Intramuscular Fat Content In Pigs Research Articles

Intramuscular Fat Deposition in pig: A Key Target for Improving Pork Quality1

Intramuscular fat (IMF) is an important economic trait for pork quality, affecting meat flavour, juiciness, and tenderness. Hence, the improvement of IMF content is one of the hotspots of animal science to provide better meat product. Here, we found that most IMF-related genes are enriched in lipid metabolism processes, including fatty acid transport and uptake, fatty acid beta oxidation, lipid synthesis, lipid storage, and lipolysis. PPAR and AMPK signalling pathways are identified to be responsible for IMF deposition. Genetics and nongenetic factors (i.e., diets, gut microbiota, age, sex, management) also positively or negatively regulate the IMF content in pigs. Taken together, this review deepens our understanding of how these factors affect pig IMF deposition and provides valuable information for moderately increasing IMF content.

Identification of a Candidate Gene Regulating Intramuscular Fat Content in Pigs through the Integrative Analysis of Transcriptomics and Proteomics Data.

Pork is a widely consumed source of animal protein worldwide, and the intramuscular fat (IMF) content in pork plays a crucial role in determining its quality. In this study, we sought to identify candidate genes that regulate IMF deposition in pigs. We performed tandem mass tags (TMT)-based quantitative proteomics analysis using Longissimus dorsi (LD) muscle samples obtained from eight pigs with extremely high and low IMF content among a group of 28 Duroc pigs and identified 50 differentially abundant proteins (DAPs). Additionally, we compared the proteomics data with RNA-sequencing data obtained in our previous study and identified TUSC5 as a differentially expressed gene corresponding to the relevant DAP. To investigate the potential role of TUSC5 in adipogenesis, we suppressed TUSC5 expression in mouse 3T3-L1 preadipocytes using short hairpin RNA (shRNA) and observed a significant reduction in the differentiation of 3T3-L1 cells into adipocytes, as indicated by Oil Red O staining and triglyceride content. Moreover, we observed a reduction in the expression of genes associated with adipogenesis (PPARG, CEBPA, FABP4, and FASN) following TUSC5 suppression. Through an integrative analysis of transcriptomics and proteomics data, our study identified TUSC5 as a crucial candidate gene associated with the regulation of IMF content in pigs.

Differentially expressed genes in the longissimus dorsi muscle between the Chinese indigenous Ningxiang pig and Large White breed using RNA sequencing

High intramuscular fat content of pigs improves pork quality and increasing intramuscular fat deposition is a long-term goal in the husbandry of pigs reared for meat production. There are significant phenotypic differences between the Ningxiang (NX) pigs (an indigenous Chinese breed) and Large White (LW) pigs (a western, lean-type breed). The present work aimed to gain insight into the longissimus dorsi muscle transcriptome between the two pig breeds. We investigated the molecular basis of these differences by comparing their transcriptome profiles. RNA-seq technology was used to identify the differentially expressed genes (DEGs) in the longissimus dorsi muscle of the NX and LW pigs. We obtained 692 million clean reads using transcriptome sequencing of muscle samples from the two pig breeds. A total of 885 DEGs were identified, including 469 upregulated and 416 downregulated genes in the NX pigs compared with the LW pigs. Using KEGG pathway analysis, it was found that the significant DEGs were mainly enriched in metabolism-related pathways, such as lipid metabolism and biosynthesis, and glucose metabolism or biosynthesis. Quantitative real-time PCR confirmed the differential expression of eight selected DEGs in both pig breeds. qPCR results showed that the RNA-seq results were reliable. Several DEGs were candidate functional genes related to the lipid metabolism, including CD36, LIPE, MCAT, LPIN1, ANGPTL4, PPARD, SCD, INSR, MOGAT, IGF1, AKT2 and JAK2. Our results provide a comprehensive basis for the investigation of the differences in transcriptional regulation of the muscles between divergent phenotypes.

Systematic Identification and Comparison of the Expressed Profiles of lncRNAs, miRNAs, circRNAs, and mRNAs with Associated Co-Expression Networks in Pigs with Low and High Intramuscular Fat.

