNAs show modest sequence conservation with human and mouse circRNAs, are flanked by long introns, exhibit low abundance, and are expressed dynamically in a spatio-temporally specific manner. S. scrofa circRNAs show the greatest abundance and complexity in the testis. Notably, 31% of circRNAs harbour well-conserved canonical miRNA seed matches, suggesting that some circRNAs act as miRNAs sponges. We identified 149 circRNAs potentially associated with muscle growth and found that their host genes were significantly involved in muscle development, contraction, chromatin modification, cation homeostasis, and ATP hydrolysis-coupled proton transport; moreover, this set of genes was markedly enriched in genes involved in tight junctions and the calcium signalling pathway. Finally, we constructed the first public S. scrofa circRNA database, allowing researchers to query comprehensive annotation, expression, and regulatory networks of circRNAs.

DNA methylation plays a pivotal role in biological processes by affecting gene expression. However, how DNA methylation mediates phenotype difference of skeletal muscle between lean-, obese-, and mini-type pigs remains unclear. We systematically carried out comparative analysis of skeletal muscle by integrating analysis of genome-wide DNA methylation, mRNA, lncRNA and miRNA profiles in three different pig breeds (obese-type Tongcheng, lean-type Landrace, and mini-type Wuzhishan pigs). We found that the differentially methylated genes (DMGs) were significantly associated with lipid metabolism, oxidative stress and muscle development. Among the identified DMGs, 253 genes were related to body-size and obesity. A set of lncRNAs and mRNAs including UCP3, FHL1, ANK1, HDAC4, and HDAC5 exhibited inversely changed DNA methylation and expression level; these genes were associated with oxidation reduction, fatty acid metabolism and cell proliferation. Gene regulatory networks involved in phenotypic variation of skeletal muscle were related to lipid metabolism, cellular movement, skeletal muscle development, and the p38 MAPK signaling pathway. DNA methylation potentially influences the propensity for obesity and body size by affecting gene expression in skeletal muscle. Our findings provide an abundant information of epigenome and transcriptome that will be useful for animal breeding and biomedical research.

Background Circular RNAs (circRNAs) represent a novel class of non‐coding RNAs formed by a covalently closed loop and play crucial roles in many biological processes. Several circRNAs associated with myogenesis have been reported. However, the dynamic expression, function, and mechanism of circRNAs during myogenesis and skeletal muscle development are largely unknown. Methods Strand‐specific RNA‐sequencing (RNA‐seq) and microarray datasets were used to profile the dynamic circRNAome landscape during skeletal muscle development and myogenic differentiation. Bioinformatics analyses were used to characterize the circRNAome and identify candidate circRNAs associated with myogenesis. Bulk and single‐cell RNA‐seq were performed to identify the downstream genes and pathways of circFgfr2. The primary myoblast cells, C2C12 cells, and animal model were used to assess the function and mechanism of circFgfr2 in myogenesis and muscle regeneration in vitro or in vivo by RT‐qPCR, western blotting, dual‐luciferase activity assay, RNA immunoprecipitation, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation. Results We profiled the dynamic circRNAome in pig skeletal muscle across 27 developmental stages and detected 52 918 high‐confidence circRNAs. A total of 2916 of these circRNAs are conserved across human, mouse, and pig, including four circRNAs (circFgfr2, circQrich1, circMettl9, and circCamta1) that were differentially expressed (|log2 fold change| > 1 and adjusted P value < 0.05) in various myogenesis systems. We further focused on a conserved circRNA produced from the fibroblast growth factor receptor 2 (Fgfr2) gene, termed circFgfr2, which was found to inhibit myoblast proliferation and promote differentiation and skeletal muscle regeneration. Mechanistically, circFgfr2 acted as a sponge for miR‐133 to regulate the mitogen‐activated protein kinase kinase kinase 20 (Map3k20) gene and JNK/MAPK pathway. Importantly, transcription factor Kruppel like factor 4 (Klf4), the downstream target of the JNK/MAPK pathway, directly bound to the promoter of circFgfr2 and affected its expression via an miR‐133/Map3k20/JNK/Klf4 auto‐regulatory feedback loop. RNA binding protein G3BP stress granule assembly factor 1 (G3bp1) inhibited the biogenesis of circFgfr2. Conclusions The present study provides a comprehensive circRNA resource for skeletal muscle study. The functional and mechanistic analysis of circFgfr2 uncovered a circRNA‐mediated auto‐regulatory feedback loop regulating myogenesis and muscle regeneration, which provides new insight to further understand the regulatory mechanism of circRNAs.

