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Abstract pH value is a crucial index used to evaluate pork quality due to its direct impact on specific meat characteristics. This study investigated the genetic mechanisms influencing pH values through measurements taken from the longissimus dorsi muscle of Beijing Black pigs at 2 h (pH2h) and 24 h (pH24h) postmortem. A total of 614 Beijing Black pigs were subsequently genotyped using the Illumina Porcine 50K SNP Chip. Heritability estimates for pH2h and pH24h were found to be 0.19 and 0.25, respectively, with a genetic correlation of 0.53. Furthermore, we conducted both a genome‐wide association study (GWAS) and an RNA sequencing (RNA‐seq) analysis, the latter of which identified differentially expressed genes (DEGs) between high and low pH groups. We identified 31, 6, and 32 single‐nucleotide polymorphisms in the pH2h, pH24h, and pH2–24h traits, respectively. The GWAS results revealed the presence of the SYT5 gene in both the pH2h and pH2–24h traits, while the SNX13 gene was simultaneously identified in the pH24h and pH2–24h traits. The RNA‐seq results also found SYT5 to be highly expressed, while SNX13 did not exhibit differential expression. Moreover, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses based on the DEGs revealed potential links between pH levels and the glycogen metabolic process as well as associations with the regulation of cell proliferation and calcium ion transmembrane transport. Ultimately, SYT5 and SNX13 emerged as key candidate genes affecting pH values at 2 and 24 h, respectively. These findings contribute to a better understanding of the genetic mechanisms affecting pork quality and safety and offer insights for enhancing meat quality through genetic improvement.

Abstract Background During approximately 10,000 years of domestication and selection, a large number of structural variations (SVs) have emerged in the genome of pig breeds, profoundly influencing their phenotypes and the ability to adapt to the local environment. SVs (≥ 50 bp) are widely distributed in the genome, mainly in the form of insertion (INS), mobile element insertion (MEI), deletion (DEL), duplication (DUP), inversion (INV), and translocation (TRA). While studies have investigated the SVs in pig genomes, genome-wide association studies (GWAS)-based on SVs have been rarely conducted. Results Here, we obtained a high-quality SV map containing 123,151 SVs from 15 Large White and 15 Min pigs through integrating the power of several SV tools, with 53.95% of the SVs being reported for the first time. These high-quality SVs were used to recover the population genetic structure, confirming the accuracy of genotyping. Potential functional SV loci were then identified based on positional effects and breed stratification. Finally, GWAS were performed for 36 traits by genotyping the screened potential causal loci in the F2 population according to their corresponding genomic positions. We identified a large number of loci involved in 8 carcass traits and 6 skeletal traits on chromosome 7, with FKBP5 containing the most significant SV locus for almost all traits. In addition, we found several significant loci in intramuscular fat, abdominal circumference, heart weight, and liver weight, etc. Conclusions We constructed a high-quality SV map using high-coverage sequencing data and then analyzed them by performing GWAS for 25 carcass traits, 7 skeletal traits, and 4 meat quality traits to determine that SVs may affect body size between European and Chinese pig breeds.

The local pig breeds in Guizhou possess exceptional meat quality, robust adaptability, and resilience to harsh feeding conditions, making them ideal for producing high-quality pork. With over 10 local pig breeds in the region, we focused on 7 specific breeds: Baixi pigs (BX), Congjiang Xiang pigs (CJX), Guanling pigs (GL), Jianhe White Xiang pigs (JHBX), Jiangkou Luobo pigs (JKLB), Kele pigs (KL), and Qiandong Hua pigs (QDH). Unfortunately, these breeds face threats such as introduced species and inbreeding, resulting in a decline in population size and numbers. To better protect and utilize these breeds, we employed genome-wide single-nucleotide polymorphism (SNP) markers to investigate the population structure, genetic diversity, and selection characteristics of 283 pigs across these seven breeds. Our findings revealed distinct ancestral sources between Chinese and Western pig breeds, as demonstrated by principal component analysis, adjacent tree analysis, and ADMIXTURE analysis. Notably, JHBX exhibited a distant genetic relationship from the other six local pig breeds in Guizhou province, showcasing unique genetic characteristics. While the genetic diversity of the six Chinese native pig populations, excluding JHBX, was generally moderate in Guizhou province, the JHBX population displayed low genetic diversity. Therefore, it is imperative to intensify selection efforts to prevent inbreeding decline in JHBX while further enhancing the protection measures for the other six pig populations. Additionally, we identified candidate genes influencing the size disparity among pigs in Guizhou province through signal selection. Our study outcomes serve as a reference for developing effective conservation and utilization plans for pig breeds in Guizhou province and deepen our understanding of the genetic mechanisms underlying pig body size.

Sows’ maternal behavior is important for improving piglet survival and growth; thus, breeding for good mothering sows is necessary for pig production. However, there is little research on the genetic mechanism of maternal behavior. In this study, a comparative analysis of piglets’ growth traits between good and bad maternal behavior groups and a genome-wide association study (GWAS) was performed to elucidate the impact of sows’ maternal behavior on piglet growth and identify candidate genes and markers of sow’s maternal behaviors. Comparing the growth traits of piglets between good and bad sows’ maternal behavior groups, the results showed that the growth traits of piglets from sows with good maternal behavior were better than those from sows with bad maternal behavior and especially for the multiparous sows group, this comparative difference was significant. For the intensive study of the genetic mechanisms of sows’ maternal behavior, a total of 452 sows were genotyped using the Illumina Porcine 50K SNP Chip, and 4 traits, including biting piglets (BP), crushing piglets (CP), trampling piglets (TP) and screaming test (ST), were examined. Using a GWAS, 20 single nucleotide polymorphisms (SNPs) were found to be associated with these traits. Within 1 Mb upstream and downstream of the significant SNPs screened, 138 genes were obtained. After pathway enrichment and gene annotation, HIP1, FZD9 and HTR7 were identified as important candidate genes affecting sows’ maternal behaviors. These findings preliminarily elucidate the genetic basis of sows’ maternal behavior traits and provide candidate genes and markers for molecular breeding in pigs.