Simple SummaryIntramuscular fat (IMF) is a key factor affecting many meat quality traits of pigs, such as pork tenderness, flavor, and many more. In this study, a systematic identification and comparison of the expressed profiles of messenger RNA (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) with associated co-expression networks longissimus dorsi muscle (LDM) in Large White × Min pigs F2 resource population were performed. The results contain high-throughput genomic data, which are helpful to clarify the regulatory role of a variety of RNAs in regulating intramuscular fat formation and lipid metabolism at the genomic level and provide new insights for studying the mechanism of fat formation and the regulation of meat quality related genes at the molecular level.Intramuscular fat (IMF) content is a complex trait that affects meat quality and determines pork quality. In order to explore the potential mechanisms that affect the intramuscular fat content of pigs, a Large white × Min pigs F2 resource populations were constructed, then whole-transcriptome profile analysis was carried out for five low-IMF and five high-IMF F2 individuals. In total, 218 messenger RNA (mRNAs), 213 long non-coding RNAs (lncRNAs), 18 microRNAs (miRNAs), and 59 circular RNAs (circRNAs) were found to be differentially expressed in the longissimus dorsi muscle. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes annotations revealed that these differentially expressed (DE) genes or potential target genes (PTGs) of DE regulatory RNAs (lncRNAs, miRNAs, and circRNAs) are mainly involved in cell differentiation, fatty acid synthesis, system development, muscle fiber development, and regulating lipid metabolism. In total, 274 PTGs were found to be differentially expressed between low- and high-IMF pigs, which indicated that some DE regulatory RNAs may contribute to the deposition/metabolism of IMF by regulating their PTGs. In addition, we analyzed the quantitative trait loci (QTLs) of DE RNAs co-located in high- and low-IMF groups. A total of 97 DE regulatory RNAs could be found located in the QTLs related to IMF. Co-expression networks among different types of RNA and competing endogenous RNA (ceRNA) regulatory networks were also constructed, and some genes involved in type I diabetes mellitus were found to play an important role in the complex molecular process of intramuscular fat deposition. This study identified and analyzed some differential RNAs, regulatory RNAs, and PTGs related to IMF, and provided new insights into the study of IMF formation at the level of the genome-wide landscape.

Dietary calcium supplementation promotes the accumulation of intramuscular fat

BackgroundIn the livestock industry, intramuscular fat content is a key factor affecting meat quality. Many studies have shown that dietary calcium supplementation is closely related to lipid metabolism. However, few studies have examined the relationship between dietary calcium supplementation and intramuscular fat accumulation.MethodsHere, we used C2C12 cells, C57BL/6 mice (n = 8) and three-way cross-breeding pigs (Duroc×Landrace×Large white) (n = 10) to study the effect of calcium addition on intramuscular fat accumulation. In vitro, we used calcium chloride to adjust the calcium levels in the medium (2 mmol/L or 3 mmol/L). Then we measured various indicators. In vivo, calcium carbonate was used to regulate calcium levels in feeds (Mice: 0.5% calcium or 1.2% calcium) (Pigs: 0.9% calcium or 1.5% calcium). Then we tested the mice gastrocnemius muscle triglyceride content, pig longissimus dorsi muscle meat quality and lipidomics.ResultsIn vitro, calcium addition (3 mmol/L) had no significant effect on cell proliferation, but promoted the differentiation of C2C12 cells into slow-twitch fibers. Calcium supplementation increased triglyceride accumulation in C2C12 cells. Calcium addition increased the number of mitochondria and also increased the calcium level in the mitochondria and reduced the of key enzymes activity involved in β-oxidation such as acyl-coenzyme A dehydrogenase. Decreasing mitochondrial calcium level can alleviate lipid accumulation induced by calcium addition. In addition, calcium addition also reduced the glycolytic capacity and glycolytic conversion rate of C2C12 cells. In vivo, dietary calcium supplementation (1.2%) promoted the accumulation of triglycerides in the gastrocnemius muscle of mice. Dietary calcium supplementation (1.5%) had no effect on pig weight, but significantly improved the flesh color of the longissimus dorsi muscle, reduced the backfat thickness and increased intramuscular fat content in pigs. Besides, calcium addition had no effect on longissimus dorsi pH, electrical conductivity and shear force.ConclusionsThese results suggest that calcium addition promotes intramuscular fat accumulation by inhibiting the oxidation of fatty acids. These findings provide a new tool for increasing intramuscular fat content and an economical strategy for improving meat quality.

