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ABSTRACT: Proteins are considered major effectors of sperm function. However, the proteins expressed in pigeon sperm have not been explored. Here, we collected semen from meat and racing pigeons using the electroejaculation method and identified proteins in pigeon sperm using the proteomics approach. A total of 1,641 proteins were identified in the sperm of domesticated pigeons. Of which, 1,541 proteins were reliably quantified, and gene ontology (GO) and associated bioinformatics analyses indicated that annotated proteins were linked to the oxidation-reduction process, integral component membrane, and protein binding, etc. Among quantified proteins, 1,515 and 1,507 proteins were respectively presented in White King pigeons and racing pigeons, and 1,481 proteins were shared between these 2 types of pigeons, including axonemal dynein, solute carrier, cilia- and flagella-associated protein, outer dense fiber protein, etc. Proteins in our constructed protein-protein interaction (PPI) network are involved in oxidative phosphorylation, sperm axoneme assembly, cilium-dependent cell motility, axonemal dynein complex assembly, flagellated sperm motility, etc. In conclusion, this study characterized the sperm proteome of pigeons and provided a foundation for the subsequent research screening markers for fertility evaluation of pigeons.

Abstract In this study, exosomes from cooked meat were extracted by ultra-high-speed centrifugation. Approximately 80% of exosome vesicles were within 20–200 nm. In addition, the surface biomarkers of isolated exosomes were evaluated using flow cytometry. Further studies showed the exosomal microRNA profiles were different among cooked porcine muscle, fat and liver. Cooked pork-derived exosomes were chronically administered to ICR mice by drinking for 80 days. The mice plasma levels of miR-1, miR-133a-3p, miR-206 and miR-99a were increased to varying degrees after drinking exosome enriched water. Furthermore, GTT and ITT results confirmed an abnormal glucose metabolism and insulin resistance in mice. Moreover, the lipid droplets were significantly increased in the mice liver. A transcriptome analysis performed with mice liver samples identified 446 differentially expressed genes (DEGs). Functional enrichment analysis found that DEGs were enriched in metabolic pathways. Overall, the results suggest that microRNAs derived form cooked pork may function as a critical regulator of metabolic disorder in mice.

Objective Pigs, an ideal biomedical model for human diseases, suffer from about 50% early embryonic and fetal death, a major cause of fertility loss worldwide. However, identifying the causal variant remains a huge challenge. This study aimed to detect single nucleotide polymorphisms (SNPs) and candidate genes for the number of mummified (NM) piglets using the imputed whole-genome sequence (WGS) and validate the potential candidate genes. Methods The imputed WGS was introduced from genotyping-by-sequencing (GBS) using a multi-breed reference population. We performed genome-wide association studies (GWAS) for NM piglets at birth from a Landrace pig populatiGWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase on. A total of 300 Landrace pigs were genotyped by GBS. The whole-genome variants were imputed, and 4,252,858 SNPs were obtained. Various molecular experiments were conducted to determine how the genes affected NM in pigs. Results A strong GWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase 8 (CDK8) gene, which plays a crucial role in embryonic retardation and lethality. Based on the molecular experiments, we found that Y-box binding protein 1 (YBX1) was a crucial transcription factor for CDK8, which mediated the effect of CDK8 in the proliferation of porcine ovarian granulosa cells via transforming growth factor beta/small mother against decapentaplegic signaling pathway, and, as a consequence, affected embryo quality, indicating that this pathway may be contributing to mummified fetal in pigs. Conclusion A powerful imputation-based association study was performed to identify genes associated with NM in pigs. CDK8 was suggested as a functional gene for the proliferation of porcine ovarian granulosa cells, but further studies are required to determine causative mutations and the effect of loci on NM in pigs.

Objective Investigation of muscle growth at different developmental stages is an appropriate strategy for studying the mechanisms underlying muscle development and differences in phenotypes. In particular, the muscle development mechanisms and the difference between the fastest and slowest growth rates. Methods In this study, we used a growth curve model to fit the growth inflection point (IP) of QingYu pigs and compared differences in the long non-coding RNA (lncRNA) transcriptome of muscle both at the growth IP and plateau phase (PP). Results The growth curve of the QingYu pig had a good fit (R2 = 0.974) relative to a typical S-curve and reached the IP at day 177.96. At the PP, marbling, intramuscular fat, and monounsaturated fatty acids had increased significantly and the percentage of lean muscle and polyunsaturated fatty acids had decreased. A total of 1,199 mRNAs and 62 lncRNAs were differentially expressed at the IP compared with the PP. Additional to gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses, these differentially expressed protein coding genes were principally related to muscle growth and lipid metabolism. Conclusion Our results suggest that the identified differentially expressed lncRNAs, could play roles in muscle growth, fat deposition and regulation of fatty acid composition at the IP and PP.