Objective This study was conducted to identify the functional long non-coding RNAs (lncRNAs) for swine lactation by RNA-seq data of mammary gland. Methods According to the RNA-seq data of swine mammary gland, we screened lncRNAs, performed differential expression analysis, and confirmed the functional lncRNAs for swine lactation by validation of genome wide association study (GWAS) signals, functional annotation and weighted gene co-expression network analysis (WGCNA). Results We totally identified 286 differentially expressed (DE) lncRNAs in mammary gland at different stages from 14 days prior to (-) parturition to day 1 after (+) parturition, and the expressions of most of lncRNAs were strongly changed from day −2 to day +1. Further, the GWAS signals of sow milk ability trait were significantly enriched in DE lncRNAs. Functional annotation revealed that these DE lncRNAs were mainly involved in mammary gland and lactation developing, milk composition metabolism and colostrum function. By performing weighted WGCNA, we identified 7 out of 12 lncRNA-mRNA modules that were highly associated with the mammary gland at day −14, day −2, and day +1, in which, 35 lncRNAs and 319 mRNAs were involved. Conclusion This study suggested that 18 lncRNAs and their 20 target genes were promising candidates for swine parturition and colostrum occurrence processes. Our research provided new insights into lncRNA profiles and their regulating mechanisms from colostrogenesis to lactogenesis in swine.

Optimizing the construction and update strategies for reference and candidate populations is the basis of the application of genomic selection (GS). In this study, we first simulated1200-purebred-pigs population that have been popular in China for 20 generations to study the effects of different population sizes and the relationship between individuals of the reference and candidate populations. The results showed that the accuracy was positively correlated with the size of the reference population within the same generation (r = 0.9366, p < 0.05), while was negatively correlated with the number of generation intervals between the reference and candidate populations (r = −0.9267, p < 0.01). When the reference population accumulated more than seven generations, the accuracy began to decline. We then simulated the population structure of 1200 purebred pigs for five generations and studied the effects of different heritabilities (0.1, 0.3, and 0.5), genotyping proportions (20, 30, and 50%), and sex ratios on the accuracy of the genomic estimate breeding value (GEBV) and genetic progress. The results showed that if the proportion of genotyping individuals accounts for 20% of the candidate population, the traits with different heritabilities can be genotyped according to the sex ratio of 1:1male to female. If the proportion is 30% and the traits are of low heritability (0.1), the sex ratio of 1:1 male to female is the best. If the traits are of medium or high heritability, the male-to-female ratio is 1:1, 1:2, or 2:1, which may achieve higher genetic progress. If the genotyping proportion is up to 50%, for low heritability traits (0.1), the proportion of sows from all genotyping individuals should not be less than 25%, and for the medium and high heritability traits, the optimal choice for the male-to-female ratio is 1:1, which may obtain the greatest genetic progress. This study provides a reference for determining a construction and update plan for the reference population of breeding pigs.

Abstract Background The development of skeletal muscle in pigs during the embryonic stage is precisely regulated by transcriptional mechanisms, which depend on chromatin accessibility. However, how chromatin accessibility plays a regulatory role during embryonic skeletal muscle development in pigs has not been reported. To gain insight into the landscape of chromatin accessibility and the associated genome-wide transcriptome during embryonic muscle development, we performed ATAC-seq and RNA-seq analyses of skeletal muscle from pig embryos at 45, 70 and 100 days post coitus (dpc). Results In total, 21,638, 35,447 and 60,181 unique regions (or peaks) were found across the embryos at 45 dpc (LW45), 70 dpc (LW70) and 100 dpc (LW100), respectively. More than 91% of the peaks were annotated within − 1 kb to 100 bp of transcription start sites (TSSs). First, widespread increases in specific accessible chromatin regions (ACRs) from embryos at 45 to 100 dpc suggested that the regulatory mechanisms became increasingly complicated during embryonic development. Second, the findings from integrated ATAC-seq and RNA-seq analyses showed that not only the numbers but also the intensities of ACRs could control the expression of associated genes. Moreover, the motif screening of stage-specific ACRs revealed some transcription factors that regulate muscle development-related genes, such as MyoG, Mef2c, and Mef2d. Several potential transcriptional repressors, including E2F6, OTX2 and CTCF, were identified among the genes that exhibited different regulation trends between the ATAC-seq and RNA-seq data. Conclusions This work indicates that chromatin accessibility plays an important regulatory role in the embryonic muscle development of pigs and regulates the temporal and spatial expression patterns of key genes in muscle development by influencing the binding of transcription factors. Our results contribute to a better understanding of the regulatory dynamics of genes involved in pig embryonic skeletal muscle development.