Genome-wide association study reveals a quantitative trait locus and two candidate genes on Sus scrofa chromosome 5 affecting intramuscular fat content in Suhuai pigs

Intramuscular fat content (IFC) is an essential quantitative trait of meat, affecting multiple meat quality indicators. A certain amount of IFC could not only improve the sensory score of pork but also increase the flavour, tenderness, juiciness and shelf-life. To dissect the genetic determinants of IFC, two methods, including genome-wide efficient mixed-model analysis (GEMMA) and linkage disequilibrium adjusted kinships (LDAKs), were used to carry out genome-wide association studies for IFC in Suhuai pig population. A total of 14 and 18 significant single nucleotide polymorphisms (SNPs) were identified by GEMMA and LDAK, respectively. The results of these two methods were highly consistent and all 14 significant SNPs in GEMMA were detected by LDAK. Seven of the 18 SNPs reached the genome-wide significance level (P < 9.85E−07) while 11 cases reached the suggestive significance level (P < 1.77E−05). These significant SNPs were mainly distributed on Sus scrofa chromosome (SSC) 5, 3, and 7. Moreover, one locus resides in a 2.27 Mb (71.37–73.64 Mb) region on SSC5 harbouring 13 significant SNPs associated with IFC, and the lead SNP (rs81302978) also locates in this region. Linkage disequilibrium (LD) analysis showed that there were four pairs of complete LD (r2 = 1) among these 13 SNPs, and the remaining 9 SNPs with incomplete LD (r2 ≠ 1) were selected for subsequent analyses of IFC. Association analyses showed that 7 out of 9 SNPs were significantly associated with IFC (P < 0.05) in 330 Suhuai pigs, and the other 2 SNPs tended to reach a significant association level with IFC (P < 0.1). The phenotypic variance explained (PVE) range of these 9 SNPs was 0.92–3.55%. Meanwhile, the lead SNP was also significantly associated (rs81302978) with IFC (P < 0.05) in 378 commercial hybrid pigs (Pietrain × Duroc) × (Landrace × Yorkshire) (PDLY), and the PVE was 1.38%. Besides, two lipid metabolism-relevant candidate genes, the leucine rich repeat kinase 2 (LRRK2) and PDZ domain containing ring finger 4 (PDZRN4) were identified in the 2.27 Mb region on SSC5. In conclusion, our results may provide a set of markers useful for genetic improvement of IFC in pigs and will advance the genome selection process of IFC on pig breeding programmes.

Evidence Against the Causal Relationship Between a Putative Cis-Regulatory Variant of MYH3 and Intramuscular Fat Content in Pigs.

Improving meat quality has become the main goal of modern pig breeding. Intramuscular fat content (IMF) is an important trait influencing meat quality of livestock, but the molecular mechanism behind this trait is still unclear. Recently, Cho et al. reported the discovery of the first causal mutation affecting IMF and red flesh color (a*) in pigs, namely XM_013981330.2:g.−1805_−1810del, a 6-bp deletion variant in the porcine MYH3 promoter region. The objective of this study was to reassess the causality of this mutation for its potential commercial application. By Sanger sequencing, we firstly identified several new variants (including a 4-bp deletion) at or near the 6-bp deletion site, which formed four haplotypes in multiple breeds. Unexpectedly, the 6-bp deletion allele, previously determined as the MYH3 Q allele because of its significantly positive effect on IMF and a*, was found not only in Chinese indigenous breeds, but also in four western commercial breeds with relatively lower IMF levels, including Duroc, Large White, Landrace and Pietrain. More surprisingly, we found that the MYH3 Q allele and the haplotypes harboring it had no significant effects on IMF, marbling and color score in three large-scale divergent pig populations: the heterogeneous F6 and F7 pigs and commercial crossbred Duroc × (Landrace × Yorkshire) pigs. Transient transfection analysis in porcine satellite cells showed that the 6-bp deletion variants had a negligible effect on transcription of reporter gene, but could attenuate the MRF (myogenesis regulatory factors)-induced increase in luciferase activity of the MYH3 promoter vector. The MYH3 protein level in muscle did not differ significantly among the haplotype groups. Therefore, our results cannot support the causal relationship between the 6-bp deletion in MYH3 and IMF trait, suggesting that the causal mutation for the IMF QTL on SSC12 needs to be further identified.