Pingxian Wu and Kai Wang et al. perform single-locus and haplotype-based GWAS in Yorkshire pigs to evaluate genetic factors involved in socially affected traits, like feed intake and weight gain. These results indicate several genetic factors that may ultimately be targeted to improve livestock breeding.

The bursa of Fabricius (BF) is the critical humoral immune organ to birds, playing an essential role in B lymphocyte differentiation. However, unlike other poultries, surgical removal of pigeon BF did not limit humoral immune responsiveness. To investigate the expression profiles and the potential role of mRNA and long non-coding RNA (LncRNA) in squab BFs, transcriptome analysis was performed by RNA-Sequencing (RNA-Seq) over three developmental stages (1-day, 13 and 26 days old). We identified 13,072 mRNAs and 19,129 lncRNAs, of which 2,752 mRNAs and 1,515 lncRNAs were differential expressed (DE) in pigeon BFs over three developmental stages. Cluster analysis presented different expression patterns in DE mRNAs and lncRNAs. Functional enrichment analysis revealed that DE lncRNAs and mRNAs with distinct expression patterns might play crucial roles in the immune system process and tissue morphogenesis. In particular, some DE genes and lncRNAs with higher expression levels in 13D or 26D are related to lymphocyte activation and differentiation, adaptive immune response, positive regulation of immune response, leukocyte migration, etc. Protein-protein interaction (PPI) network and Molecular Complex Detection (MCODE) analysis sreened six significant modules containing 37 genes from immune-related DE gene cluster, which is closely linked in B cell activation, lymphocyte differentiation, B cell receptor signaling pathway, etc. Our study characterizes mRNA and lncRNA transcriptomic variability in pigeon BFs over different developmental stages and enhances understanding of the mechanisms underlying physiological functions of pigeon BF.

ObjectiveWe present a case of autoimmune cerebellar ataxia (ACA) associated with Homer protein homolog 3 (Homer-3) antibodies. Then, a review of the literature was conducted to summarize its clinical spectrum to improve clinicians' understanding of this rare entity.Case presentationA 25-year-old man suffered from the subacute onset of cerebellar ataxia and psychiatric symptoms with abnormalities in the cerebellum on initial brain MRI and Homer-3 antibodies titers of 1:100 in the serum. His neurological symptoms did not improve after intravenous methylprednisolone but significantly improved following plasma exchange with a modified Rankin Scale (mRS) score of 1. However, 5 months later, he experienced relapse during oral prednisone tapering with enhanced cerebellar lesions and obvious cerebellar atrophy on repeated MRI. Various immunomodulatory approaches, including corticosteroids and plasma exchange, were utilized with no improvement. Then rituximab was given for the first time to treat Homer-3 autoimmunity with partial improvement of symptoms. However, the patient remained profoundly disabled with an mRS score of 4.ConclusionACA associated with Homer-3 antibodies may have a suboptimal response to corticosteroid therapy. More intense immunotherapy such as rituximab may contribute to the improvement of cerebellar syndrome. Relapsing courses and presentation of cerebellar atrophy may suggest a poor prognosis in this entity.

The liver is the central organ for metabolism and influence the growth and development of the animals. To date, little is known about the microRNA (miRNA) in pigeon livers, particularly in different developmental stages. A comprehensive investigation into miRNA transcriptomes in livers across 3 pigeon developmental stages (1, 14, 28 d old) and an adult stage (2 y old) was performed by small RNA sequencing. We identified 312 known miRNA, 433 conserved miRNA, and 192 novel miRNA in pigeon livers. A set of differentially expressed (DE) miRNA in livers were screened out during pigeon development. This set of miRNA might be involved in hepatospecific phenotype and liver development. A Short Time-series Expression Miner analysis indicated significant expression variations in DE miRNA during liver development of pigeons. These DE miRNA with different expression patterns might play essential roles in response to growth factor, cell morphogenesis, and gland development, etc. Protein–protein interaction network and Molecular Complex Detection analysis identified several vital target genes (e.g., TNRC6B, FRS2, PTCH1, etc.) of DE miRNA, which is closely linked in liver development and enriched in PI3K cascade and regulation of growth. Our results expanded the repertoire of pigeon miRNA and may be of help in better understanding the mechanism of squab's rapid development from the perspective of liver development.