Pig diseases seriously threaten the health of pigs and the benefits of pig production. Previous research has indicated that Chinese native pigs, such as the Min (M) pig, has a better disease resistance ability than Large White (LW) pigs. However, the molecular mechanism of this resistance is still unclear. In our study, we used serum untargeted metabolomics and proteomics, interrogated to characterize differences in the molecular immunities between six resistant and six susceptible pigs raised in the same environment. A total of 62 metabolites were identified as being significantly exhibited in M and LW pigs. Ensemble feature selection (EFS) machine learning methods were used to predict biomarkers of metabolites and proteins, and the top 30 were selected and retained. Weighted gene co-expression network analysis (WGCNA) confirmed that four key metabolites, PC (18:1 (11 Z)/20:0), PC (14:0/P-18: 0), PC (18:3 (6 Z, 9 Z, 12 Z)/16:0), and PC (16:1 (9 Z)/22:2 (13 Z, 16 Z)), were significantly associated with phenotypes, such as cytokines, and different pig breeds. Correlation network analysis showed that 15 proteins were significantly correlated with the expression of both cytokines and unsaturated fatty acid metabolites. Quantitative trait locus (QTL) co-location analysis results showed that 13 of 15 proteins co-localized with immune or polyunsaturated fatty acid (PUFA)-related QTL. Moreover, seven of them co-localized with both immune and PUFA QTLs, including proteasome 20S subunit beta 8 (PSMB8), mannose binding lectin 1 (MBL1), and interleukin-1 receptor accessory protein (IL1RAP). These proteins may play important roles in regulating the production or metabolism of unsaturated fatty acids and immune factors. Most of the proteins could be validated with parallel reaction monitoring, which suggests that these proteins may play an essential role in producing or regulating unsaturated fatty acids and immune factors to cope with the adaptive immunity of different pig breeds. Our study provides a basis for further clarifying the disease resistance mechanism of pigs.

Abstract Background Intramuscular fat (IMF) is associated with meat quality and insulin resistance in animals. Research on genetic mechanism of IMF decomposition has positive meaning to pork quality and diseases such as obesity and type 2 diabetes treatment. In this study, an IMF trait segregation population was used to perform RNA sequencing and to analyze the joint or independent effects of genes and long intergenic non-coding RNAs (lincRNAs) on IMF. Results A total of 26 genes including six lincRNA genes show significantly different expression between high- and low-IMF pigs. Interesting, one lincRNA gene, named IMF related lincRNA (IRLnc) not only has a 292-bp conserved region in 100 vertebrates but also has conserved up and down stream genes (

Alternative splicing (AS) is a key step in the post-transcriptional regulation of gene expression that can affect intramuscular fat (IMF). In this study, longissimus dorsi muscles from 30 pigs in high- and low- IMF groups were used to perform Oxford Nanopore Technologies (ONT) full-length sequencing and Illumina strand-specific RNA-seq. A total of 43,688 full-length transcripts were identified, with 4,322 novel genes and 30,795 novel transcripts. Using AStalavista, a total of 14,728 AS events were detected in the longissimus dorsi muscle. About 17.79% of the genes produced splicing isoforms, in which exon skipping was the most frequent AS event. By analyzing the expression differences of mRNAs and splicing isoforms, we found that differentially expressed mRNAs with splicing isoforms could participate in skeletal muscle development and fatty acid metabolism, which might determine muscle-related traits. SERBP1, MYL1, TNNT3, and TNNT1 were identified with multiple splicing isoforms, with significant differences in expression. AS events occurring in IFI6 and GADD45G may cause significant differences in gene expression. Other AS events, such as ONT.15153.3, may regulate the function of ART1 by regulating the expression of different transcripts. Moreover, co-expression and protein-protein interaction (PPI) analysis indicated that several genes (MRPL27, AAR2, PYGM, PSMD4, SCNM1, and HNRNPDL) may be related to intramuscular fat. The splicing isoforms investigated in our research provide a reference for the study of alternative splicing regulation of intramuscular fat deposition.

Abstract Background Intrauterine growth restriction (IUGR) remains a major problem associated with swine production. Thus, understanding the physiological changes of postnatal IUGR piglets would aid in improving growth performance. Moreover, liver metabolism plays an important role in the growth and survival of neonatal piglets. Results By profiling the transcriptome of liver samples on postnatal Days 1, 7, and 28, our study focused on characterizing the growth, function, and metabolism in the liver of IUGR neonatal piglets. Our study demonstrates that the livers of IUGR piglets were associated with a series of complications, including inflammatory stress and immune dysregulation; cytoskeleton and membrane structure disorganization; dysregulated transcription events; and abnormal glucocorticoid metabolism. In addition, the abnormal liver function index in the serum [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total protein (TP)], coupled with hepatic pathological and ultrastructural morphological changes are indicative of liver damage and dysfunction in IUGR piglets. Moreover, these results reveal the sex-biased developmental dynamics between male and female IUGR piglets, and that male IUGR piglets may be more sensitive to disrupted metabolic homeostasis. Conclusions These observations provide a detailed reference for understanding the mechanisms and characterizations of IUGR liver functions, and suggest that the potential strategies for improving the survival and growth performance of IUGR offspring should consider the balance between postnatal catch-up growth and adverse metabolic consequences. In particular, sex-specific intervention strategies should be considered for both female and male IUGR piglets.