Investigation on the Effect of Two Fat Metabolism Related Pathways on Intramuscular Fat Content in Pigs

Investigation on the Effect of Two Fat Metabolism Related Pathways on Intramuscular Fat Content in Pigs

Muscle Transcriptome Analysis Reveals Potential Candidate Genes and Pathways Affecting Intramuscular Fat Content in Pigs.

Intramuscular fat (IMF) content plays an essential role in meat quality. For identifying potential candidate genes and pathways regulating IMF content, the IMF content and the longissimus dorsi transcriptomes of 28 purebred Duroc pigs were measured. As a result, the transcriptome analysis of four high- and four low-IMF individuals revealed a total of 309 differentially expressed genes (DEGs) using edgeR and DESeq2 (p < 0.05, |log2(fold change)| ≥ 1). Functional enrichment analysis of the DEGs revealed 19 hub genes significantly enriched in the Gene Ontology (GO) terms and pathways (q < 0.05) related to lipid metabolism and fat cell differentiation. The weighted gene coexpression network analysis (WGCNA) of the 28 pigs identified the most relevant module with 43 hub genes. The combined results of DEGs, WGCNA, and protein–protein interactions revealed ADIPOQ, PPARG, LIPE, CIDEC, PLIN1, CIDEA, and FABP4 to be potential candidate genes affecting IMF. Furthermore, the regulation of lipolysis in adipocytes and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were significantly enriched for both the DEGs and genes in the most relevant module. Some DEGs and pathways detected in our study play essential roles and are potential candidate genes and pathways that affect IMF content in pigs. This study provides crucial information for understanding the molecular mechanism of IMF content and would be helpful in improving pork quality.

Promoter CpG methylation status affects ADRP gene expression level and intramuscular fat content in pigs

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed early during adipose differentiation. However, the underlying mechanism is still unclear. In this study, the porcine ADRP (pADRP) gene regulatory mechanism and its relationship to intramuscular fat (IMF) content are analysed. We investigated the characteristics of the pADRP gene prompter as well as its transcription level, core promoter and epigenetic modification status using real-time PCR, Dual-luciferase reporter assays and bisulphite sequencing. The results showed that pADRP mRNA expression in Longissimus Dorsi (LD) muscle of the low IMF (LEAN) group was lower than that in the high IMF (FAT) group (p<.01). Maximum promoter activity was found in the −926 bp to −235 bp region. The methylation level of the pADRP of this region (mainly 7th, 10th, 12th CpG sites) in the low IMF group (86.67%) was significantly higher than that in the high IMF group (46.7%) (p<.01). Our result plays an important role in pADRP transcriptional regulation and provides a good prospect for the marbling traits improvement. Highlights pADRP expression differs in divergent IMF content pigs. Promoter region located −929 to −235 bp, and its methylation contributes to gene expression.