Epigenetic factors, including non-coding RNA regulation, play a vital role in the development of obesity and have been well researched. Transfer RNA-derived small RNA (tsRNA) is a class of non-coding RNA proven to be involved in various aspects of mammalian biology. Here we take pigs as a model for obesity research and use tsRNA-seq to investigate the difference in tsRNA expression in the subcutaneous adipose tissue of obese and lean pigs to elucidate the role of tsRNA in obesity development. A total of 482 tsRNAs were identified in pig adipose tissue, of which 123 were significantly differentially accumulated tsRNAs compared with the control group. The tRF-5c was the main type of these tsRNAs. The largest number of tsRNAs produced was the Gly-carrying tRNA, which produced 81 tsRNAs. Functional enrichment analysis revealed that differential tsRNAs indirectly participated in MAPK, AMPK, insulin resistance, the TNF signaling pathway, adipocytokine signaling pathway, and other signaling pathways by interacting with target genes. These are involved in bioenergetic metabolic regulatory processes, suggesting that tsRNAs may influence these pathways to mediate the regulation of energy metabolism in porcine adipocytes to promote lipid deposition, thus contributing to obesity. Our findings suggest a potential function of tsRNA in regulating obesity development.

This study aimed to identify biomarkers for pork quality evaluation. Firstly, the correlation between indicators of pork quality evaluation was investigated. The pH of pork meat at 45 min post slaughter showed a significant negative correlation with meat color indicators (r: −0.4868–−0.3040). Subsequently, porcine plasma samples were further divided into low pH (pH = 6.16 ± 0.22) or high pH (pH = 6.75 ± 0.08) groups. Plasma metabolites in both sample groups were investigated using untargeted metabolomics. In total, 90 metabolites were recognized as differential metabolites using partial least squares discriminant analysis. Pathway enrichment analysis indicated these differential metabolites were enriched in amino acid metabolism and energy metabolism. Correlation analysis revealed that creatinine, L-carnitine, D-sphingosine, citraconic acid, and other metabolites may constitute novel plasma biomarkers with the pH value of pork meat. The current study provides important insights into plasma biomarkers for predicting pork quality based on pH value.

The breast muscle is essential for flight and determines the meat yield and quality of the meat type in pigeons. At present, studies about long non-coding RNA (lncRNA) expression profiles in skeletal muscles across the postnatal development of pigeons have not been reported. Here, we used transcriptome sequencing to examine the White-King pigeon breast muscle at four different ages (1 day, 14 days, 28 days, and 2 years old). We identified 12,918 mRNAs and 9158 lncRNAs (5492 known lncRNAs and 3666 novel lncRNAs) in the breast muscle, and 7352 mRNAs and 4494 lncRNAs were differentially expressed in the process of development. We found that highly expressed mRNAs were mainly related to cell-basic and muscle-specific functions. Differential expression and time-series analysis showed that differentially expressed genes were primarily associated with muscle development and functions, blood vessel development, cell cycle, and energy metabolism. To further predict the possible role of lncRNAs, we also conducted the WGCNA and trans/cis analyses. We found that differentially expressed lncRNAs such as lncRNA-LOC102093252, lncRNA-G12653, lncRNA-LOC110357465, lncRNA-G14790, and lncRNA-LOC110360188 might respectively target UBE2B, Pax7, AGTR2, HDAC1, Sox8 and participate in the development of the muscle. Our study provides a valuable resource for studying the lncRNAs and mRNAs of pigeon muscles and for improving the understanding of molecular mechanisms in muscle development.

MicroRNAs (miRNAs) are a group of crucial regulators in the process of animal growth and development. However, little is known about the expression and function of miRNAs in pigeon muscles. To identify the miRNAs participating in the rapid development of pigeon pectoral muscles and quantitate their expression levels of pectoral muscles in different age stages, we performed miRNA transcriptome analysis in pigeon pectoral muscles by sequencing small RNAs over three different age stages (1-day old, 28 days old, and 2 years old). Dual-luciferase reporter assay was applied to validate the interaction between miRNA and its target gene. We identified 304 known miRNAs, 201 conserved miRNAs, and 86 novel miRNAs in pigeon pectoral muscles. 189 differentially expressed (DE) miRNAs were screened out during pigeon development. A short time-series expression miner (STEM) analysis indicated 89 DE miRNAs were significantly clustered in a progressively decreasing expression profile, and mainly enriched in biosynthesis-related GO categories and signaling pathways for MAPK and TGF-β. Dual-luciferase reporter assay indicated that a progressively down-regulated miRNA (miR-20b-5p) could directly target Krüppel-like factor 3 (KLF3) gene. To sum-up, our data expand the repertoire of pigeon miRNAs and enhance understanding of the mechanisms underlying rapid development in squabs.