Abstract Background Runs of homozygosity (ROHs) are homozygous segments of the genome where the two haplotypes inherited from the parents are identical. The current availability of genotypes for a very large number of single nucleotide polymorphisms (SNPs) is leading to more accurate characterization of ROHs in the whole genome. Here, we investigated the occurrence and distribution of ROHs in 3,692 Large White pigs and compared estimates of inbreeding coefficients calculated based on ROHs (F ROH), homozygosity (F HOM), genomic relationship matrix (F GRM) and pedigree (F PED). Furthermore, we identified genomic regions with high ROH frequencies and annotated their candidate genes. Results In total, 176,182 ROHs were identified from 3,569 animals, and all individuals displayed at least one ROH longer than 1 Mb. The ROHs identified were unevenly distributed on the autosomes. The highest and lowest coverages of Sus scrofa chromosomes (SSC) by ROH were on SSC14 and SSC13, respectively. The highest pairwise correlation among the different inbreeding coefficient estimates was 0.95 between F ROH_total and F HOM, while the lowest was − 0.083 between F GRM and F PED. The correlations between F PED and F ROH using four classes of ROH lengths ranged from 0.18 to 0.37 and increased with increasing ROH length, except for ROH > 10 Mb. Twelve ROH islands were located on four chromosomes (SSC1, 4, 6 and 14). These ROH islands harboured genes associated with reproduction, muscular development, fat deposition and adaptation, such as SIRT1, MYPN, SETDB1 and PSMD4. Conclusion F ROH can be used to accurately assess individual inbreeding levels compared to other inbreeding coefficient estimators. In the absence of pedigree records, F ROH can provide an alternative to inbreeding estimates. Our findings can be used not only to effectively increase the response to selection by appropriately managing the rate of inbreeding and minimizing the negative effects of inbreeding depression but also to help detect genomic regions with an effect on traits under selection.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals.

Sow milk production ability is an important limiting factor impacting suboptimal growth and the survival of piglets. Through pig genetic improvement, litter sizes have been increased. Larger litters need more suckling mammary glands, which results in increased milk from the lactating sow. Hence, there is much significance to exploring sow lactation performance. For milk production ability, it is not practical to directly measure the milk yield, we used litter weight gain (LWG) throughout sow lactation as an indicator. In this study, we estimated the heritability of LWG, namely, 0.18 ± 0.07. We then performed a GWAS, and detected seven significant SNPs, namely, Sus scrofa Chromosome (SSC) 2: ASGA0010040 (p = 7.73E-11); SSC2:MARC0029355 (p = 1.30E-08), SSC6: WU_10.2_6_65751151 (p = 1.32E-10), SSC7: MARC0058875 (p = 4.99E-09), SSC10: WU_10.2_10_49571394 (p = 6.79E-08), SSC11: M1GA0014659 (p = 1.19E-07), and SSC15: MARC0042106 (p = 1.16E-07). We performed the distribution of phenotypes corresponding to the genotypes of seven significant SNPs and showed that ASGA0010040, MARC0029355, MARC0058875, WU_10.2_10_49571394, M1GA0014659, and MARC0042106 had extreme phenotypic values that corresponded to the homozygous genotypes, while the intermediate values corresponded to the heterozygous genotypes. We screened for flanking regions ± 200 kb nearby the seven significant SNPs, and identified 38 genes in total. Among them, 28 of the candidates were involved in lactose metabolism, colostrum immunity, milk protein, and milk fat by functional enrichment analysis. Through the combined analysis between 28 candidate genes and transcriptome data of the sow mammary gland, we found nine commons (ANO3, MUC15, DISP3, FBXO6, CLCN6, HLA-DRA, SLA-DRB1, SLA-DQB1, and SLA-DQA1). Furthermore, by comparing the chromosome positions of the candidate genes with the quantitative trait locus (QTLs) as previously reported, a total of 17 genes were found to be within 0.86–94.02 Mb of the reported QTLs for sow milk production ability, in which, NAV2 was found to be located with 0.86 Mb of the QTL region ssc2: 40936355. In conclusion, we identified seven significant SNPs located on SSC2, 6, 7, 10, 11, and 15, and propose 28 candidate genes for the ability to produce milk in Yorkshire pigs, 10 of which were key candidates.

Long-term selection or evolution is an important factor governing the development of disease resistance in pigs. To better clarify the molecular mechanisms underlying different levels of disease resistance, we used transcriptomics and proteomics analysis to characterize differences in the immunities between six resistant (Min pig) and six susceptible (Large White, LW) pigs which were raised in the same environment. A total of 135 proteins and 791 genes were identified as being differentially expressed between the Large White and Min pig groups. Protein expression clustering and functional analysis revealed that proteins related to immune system process, humoral immune response, the B cell receptor signaling pathway, lymphocyte-mediated immunity, and innate immune responses were more highly expressed in Min pigs. Transcriptome gene set enrichment analysis was used to reveal that pathways of cell adhesion molecules and antigen processing and presentation are significantly enriched in Min pigs. Integrated proteomics and transcriptomics data analysis identified 16 genes that are differentially expressed at both the mRNA and protein levels. In addition, 13 out of these 16 genes were related to the quantitative trait loci of immune diseases, including neural EGFL-like 2 (NELL2) and lactate dehydrogenase B (LDHB), which are involved in innate immunity. Correlation analysis between the genes/proteins and cytokines shows upregulated proteins in LW pigs in association with immunosuppressive/pro-inflammatory cytokines, such as interleukin (IL) 10, IL6, and tumor necrosis factor alpha. This was further validated using parallel reaction monitoring analysis. In summary, we discovered several potential candidate pathways and key genes/proteins involved in determining differences in disease resistance between the two studied pig breeds, which could provide new insights into the breeding of pigs for disease resistance.