MeiShan and Large White pigs differ in their female fecundity. However, the mechanisms behind the gene expression and regulation that cause these differences remain unclear. In this study, we profiled circRNAs and identified 5,879 circRNAs from the ovaries of MeiShan and Large White pigs. Eighty-five circRNAs were differentially expressed between the two pig breeds. Of these, 37 were up-regulated and 48 were down-regulated in MeiShan pigs. Gene ontology enrichment analysis suggested that the differentially expressed circRNA were involved in the hormone-mediated signaling pathway. We verified that circSCIN and its parent gene, scinderin (SCIN), were differentially expressed by reverse transcription and quantitative PCR (RT-qPCR). Luciferase assays demonstrated that circSCIN can target and sponge miR-133 and miR-148a/b. The identification of differentially expressed circRNAs (DECs) and their regulatory functions increased our understanding of the differences in reproductive efficiency between MeiShan and Large White pigs.

Intestinal microbiota significantly influences the intake, storage, and utilization of body nutrients, as well as animal growth and development. The establishment of microbiota is affected by many factors, such as delivery and feeding modes, antibiotics, disease, and the surrounding environment. In this study, we selected Chinese indigenous Mashen and Jinfen White pigs as the study subjects. To explore the source and factors affecting the piglet intestinal microbiota, 16S rRNA gene sequencing was performed to analyze the microbial composition of the feces, saliva, vaginal secretions, and colostrum of parturient sows, feces and saliva of newborn piglets, and surrounding environment samples. The results showed that the microbiota of the saliva of sows and piglets is structurally similar to that of the environment and is dominated by the phylum Proteobacteria, including Acinetobacter, Actinomyces, and Pseudomonas. The core genus in the vaginal secretions and colostrum of sows was Pseudomonas. Among the fecal samples, the core bacterial genera in sows before and after delivery were Clostridium sensu_stricto_1 and Christensenellaceae_R-7_group, while in piglets at 1 d of age, Pseudomonas and Escherichia-Shigella were most abundant. These results indicate that microbiota in feces, colostrum, and vaginal secretions of sows more easily colonized piglet intestines through a symbiotic effect. The environmental and salivary microbiota could also affect the early colonization and succession of the intestinal microbiota of piglets to some extent. This study provides a theoretical basis for sow delivery protection and early nursing of piglets and background for the research and development of microbial agents to improve piglet intestinal health.

Intestinal microbiota can affect the intake, storage, and absorption of nutrients in the body, thereby greatly impacting the growth and development of animals. In addition to diet, the breed and growth stages of pigs could also affect changes in the intestinal microbiota. However, research on the developmental changes in the ileum microbiota of piglets remains unclear. In this study, the ileum microbiota of Jinfen White and Mashen piglets at different developmental stages were investigated using 16S rRNA sequencing. Physiologically, the villus height of the ileum decreased, and the crypt depth increased during the development of the two pig breeds. Additionally, the serum antioxidant factors in the Jinfen White piglets were significantly higher than in the Mashen piglets at the end of the nursing stage. A total of 690 operational taxonomic units (OTUs) belonging to 21 phyla and 286 genera were identified, of which Firmicutes and Proteobacteria were the dominant phyla during the development of both the Jinfen White and Mashen piglets, accounting for ∼90% of all OTUs. Further research revealed differences in dominant bacteria between the two breeds. With increasing age, the ileum microbial diversity increased, and in both the pig breeds, the proportion of Firmicutes increased, whereas the proportion of Proteobacteria decreased. Additionally, different samples were characterized by specific genera, and different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were predicted at certain developmental stages. Finally, the correlation between the ileum microbiota and physiological features was analyzed, and it was suggested that the host and environmental factors play important roles in the formation of the microbial community structure in piglets. In summary, we delineated the structure, function, and differences in ileum microbiota between Jinfen White and Mashen piglets during different growth stages. This study helps to understand the development of the intestinal microbiota in local and hybrid pig breeds.

Objective: In this study, we aimed to identify long non-coding RNAs (lncRNAs) that play important roles in starvation stress, analyze their functions, and discover potential molecular targets to alleviate starvation stress, to provide a theoretical reference for subsequent in-depth research. Methods:We generated a piglet hunger stress animal model. Nine Yorkshire weaned piglets were randomly divided into a long-term hunger stress group (starved for 72 h), short-term hunger stress group (starved for 48 h), and the control group. LncRNA libraries were constructed using high-throughput sequencing of piglet ileums. Results:We obtained 11,792 lncRNAs, among which, 2500 lncRNAs were novel. In total, 509 differentially expressed (DE)lncRNAs were identified in this study. Target genes of DElncRNAs were predicted via cis and trans interactions, and functional and pathway analyses were performed. Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that lncRNA-targeted genes mainly participated in metabolic pathways, cellular processes, immune system processes, digestive systems, and transport activities. To reveal the mechanism underlying starvation stress, the interaction network between lncRNAs and their targets was constructed based on 26 DElncRNAs and 72 DE mRNAs. We performed an interaction network analysis of 121 DELncRNA-DEmRNA pairs with a Pearson correlation coefficient greater than 0.99.

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