Abstract Background The number of animals born dead, which includes the number of mummified (NM) and stillborn (NS) animals, is the most important trait to directly quantify the reproductive loss in domestic pigs. In this study, 282 Landrace sows and 250 Large White sows were genotyped by sequencing (GBS). A total of 816 and 1068 litter records for NM and NS were collected from them. A genome-wide association study (GWAS) was conducted to reveal the genetic difference between NM and NS. Results A total of 248 and 10 genome-wide significant SNPs were detected for NM and NS across numerous parities in Landrace pigs. The corresponding numbers for Large White pigs were 175 and 6, respectively. All of the detected SNPs were parity specific for both NM and NS in two breeds. Based on significant SNPs, in total 242 (146 for Landrace pig, 96 for Large White pig) and 10 significant chromosome regions (8 for Landrace pigs, 2 for Large White pigs) were found for NM and NS, respectively. Among them, 237 (142 for Landrace pig, 95 for Large White pig) and 8 significant chromosome regions (6 for Landrace pigs, 2 for Large White pigs) for NM and NS were not reported in previous studies. A list of candidate genes at the identified loci was proposed, including HMGB1, SOX5, KCNJ8, ABCC9 and YY1 for NM, ASTN1 for NS. Conclusion This is the first time when GBS data was used to identify genetic regions affecting NM and NS in Landrace and Large White pigs. Many identified informative SNPs and candidate genes advance our understanding of the genetic architecture of NM and NS in pigs. However, further studies are needed to validate using larger populations with more breeds.

Abstract Background Pigeon crop has the unique ability to produce a nutrient rich substance termed pigeon ‘milk’ (PM), which has functional resemblance with the mammalian milk. Previous researches have demonstrated that a large number of exosomes and exosomal miRNAs exist in mammalian milk, and many of them are associated with immunity, growth and development. However, to date, little is known about the exosomes and exosomal miRNAs in PM. Results In this study, we isolated the exosomes from PM and used small RNA sequencing to investigate the distribution and expression profiles of exosomal miRNAs. A total of 301 mature miRNAs including 248 conserved and 53 novel miRNAs were identified in five lactation stages i.e. 1d, 5d, 10d, 15d, and 20d. From these, four top 10 conserved miRNAs (cli-miR-21-5p, cli-miR-148a-3p, cli-miR-10a-5p and cli-miR-26a-5p) were co-expressed in all five stages. We speculate that these miRNAs may have important role in the biosynthesis and metabolism of PM. Moreover, similar to the mammalian milk, a significant proportion of immune and growth-related miRNAs were also present and enriched in PM exosomes. Furthermore, we also identified 41 orthologous miRNAs group (giving rise to 81 mature miRNA) commonly shared with PM, human, bovine and porcine breast milk. Additionally, functional enrichment analysis revealed the role of exosomal miRNAs in organ development and in growth-related pathways including the MAPK, Wnt and insulin pathways. Conclusions To sum-up, this comprehensive analysis will contribute to a better understanding of the underlying functions and regulatory mechanisms of PM in squabs.

Betaine is a natural compound present in commonly consumed foods and may have a potential role in the regulation of glucose and lipids metabolism. However, the underlying molecular mechanism of its action remains largely unknown. Here, we show that supplementation with betaine contributes to improved high-fat diet (HFD)-induced gut microbiota dysbiosis and increases anti-obesity strains such as Akkermansia muciniphila, Lactobacillus, and Bifidobacterium. In mice lacking gut microbiota, the functional role of betaine in preventing HFD-induced obesity, metabolic syndrome, and inactivation of brown adipose tissues are significantly reduced. Akkermansia muciniphila is an important regulator of betaine in improving microbiome ecology and increasing strains that produce short-chain fatty acids (SCFAs). Increasing two main members of SCFAs including acetate and butyrate can significantly regulate the levels of DNA methylation at host miR-378a promoter, thus preventing the development of obesity and glucose intolerance. However, these beneficial effects are partially abolished by Yin yang (YY1), a common target gene of the miR-378a family. Taken together, our findings demonstrate that betaine can improve obesity and associated MS via the gut microbiota-derived miR-378a/YY1 regulatory axis, and reveal a novel mechanism by which gut microbiota improve host health.

High-altitude acclimatization is a representative example of vertebrates’ acclimatization to harsh and extreme environments. Previous studies reported sufficient evidence for a molecular genetic basis of high-altitude acclimatization, and genomic patterns of genetic variation among populations and species have been widely elucidated in recent years. However, understanding of the miRNA role in high-altitude acclimatization have lagged behind, especially in non-model species. To investigate miRNA expression alterations of goats that were induced by high-altitude stress, we performed comparative miRNA transcriptome analysis on six hypoxia-sensitive tissues (heart, kidney, liver, lung, skeletal muscle, and spleen) in two goat populations from distinct altitudes (600 and 3000 m). We obtained the expression value of 1391 mature miRNAs and identified 138 differentially expressed (DE) miRNAs between high and low altitudes. Combined with tissue specificity analysis, we illustrated alterations of expression levels among altitudes and tissues, and found that there were coexisting tissue-specific and -conserved mechanisms for hypoxia acclimatization. Notably, the interplay between DE miRNA and DE target genes strongly indicated post-transcriptional regulation in the hypoxia inducible factor 1, insulin, and p53 signaling pathways, which might play significant roles in high-altitude acclimatization in domestic goats. It’s also worth noting that we experimentally confirmed miR-106a-5p to have a negative regulation effect on angiogenesis by directly targeting FLT-1. These results provide insight into the complicated miRNA expression patterns and regulatory mechanisms of high-altitude acclimatization in domestic goats.