Pigs’ backfat quality has an important impact on the quality of pork and pork products and has a strong relationship with nutrition and sensory characteristics. This study aimed to identify the related candidate genes of backfat quality and to preliminary clarify the molecular regulatory mechanism underlying pig backfat quality phenotypes. Expression assessments of long non-coding RNA (lncRNA) and mRNA profiling in backfat from high-quality (firm and white) and low-quality (soft and yellow) Beijing Black pigs were performed by RNA sequencing. Significantly different expressions were observed in 610 protein-coding genes and 290 lncRNAs between the two groups. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway annotation showed that some candidate differentially expressed genes that participate in lipid-related pathways and pigmentation terms may play a role in backfat quality in pigs. The cis-target and trans-target genes were predicted to explore the regulatory function of lncRNAs, and integrative analyses of different expression lncRNAs targets and different expression genes were performed. The results showed the regulatory networks of lncRNA-mRNA related to backfat quality, and our study obtained strong candidate genes for backfat quality: ELOVL5, SCD, DGAT2, SLC24A5, and TYRP1, which were involved in fat metabolism, adipogenesis regulation, and pigmentation. To our knowledge, this study is the first to demonstrate the molecular genetic mechanisms of backfat quality in pigs, and these findings improve the current understanding of backfat quality mechanisms and provide a foundation for further studies.

Loin muscle area (LMA) is an important meat production trait and plays a key role in determining carcass leanness. Genome-wide association study (GWAS) and RNA sequencing (RNA-seq) analysis were used to identify candidate LMA genes in Beijing Black pigs, a popular breed among consumers in northern China. Ten single nucleotide polymorphisms (SNPs) in sus scrofa chromosome (SSC) 9 were significantly associated with LMA. These SNPs were mapped to a 2.90 Mb (84.94–87.84 Mb) region. A total of 11 annotated genes were mapped on this region, namely MEOX2, CRPPA, SOSTDC1, LRRC72, ANKMY2, BZW2, TSPAN13, AGR2, AHR, SNX13, and HDAC9. In addition, RNA-seq analysis was performed between the high- and low-LMA groups, and 329 differentially expressed genes (DEGs) were identified. Further, Kyoto Encyclopedia of Genes and Genomes analysis based on DEGs revealed that the JAK/STAT signaling pathway and oxytocin signaling pathway may be responsible for LMA. Both GWAS and RNA-seq analysis identified the HDAC9 gene, indicating that it may be an important candidate gene affecting LMA in Beijing Black pigs. The findings provide valuable molecular insights into the mechanisms that influence LMA content in pigs, which can be utilized in targeted approaches to enhance meat quality and commercial profitability.

Frozen storage is an important strategy to maintain meat quality for long-term storage and transportation. Lipid oxidation is one of the predominant causes of the deterioration of meat quality during frozen storage. Untargeted lipidomic and targeted metabolomics were employed to comprehensively evaluate the effect of frozen duration on pork lipid profiles and lipid oxidative products including free fatty acids and fatty aldehydes. A total of 688 lipids, 40 fatty acids and 14 aldehydes were successfully screened in a pork sample. We found that ether-linked glycerophospholipids, the predominant type of lipids, gradually decreased during frozen storage. Of these ether-linked glycerophospholipids, ether-linked phosphatidylethanolamine and phosphatidylcholine containing more than one unsaturated bond were greatly influenced by frozen storage, resulting in an increase in free polyunsaturated fatty acids and fatty aldehydes. Among these lipid oxidative products, decanal, cis-11,14-eicosenoic acid and cis-5,8,11,14,17-dicosapentaenoic acid can be considered as potential indicators to calculate the freezing time of unknown frozen pork samples. Moreover, over the three-month frozen storage, the first month was a rapid oxidation stage while the other two months were a slow oxidation stage.

The correlation between oxidative stress and liver metabolic dysfunction in piglets with intrauterine growth restriction (IUGR) remains limited. Therefore, the objective of the present study was to investigate potential mechanisms of metabolic characteristics induced by oxidative stress in the livers of IUGR piglets using metabolomic and transcriptomic analysis. Analysis of the phenotypic characteristics showed that the liver weight of the intrauterine growth restriction piglets was significantly lower than that of normal birth weight piglets. Intrauterine growth restriction piglets exhibited disordered hepatic cord arrangement and vacuolization as well as excessive lipid accumulation in hepatocytes. In addition, the activities of antioxidant enzymes were significantly decreased in the liver of the intrauterine growth restriction piglets, whereas the level of the lipid peroxidation marker MDA was significantly increased. Finally, our findings revealed that intrauterine growth restriction piglets were involved in a variety of metabolic abnormalities, including mitochondrial dysfunction, imbalance of fatty acid composition, disruption to sources of one-carbon unit supply, and abnormal galactose conversion, which may be responsible for oxidative stress in the liver. In summary, these data provided a detailed theoretical reference for revealing the hepatic metabolic characteristics of intrauterine growth restriction piglets.

Abstract Background RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited. Results Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver, muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine (A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions (CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155 (muscle) to 25001 (brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissue-specific editing sites in each tissue revealed that RNA editing might play important roles in tissue function. Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle. Conclusions This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

Intramuscular fat (IMF) is a key meat quality trait. Research on the genetic mechanisms of IMF decomposition is valuable for both pork quality improvement and the treatment of obesity and type 2 diabetes. Copy number variations (CNVs) are a type of variant that may influence meat quality. In this study, a total of 1185 CNV regions (CNVRs) including 393 duplicated CNVRs, 432 deleted CNVRs, and 361 CNVRs with both duplicated and deleted status were identified in a pig F2 resource population using next-generation sequencing data. A genome-wide association study (GWAS) was then performed between CNVs and IMF, and a total of 19 CNVRs were found to be significantly associated with IMF. QTL colocation analysis indicated that 3 of the 19 CNVRs overlapped with known QTLs. RNA-seq and qPCR validation results indicated that CNV150, which is located on the 3′UTR end of the proline, as well as glutamate and the leucine rich protein 1 (PELP1) gene may affect the expression of PELP1 alternative splices. Sequence alignment and Alphafold2 structure prediction results indicated that the two alternative splices of PELP1 have a 23 AA sequence variation and a helix-fold structure variation. This region is located in the region of interaction between PELP1 and other proteins which have been reported to be significantly associated with fat deposition or insulin resistance. We infer that the CNVR may influence IMF content by regulating the alternative splicing of the PELP1 gene and ultimately affects the structure of the PELP1 protein. In conclusion, we found some CNVRs, especially CNV150, located in PELP1 that affect IMF. These findings suggest a novel mechanistic approach for meat quality improvement in animals and the potential treatment of insulin resistance in human beings.

RNA-Seq technology is widely used to analyze global changes in the transcriptome and investigate the influence on relevant phenotypic traits. Beijing Black pigs show differences in growth rate and meat quality compared to western pig breeds. However, the molecular mechanisms responsible for such phenotypic differences remain unknown. In this study, longissimus dorsi muscles from Beijing Black and Yorkshire pigs were used to construct RNA libraries and perform RNA-seq. Significantly different expressions were observed in 1051 mRNAs, 322 lncRNAs, and 82 circRNAs. GO and KEGG pathway annotation showed that differentially expressed mRNAs participated in skeletal muscle development and fatty acid metabolism, which determined the muscle-related traits. To explore the regulatory role of lncRNAs, the cis and trans-target genes were predicted and these lncRNAswere involved in the biological processes related to skeletal muscle development and fatty acid metabolismvia their target genes. CircRNAs play a ceRNA role by binding to miRNAs. Therefore, the potential miRNAs of differentially expressed circRNAs were predicted and interaction networks among circRNAs, miRNAs, and key regulatory mRNAs were constructed to illustrate the function of circRNAs underlying skeletal muscle development and fatty acid metabolism. This study provides new clues for elucidating muscle phenotypic variation in pigs.

Being in a confined environment causes chronic stress in gestating sows, which is detrimental for sow health, welfare and, consequently, offspring physiology. This study assessed the health and welfare of gestating sows housed in a group housing system compared to individual gestation stalls. After pregnancy was confirmed, experimental sows were divided randomly into two groups: the group housing system (GS), with the electronic sow feeding (ESF) system; or individual stall (IS). The behavior of sows housed in the GS or IS was then compared; throughout pregnancy, GS sows displayed more exploratory behavior, less vacuum chewing, and less sitting behavior (p < 0.05). IS sows showed higher stress hormone levels than GS sows. In particular, at 41 days of gestation, the concentration of the adrenocorticotropic hormone (ACTH) and adrenaline (A) in IS sows was significantly higher than that of GS sows, and the A level of IS sows remained significantly higher at 71 days of gestation (p < 0.01). The lipopolysaccharide (LPS) test was carried out in the weaned piglets of the studied sows. Compared with the offspring of gestating sows housed in GS (PG) or IS (PS), PG experienced a shorter period of high temperature and showed a quicker return to the normal state (p < 0.05). Additionally, their lower levels of stress hormone (p < 0.01) suggest that PG did not suffer from as much stress as PS. These findings suggested that gestating sows housed in GS were more able to carry out their natural behaviors and, therefore, had lower levels of stress and improved welfare. In addition, PG also showed better disease resistance and resilience. These results will provide a research basis for the welfare and breeding of gestating sows.

Longer porcine carcasses may be expected to have more vertebrae. Therefore, vertebrae number in pigs is an economically important trait. To examine the genetic basis of this trait, we genotyped 578 F2 Large White × Minzhu pigs using the Porcine SNP60K BeadChip. A genome-wide association study (GWAS) identified 36 significant single nucleotide polymorphisms (SNPs) on the chromosomes SSC1 (294.28–300.32 Mb) and SSC7 (102.22–109.39 Mb). A 6.04-Mb region that contained all 13 significant SNPs on SSC1 also contained the gene NR6A1, previously reported to influence the number of vertebrae in pigs. However, the reported putative casual mutation of NR6A1 c.748C>T showed no genome-wide significant association with the trait, suggesting it was not a causal mutation in our population. The remaining 23 significant SNPs on SSC7 were concentrated in a 7.17-Mb region, which was within a quantitative trait locus interval for number of vertebrae. TMED10 was the closest gene to the most significant SNP and might be a candidate. Haplotype sharing and block analysis refined the QTL to an interval of about 3 Mb containing 29 candidate genes. Of these 29 genes, the previously reported possible casual mutation of VRTN g.19034A>C was not found to be a causal mutation in our population. Exploration of these genes via additional genetic and functional studies in mammals revealed that TGFβ3 could be a good candidate on SSC7. A mutation of TGFβ3 c.1749G>A was detected by GWAS and could be proposed as a candidate causal mutation, or as closely linked to a causal mutation, for the number of vertebrae in pigs.