The whole-genome sequencing (WGS) data can potentially discover all genetic variants. Studies have shown the power of WGS for genome-wide association study (GWAS) lies in the ability to identify quantitative trait loci and nucleotides (QTNs). However, the resequencing of thousands of target individuals is expensive. Genotype imputation is a powerful approach for WGS and to identify causal mutations. This study aimed to evaluate the imputation accuracy from genotyping-by-sequencing (GBS) to WGS in two pig breeds using a resequencing reference population and to detect single-nucleotide polymorphisms (SNPs) and candidate genes for farrowing interval (FI) of different parities using the data before and after imputation for GWAS. Six hundred target pigs, 300 Landrace and 300 Large White pigs, were genotyped by GBS, and 60 reference pigs, 20 Landrace and 40 Large White pigs, were sequenced by whole-genome resequencing. Imputation for pigs was conducted using Beagle software. The average imputation accuracy (allelic R2) from GBS to WGS was 0.42 for Landrace pigs and 0.45 for Large White pigs. For Landrace pigs (Large White pigs), 4,514,934 (5,533,290) SNPs had an accuracy >0.3, resulting an average accuracy of 0.73 (0.72), and 2,093,778 (2,468,645) SNPs had an accuracy >0.8, resulting an average accuracy of 0.94 (0.93). Association studies with data before and after imputation were performed for FI of different parities in two populations. Before imputation, 18 and 128 significant SNPs were detected for FI in Landrace and Large White pigs, respectively. After imputation, 125 and 27 significant SNPs were identified for dataset with an accuracy >0.3 and 0.8 in Large White pigs, and 113 and 18 SNPs were found among imputed sequence variants. Among these significant SNPs, six top SNPs were detected in both GBS data and imputed WGS data, namely, SSC2: 136127645, SSC5: 103426443, SSC6: 27811226, SSC10: 3609429, SSC14: 15199253, and SSC15: 150297519. Overall, many candidate genes could be involved in FI of different parities in pigs. Although imputation from GBS to WGS data resulted in a low imputation accuracy, association analyses with imputed WGS data were optimized to detect QTNs for complex trait. The obtained results provide new insight into genotype imputation, genetic architecture, and candidate genes for FI of different parities in Landrace and Large White pigs.

Abstract The physiological role of miRNAs is widely understood to include fine-tuning the post-transcriptional regulation of a wide array of biological processes. Extensive studies have indicated that exosomal miRNAs in the bodily fluids of various organisms can be transferred between living cells for the delivery of gene silencing signals. Here, we illustrated the expression characteristics of exosomal miRNAs in giant panda breast milk during distinct lactation periods and highlighted the enrichment of immune- and development-related endogenous miRNAs in colostral and mature giant panda milk. These miRNAs are stable, even under certain harsh conditions, via the protection of extracellular vesicles. These findings indicate that breast milk may facilitate the dietary intake of maternal miRNAs by infants for the regulation of postnatal development. We also detected exogenous plant miRNAs from the primary food source of the giant panda (bamboo) in the exosomes of giant panda breast milk that were associated with regulatory roles in basic metabolism and neuron development. This result suggested that dietary plant miRNAs are absorbed by host cells and subsequently secreted into bodily fluids as potential cross-kingdom regulators. In conclusion, exosomal miRNAs in giant panda breast milk may be crucial maternal regulators for the development of intrinsic ‘slink’ newborn cubs.

Abstract MicroRNAs are a class of small RNAs that are important in post-transcriptional gene regulation in animals and plants. These single-stranded molecules are widely distributed in organisms and influence fundamental biological processes. Interestingly, recent studies have reported that diet-derived plant miRNAs could regulate mammalian gene expression, and these studies have broadened our view of cross-kingdom communication. In the present study, we evaluated miRNA levels in cooked maize-containing chow diets, and found that plant miRNAs were resistant to the harsh cooking conditions to a certain extent. After feeding fresh maize to pigs (7 days), maize-derived miRNAs could be detected in porcine tissues and serum, and the authenticity of these plant miRNAs was confirmed by using oxidization reactions. Furthermore, in vivo and in vitro experiments demonstrated that dietary maize miRNAs could cross the gastrointestinal tract and enter the porcine bloodstream. In the porcine cells, we found that plant miRNAs are very likely to specifically target their endogenous porcine mRNAs and influence gene expression in a fashion similar to that of mammalian miRNAs. Our results indicate that maize-derived miRNAs can cross the gastrointestinal tract and present in pigs, and these exogenous miRNAs have the potential to regulate mammalian gene expression.