The number of vertebrae (NV), especially the number of thoracic vertebrae (NTV), varies among pig breeds. The NTV is controlled by vertebral segmentation and the number of somites during embryonic development. Although there is a high correlation between the NTV and NV, studies on a fixed NV have mainly considered the absolute numbers of thoracic vertebrae instead of vertebral segmentation. Therefore, this study aimed to discover variants associated with the NTV by considering the effect of the NV in pigs. The NTV and NV of 542 F2 individuals from a Large White × Minzhu pig crossbreed were recorded. All animals were genotyped for VRTN g.19034 A > C, LTBP2 c.4481A > C, and 37 missense or splice variants previously reported in a 951-kb interval on SSC7 and 147 single nucleotide polymorphisms (SNPs) on SSC14. To identify NTV-associated SNPs, we firstly performed a genome-wide association study (GWAS) using the Q + K (population structure + kinship matrix) model in TASSEL. With the NV as a covariate, the obtained data were used to identify the SNPs with the most significant genome-wide association with the NTV by performing a GWAS on a PorcineSNP60K Genotyping BeadChip. Finally, a conditional GWAS was performed by fixing this SNP. The GWAS showed that 31 SNPs on SSC7 have significant genome-wide associations with the NTV. No missense or splice variants were found to be associated with the NTV significantly. A linkage disequilibrium analysis suggested the existence of quantitative trait loci (QTL) in a 479-Kb region on SSC7, which contained a critical candidate gene FOS for the NTV in pigs. Subsequently, a conditional GWAS was performed by fixing M1GA0010658, the most significant of these SNPs. Two SNPs in BMPR1A were found to have significant genome-wide associations and a significant dominant effect. The leading SNP, S14_87859370, accounted for 3.86% of the phenotypic variance. Our study uncovered that regulation variants in FOS on SSC7 and in BMPR1A on SSC14 might play important roles in controlling the NTV, and thus these genetic factors may be harnessed for increasing the NTV in pigs.

Intramuscular fat (IMF) is essential for improving the palatability and flavor of meat, and it is strongly associated with human insulin resistance. RNA editing is a widespread regulating event in different tissues. Here, we investigated the global RNA editing difference of two groups of pig with different IMF contents to find the potential editing sites affecting IMF. In this research, RES-Scanner and REDItools were used to identify RNA editing sites based on the whole genome and transcriptome sequencing data of the high and low groups composed of three full-sib pairs with opposite IMF phenotypes. A total of 295 RNA editing sites were investigated in the longissimus dorsi muscle, and 90.17% of these sites caused A to G conversion. After annotation, most editing sites were located in noncoding regions (including five sites located on the 3′ UTR regions). Five editing sites (including two sites that could lead to nonsynonymous amino acid changes) were located in the exons of genes. A total of 36 intergroup (high and low IMF) differential RNA editing sites were found in 33 genes. Some candidate editing sites, such as sites in acyl-coenzymeA: cholesterol acyltransferase 1 (ACAT1), coatomer protein, subunit alpha (COPA), and nuclear receptor coactivator 3 (NCOA3), were selected as candidate RNA editing sites associated with IMF. One site located on the 3′ UTR region of growth hormone secretagogue receptor (GHSR) may regulate GHSR expression by affecting the interaction of miRNA and mRNA. In conclusion, we identified a total of 36 nonredundant RNA editing sites in the longissimus dorsi muscle, which may reveal the potential importance of RNA editing in IMF. Four were selected as candidate sites associated with IMF. Our findings provide some new insights of RNA editing function in pig longissimus dorsi muscle which useful for pig IMF breeding or human insulin resistances research.

Intramuscular fat (IMF) content is an important economic trait for pork quality. Our previous results regarding the genome-wide association between IMF content and copy number variations (CNVs) indicated that the CNV within Netrin-1(NTN1-CNV) was significantly associated with IMF. In order to validate the effect of NTN1-CNV, we detected the Netrin-1 (NTN1) gene dose and protein expression content in the longissimus dorsi of different IMF content pigs using Western blotting and investigated the expression of NTN1 RNA in different tissues using real-time quantitative polymerase chain reaction (qPCR). The knock-down of the NTN1 gene in C2C12 and 3T3-L1 cells and over-expression in C2C12 cells during the proliferation and differentiation stage were also investigated to explore the possible pathway of action of NTN1. The results showed that in individuals with IMF content differences, the gene dose of NTN1 and the expression of NTN1 protein were also significantly different, which indicated that NTN1-CNV may directly affect IMF by its coding protein. NTN1 had the highest expression in pig longissimus dorsi and backfat tissues, which indicates that NTN1 may play an important role in muscle and fat tissues. The in vitro validation assay indicated that NTN1 silencing could promote the proliferation and inhibit the differentiation of C2C12 cells, with no effect on 3T3-L1 cells. Additionally, NTN1 over-expression could inhibit the proliferation and promote the differentiation of C2C12 cells. Combined with previous research, we conclude that NTN1-CNV may affect IMF by its gene dose, and the expression of NTN1 may affect the proliferation and differentiation of muscle cells by the AMP-activated protein kinase (AMPK) pathway and finally influence the IMF.

Backfat deposition is strongly related to carcass traits, growth rate, feed conversion rate, and reproductive performance in pig production. To understand the molecular mechanisms underlying porcine backfat thickness phenotypes, transcriptome and miRNA profiling of backfat from high-backfat thickness and low-backfat thickness pigs were performed by RNA sequencing. Twenty genes encoding for miRNAs and 126 genes encoding for protein-coding genes were found to be differentially expressed between the two libraries. After integrative analysis of DEMs targets and DEGs, a total of 33 mRNA‒miRNA interaction pairs were identified, and the regulatory networks of these pairs were determined. Among these genes, five (AQP9, DKK3, GLYCTK, GLIPR1, and DUSP2) related to fat deposition were found to be strong candidate genes, and mir-31-5p/AQP9 and mir-31-5p/GLIPR1 may play important roles in fat deposition. Additionally, potential adipogenesis-related genes and miRNAs were identified. These findings improve the current understanding of the molecular genetic mechanisms of subcutaneous fat deposition in pigs and provide a foundation for further studies.

Pyrano[2,3-c]pyrazole derivatives have been reported as exerting various biological activities. One compound with potential anti-tumor activity was screened out by MTT assay from series of dihydropyrazopyrazole derivatives we had synthesized before using a one-pot, four-component reaction, and was named as 6-amino-4-(2-hydroxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (hereinafter abbreviated as AMDPC). The IC50 of AMDPC against Bcap-37 breast cancer cells was 46.52 μg/mL. Then the hydrophobic AMDPC was encapsulated in PEG-PLGA block copolymers, and then self-assembled as polymeric micelle (mPEG-PLGA/AMDPC) to improve both physiochemical and release profiles. The effect of mPEG-PLGA/AMDPC on BCAP-37 cancer cells showed similar anti-tumor effects as AMDPC. Furthermore, the anti-tumor mechanism of mPEG-PLGA/AMDPC was investigated, which can probably be attributed to stimulating the expression of P21 gene and therefore protein production on BCAP-37 cells, and then blocked the cell cycle through the P53-independent pathway both in S phase and G2 phase. Thus, mPEG-PLGA/AMDPC is a promising therapeutic agent for cancer treatment, and further in vivo studies will be developed.

As one of the main breeding selection criteria, external appearance has special economic importance in the hog industry. In this study, an Illumina Porcine SNP60 BeadChip was used to conduct a genome-wide association study (GWAS) in 605 pigs of the F2 generation derived from a Large White × Minzhu intercross. Traits under study were abdominal circumference (AC), body height (BH), body length (BL), cannon bone circumference (CBC), chest depth (CD), chest width (CW), rump circumference (RC), rump width (RW), scapula width (SW), and waist width (WW). A total of 138 SNPs (the most significant being MARC0033464) on chromosome 7 were found to be associated with BH, BL, CBC, and RC (P-value= 4.15E-6). One SNP on chromosome 1 was found to be associated with CD at genome-wide significance levels. The percentage phenotypic variance of these significant SNPs ranged from 0.1-25.48%. Moreover, a conditional analysis revealed that the significant SNPs were derived from a single quantitative trait locus (QTL) and indicated additional chromosome-wide significant association for 25 SNPs on SSC4 (BL, CBC) and 9 SNPs on SSC7 (RC). Linkage analysis revealed two complete linkage disequilibrium haplotype blocks that contained seven and four SNPs, respectively. In block 1, the most significant SNP, MARC0033464, was present. Annotations from pig reference genome suggested six genes (GRM4, HMGA1, NUDT3, RPS10, SPDEF and PACSIN1) in block 1 (495 kb), and one gene (SCUBE3) in block 3 (124 kb). Functional analysis indicated that HMGA1 and SCUBE3 genes are the potential genes controlling BH, BL, and RC in pigs, with an application in breeding programs. We screened several candidate intervals and genes based on SNP location and gene function, and predicted their function using bioinformatics analyses.

The quantitative trait loci (QTL) for porcine ear size was previously reported to mainly focus on SSC5 and SSC7. Recently, a missense mutation, G32E, in PPARD in the QTL interval on SSC7 was identified as the causative mutation for ear size. However, on account of the large interval of QTL, the responsible gene on SSC5 has not been identified. In this study, an intercross population was constructed from the large-eared Minzhu, an indigenous Chinese pig breed, and the Western commercial Large White pig to examine the genetic basis of ear size diversity. A GWAS was performed to detect SNPs significantly associated with ear size. Thirty-five significant SNPs defined a 10.78-Mb (30.14-40.92 Mb) region on SSC5. Further, combining linkage disequilibrium and haplotype sharing analysis, a reduced region of 3.07-Mb was obtained. Finally, by using a selective sweep analysis, a critical region of about 450-kb interval containing two annotated genes LEMD3 and WIF1 was refined in this work. Functional analysis indicated that both represent biological candidates for porcine ear size, with potential application in breeding programs. The two genes could also be used as novel references for further study of the mechanism underlying human microtia.

Copy number variations (CNVs) are one of the main contributors to genetic diversity in animals and are broadly distributed in the genomes of swine. Investigating the performance and evolutionary impacts of pig CNVs requires comprehensive knowledge of their structure and function within and between breeds. In the current study, 4 different programs (i.e., GADA, PennCNV, QuantiSNP, and cnvPartition) were used to analyze Porcine SNP60 genotyping data of 585 pigs from one Large White × Minzhu intercross population to detect copy number variant regions (CNVRs). Overlapping CNVRs recalled by at least 2 programs were used to construct a powerful and comprehensive CNVR map, which contained 249 CNVRs (i.e., 70 gains, 43 losses, and 136 gains/losses) and covered 26.22% of the regions in the swine genome. Ten CNVRs, representing different predicted statuses, were selected for validation via quantitative real-time PCR (QPCR); 9/10 CNVRs (i.e., 90%) were validated. When being traced back to the F0 generation, 58 events were identified in only Minzhu F0 parents and 2 events were identified in only Large White F0 parents. A series of CNVR function analyses were performed. Some of the CNVRs functions were predicted, and several interesting CNVRs for meat quality traits and hematological parameters were obtained. A comprehensive and lower false rate genome-wide CNV map was constructed for Large White and Minzhu pig genomes in this study. Our results may provide an important basis for determining the relationship between CNVRs and important qualitative and quantitative traits. In addition, it can help to further understand genetic processes in pigs.