Abstract Background N 6 -methyladenosine (m6A) is the most prevalent internal form of modification in messenger RNA in higher eukaryotes and potential regulatory functions of reversible m6A methylation on mRNA have been revealed by mapping of m6A methylomes in several species. m6A modification in active gene regulation manifests itself as altered methylation profiles in a tissue-specific manner or in response to changing cellular or species living environment. However, up to date, there has no data on m6A porcine transcriptome-wide map and its potential biological roles in adipose deposition and muscle growth. Methods In this work, we used methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) technique to acquire the first ever m6A porcine transcriptome-wide map. Transcriptomes of muscle and adipose tissues from three different pig breeds, the wild boar, Landrace, and Rongchang pig, were used to generate these maps. Results Our findings show that there were 5,872 and 2,826 m6A peaks respectively, in the porcine muscle and adipose tissue transcriptomes. Stop codons, 3′-untranslated regions, and coding regions were found to be mainly enriched for m6A peaks. Gene ontology analysis revealed that common m6A peaks in nuclear genes are associated with transcriptional factors, suggestive of a relationship between m6A mRNA methylation and nuclear genome transcription. Some genes showed tissue- and breed-differential methylation, and have novel biological functions. We also found a relationship between the m6A methylation extent and the transcript level, suggesting a regulatory role for m6A in gene expression. Conclusion This comprehensive map provides a solid basis for the determination of potential functional roles for RNA m6A modification in adipose deposition and muscle growth.

Gene transcripts or mRNAs and long noncoding RNAs (lncRNAs) are differentially expressed during porcine skeletal muscle development. However, only a few studies have been conducted on skeletal muscle transcriptome in pigs based on timepoints according to the growth curve for porcine. Here, we investigated gene expression in Qingyu pigs at three different growth stages: the inflection point with the maximum growth rate (MGI), the inflection point of the gradually increasing stage to the rapidly increasing stage (GRI), and the inflection point of the rapidly increasing stage to the slowly increasing stage (RSI). Subsequently, we explored gene expression profiles during muscle development at the MGI, GRI and RSI stages by Ribo-Zero RNA sequencing. Qingyu pigs reached the MGI, GRI and RSI stages at 156.40, 23.82 and 288.97 days of age with 51.73, 3.14 and 107.03 kg body weight, respectively. A total of 14,530 mRNAs and 11,970 lncRNAs were identified at the three stages, and 645, 323 differentially expressed genes (DEGs) and 696, 760 differentially expressed lncRNAs (DELs) were identified in the GRI vs. MGI, and RSI vs. MGI, comparisons. Functional enrichment analysis revealed that genes involved in immune system development and energy metabolism (mainly relate to amino acid, carbohydrate and lipid) were enriched at the GRI and MGI stages, respectively, whereas genes involved in lipid metabolism were enriched at the RSI stage. We further characterized G1430, an abundant lncRNA. The full-length sequence (316 nt) of lncRNA G1430 was determined by rapid amplification of cDNA ends (RACE). Subcellular distribution analysis by quantitative real-time PCR (qRT-PCR) revealed that G1430 is a cytoplasmic lncRNA. Binding site prediction and dual luciferase assay showed that lncRNA G1430 directly binds to microRNA 133a (miR-133a). Our findings provide the basis for further investigation of the regulatory mechanisms and molecular genetics of muscle development in pigs.

The domestic pigs have been undergone intense selection pressures for these development of interested traits following domestication and modern breeding. This has altered many traits in most of pig breeds, such as growth rate, body weight, fertility, and immunity. Thus, the objectives of this study were to (1) detect these selection signatures and identify the candidate genes that show evidences of recent artificial selection at the level of whole genome, (2) be beneficial to understand the relationship between genomic structure and phenotypic diversity, and (3) highlight the key roles of these candidate genes in growth and development in the two breeds. The data consisted of total raw number of 345570 single nucleotide polymorphisms (SNPs) in 1200 individuals from the Chinese Landrace pigs (L, n = 600) and Yorkshire pigs (Y, n = 600). Based on these SNPs data, two complementary methods, population differentiation (Fst) and composite likelihood ratio test (CLR), were carried out to detect the selection signatures in this study. A total of 540 potential selection regions (50 kb) which contained 111 candidate genes were detected for Landrace-Yorkshire pair (L-Y) by Fst. In addition, 73 and 125 candidate genes were found for Landrace pigs and Yorkshire pigs by CLR test based on 321 and 628 potential selection regions, respectively. Some candidate genes are associated with important traits and signaling pathways including the ACACA, MECR, COL11A1, GHR, IGF1R, IGF2R, IFNG, and MTOR gene. The ACACA and MECR gene are related to fatty acid biosynthesis. The COL11A1 gene is essential for the development of the normal differentiation. The GHR, IGF1R, and IGF2R gene are significant candidate genes which play major roles in the growth and development in animals. The IFNG gene is associated with some aspects of immune response. The MTOR gene regulates many signaling pathways and signaling transduction pathway.

Genome-wide transcriptomic studies in humans and mice have become extensive and mature. However, a comprehensive and systematic understanding of protein-coding genes and long non-coding RNAs (lncRNAs) expressed during pig spleen development has not been achieved. LncRNAs are known to participate in regulatory networks for an array of biological processes. Here, we constructed 18 RNA libraries from developing fetal pig spleen (55 days before birth), postnatal pig spleens (0, 30, 180 days and 2 years after birth), and the samples from the 2-year-old Wild Boar. A total of 15,040 lncRNA transcripts were identified among these samples. We found that the temporal expression pattern of lncRNAs was more restricted than observed for protein-coding genes. Time-series analysis showed two large modules for protein-coding genes and lncRNAs. The up-regulated module was enriched for genes related to immune and inflammatory function, while the down-regulated module was enriched for cell proliferation processes such as cell division and DNA replication. Co-expression networks indicated the functional relatedness between protein-coding genes and lncRNAs, which were enriched for similar functions over the series of time points examined. We identified numerous differentially expressed protein-coding genes and lncRNAs in all five developmental stages. Notably, ceruloplasmin precursor (CP), a protein-coding gene participating in antioxidant and iron transport processes, was differentially expressed in all stages. This study provides the first catalog of the developing pig spleen, and contributes to a fuller understanding of the molecular mechanisms underpinning mammalian spleen development.

Extensive and in-depth investigations of high-altitude adaptation have been carried out at the level of morphology, anatomy, physiology and genomics, but few investigations focused on the roles of microRNA (miRNA) in high-altitude adaptation. We examined the differences in the miRNA transcriptomes of two representative hypoxia-sensitive tissues (heart and lung) between yak and cattle, two closely related species that live in high and low altitudes, respectively. In this study, we identified a total of 808 mature miRNAs, which corresponded to 715 pre-miRNAs in the two species. The further analysis revealed that both tissues showed relatively high correlation coefficient between yak and cattle, but a greater differentiation was present in lung than heart between the two species. In addition, miRNAs with significantly differentiated patterns of expression in two tissues exhibited co-operation effect in high altitude adaptation based on miRNA family and cluster. Functional analysis revealed that differentially expressed miRNAs were enriched in hypoxia-related pathways, such as the HIF-1α signaling pathway, the insulin signaling pathway, the PI3K-Akt signaling pathway, nucleotide excision repair, cell cycle, apoptosis and fatty acid metabolism, which indicated the important roles of miRNAs in high altitude adaptation. These results suggested the diverse degrees of miRNA transcriptome variation in different tissues between yak and cattle, and suggested extensive roles of miRNAs in high altitude adaptation.

MicroRNAs (miRNAs) are small non-coding RNAs that can regulate their target genes at the post-transcriptional level. Skeletal muscle comprises different fiber types that can be broadly classified as red, intermediate, and white. Recently, a set of miRNAs was found expressed in a fiber type-specific manner in red and white fiber types. However, an in-depth analysis of the miRNA transcriptome differences between all three fiber types has not been undertaken. Herein, we collected 15 porcine skeletal muscles from different anatomical locations, which were then clearly divided into red, white, and intermediate fiber type based on the ratios of myosin heavy chain isoforms. We further illustrated that three muscles, which typically represented each muscle fiber type (i.e., red: peroneal longus (PL), intermediate: psoas major muscle (PMM), white: longissimus dorsi muscle (LDM)), have distinct metabolic patterns of mitochondrial and glycolytic enzyme levels. Furthermore, we constructed small RNA libraries for PL, PMM, and LDM using a deep sequencing approach. Results showed that the differentially expressed miRNAs were mainly enriched in PL and played a vital role in myogenesis and energy metabolism. Overall, this comprehensive analysis will contribute to a better understanding of the miRNA regulatory mechanism that achieves the phenotypic diversity of skeletal muscles.

Intermuscular adipose tissue is located between the muscle fiber bundles in skeletal muscles, and has similar metabolic features to visceral adipose tissue, which has been found to be related to a number of obesity-related diseases. Although various miRNAs are known to play crucial roles in adipose deposition and adipogenesis, the microRNA transcriptome of intermuscular adipose tissue has not, until now, been studied. Here, we sequenced the miRNA transcriptomes of porcine intermuscular adipose tissue by small RNA-sequencing and compared it to a representative subcutaneous adipose tissue. We found that the inflammation- and diabetes-related miRNAs were significantly enriched in the intermuscular rather than in the subcutaneous adipose tissue. A functional enrichment analysis of the genes predicted to be targeted by the enriched miRNAs also indicated that intermuscular adipose tissue was associated mainly with immune and inflammation responses. Our results suggest that the intermuscular adipose tissue should be recognized as a potential metabolic risk factor of obesity.

Adipose tissue is not only a storage organ involved in fuel metabolism, but also an endocrine organ involved in the regulation of insulin sensitivity, thermogenesis, immunity, and inflammation. There are anatomical, cellular, molecular and physiological differences among adipose tissues deposited in different body sites. However, current understanding of the intrinsic differences between the sub-compartments of the subcutaneous adipose tissue remains rudimentary. Here, we analyzed the genome-wide DNA methylation differences between the porcine superficial and deep backfat tissues using methylated DNA immunoprecipitation combined with high-throughput sequencing. We show that the genes with differentially methylated regions in their promoter are mainly involved in the processes of “lipid metabolism” and “regulation of immune-related cytokines”. Compared with the deep backfat tissue, the promoters of genes related to the ‘positive regulation of cytokine production’ were significantly hypermethylated in the superficial backfat tissue, which reflects the intrinsic functional and metabolic differences between the sub-compartments of the subcutaneous adipose tissue. This study provides epigenetic evidence for functionally relevant methylation differences between different layers of porcine backfat tissues.

Excessive accumulation of adipose tissue is a main cause of obesity or overweight, which is significantly involved in increasing the risk of diseases. Recently, numerous studies have proved that microRNAs (miRNAs) play important roles in adipogenesis by negatively regulating gene expression at posttranscriptional levels. In this study, we showed that miR-125a-5p was expressed at lower levels in the adipose tissues of high-fat diet (HFD)-fed mice than the normal chow (NCW)-fed mice. MiR-125a-5p expression were strongly up-regulated by nearly five-fold, when 3T3-L1 preadipocyte were induced and differentiated into mature adipocytes. Functional analysis indicated that overexpression of miR-125a-5p promoted 3T3-L1 preadipocyte proliferation and inhibited its differentiation. By contrast, inhibition of miR-125a-5p repressed 3T3-L1 preadipocyte proliferation and accelerated its differentiation. Furthermore, a dual-luciferase reporter assay demonstrated that signal transducer and activator of transcription 3 (STAT3) is a direct target gene of miR-125a-5p during 3T3-L1 preadipocyte differentiation. Further analysis confirmed that the process of miR-125a-5p inhibiting 3T3-L1 preadipocyte differentiation might be associated with the regulation of fatty acid metabolism related genes. Taken together, our results indicated that miR-125a-5p might promote 3T3-L1 preadipocyte proliferation, whereas inhibiting 3T3-L1 preadipocyte differentiation by negatively regulating STAT3.

Cebpa is a master transcription factor gene for adipogenesis. However, the mechanisms of enhancer-promoter chromatin interactions controlling Cebpa transcriptional regulation during adipogenic differentiation remain largely unknown. To reveal how the threedimensional structure of Cebpa changes during adipogenesis, we generated highresolution chromatin interactions of Cebpa in 3T3-L1 preadipocytes and 3T3-L1 adipocytes using circularized chromosome conformation capture sequencing (4C-seq). We revealed dramatic changes in chromatin interactions and chromatin status at interaction sites during adipogenic differentiation. Based on this, we identified five active enhancers of Cebpa in 3T3-L1 adipocytes through epigenomic data and luciferase reporter assays. Next, epigenetic repression of Cebpa-L1-AD-En2 or -En3 by the dCas9-KRAB system significantly down-regulated Cebpa expression and inhibited adipocyte differentiation. Furthermore, experimental depletion of cohesin decreased the interaction intensity between Cebpa-L1-AD-En2 and the Cebpa promoter and down-regulated Cebpa expression, indicating that long-range chromatin loop formation was mediated by cohesin. Two transcription factors, RXRA and PPARG, synergistically regulate the activity of Cebpa-L1-AD-En2. To test whether Cebpa-L1-AD-En2 plays a role in adipose tissue development, we injected dCas9-KRAB-En2 lentivirus into the inguinal white adipose tissue (iWAT) of mice to suppress the activity of Cebpa-L1-AD-En2. Repression of Cebpa-L1-AD-En2 significantly decreased Cebpa expression and adipocyte size, altered iWAT transcriptome, and affected iWAT development. We identified functional enhancers regulating Cebpa expression and clarified the crucial roles of Cebpa-L1-AD-En2 and Cebpa promoter interaction in adipocyte differentiation and adipose tissue development. [ABSTRACT FROM AUTHOR]