TMT-labeled quantitative proteomic analyses on the longissimus dorsi to identify the proteins underlying intramuscular fat content in pigs

The Laiwu pig is famous for its excessively extremely high level of intramuscular fat content (IMF), however, the exact regulatory mechanism underlying intramuscular fat deposition in skeletal muscle is still unknown. As an economically important trait in pigs, IMF is controlled by multiple genes and biological pathways. In this study, we performed an integrated transcriptome-assisted TMT-labeled quantitative proteomic analysis of the longissimus dorsi (LD) muscle in Laiwu pigs at the fastest IMF deposition stage and identified 5074 unique proteins and 52 differentially abundant proteins (DAPs) (>1.5-fold cutoff, p < .05). These DAPs were hierarchically clustered in the LD muscle over two developmental stages from 120 d to 240 d. A comparison between transcriptomic (mRNA) and proteomic data revealed two differentially expressed genes corresponding to the DAPs. Changes in the levels of the nine proteins were further analyzed using RT-qPCR and parallel reaction monitoring (PRM). The proteins identified in this study could serve as candidates for elucidating the molecular mechanism of IMF deposition in pigs. SignificanceThe intramuscular fat content (IMF) refers to the amount of fat within muscles and plays an important role in meat quality by affecting meat quality-related traits, such as tenderness, juiciness and flavor. Using the integrated transcriptome-assisted TMT-labeled quantitative proteomic approach to characterize changes in the proteomic profile of the longissimus dorsi muscle, we identified differentially abundant proteins, such as ALDH1B1, OTX2, AnxA6 and Zfp512, that are associated with intramuscular fat deposition and fat biosynthesis in pigs. These proteins could serve as candidates for elucidating the molecular mechanism of IMF deposition in pigs.

A functional regulatory variant of MYH3 influences muscle fiber-type composition and intramuscular fat content in pigs.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5′-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs.

Expression of NR3C1, INSR and SLC2A4 genes in skeletal muscles and CBG in liver depends on age and breed of pigs

The genes encoding glucose transporter 4 (SLC2A4, GLUT4), insulin receptor (INSR) and glucocorticoid receptor (NR3C1) are considered regulators of energy metabolism that may influence fat content in skeletal muscle at different ages and breeds of pigs. In the study we performed analysis of expression of NR3C1, INSR and SLC2A4 genes in two skeletal muscle tissues: semimembranosus and longissimus dorsi muscle in gilts from three breeds of pigs that differed in intramuscular fat content: Duroc (DU), Pulawska (PUL) and Polish Large White (PLW) at 60, 90, 120, 150, 180 and 210 days after weaning. We also analyzed the expression of cortisol binding globulin (CBG) in the liver. Expression was analyzed with real time PCR (qPCR) using a relative quantification method. In blood, the concentration of cortisol, insulin and leptin were evaluated with radioimmunoassay. The concentration of metabolites (triglycerides and glucose) related to the changes of lipids content was also measured. We observed a significant relationship between the breed and the age of pigs and the expression of analyzed genes in muscle tissues (P &amp;lt; 0.01 for all analyzed genes) as well as with two hormones (for insulin P = 0.001; for cortisol P &amp;lt; 0.0001). Leptin level and metabolites in serum were not significant in the model (P &amp;gt; 0.05). The expression of analyzed genes (SLC2A4, INSR and NR3C1 in muscles and CBG in liver) strongly correlates with intramuscular fat content in pigs and is associated with age and breed.

Linoleic acid metabolic pathway allows for an efficient increase of intramuscular fat content in pigs

BackgroundIntramuscular fat (IMF) content is a relevant trait for high-quality meat products such as dry-cured ham, but increasing IMF has the undesirable correlated effect of decreasing lean growth. Thus, there is a need to find selection criteria for IMF independent from lean growth. In pigs, the proportion of linoleic (C18:2) and arachidonic (C20:4) acids decline with fat deposition and therefore they can be considered as indicators of fatness. The aim of this research was to estimate the genetic variation for C18:2 and C20:4 in IMF and their genetic correlations with IMF and lean growth traits, with the objective to assess their potential as specific biomarkers of IMF. The analysis was conducted using a full-pedigreed Duroc resource line with 91,448 records of body weight and backfat thickness (BT) at 180 days of age and 1371 records of fatty acid composition in the muscle gluteus medius.ResultsThe heritability estimates for C18:2 and C20:4 in IMF, whether expressed in absolute (mg/g of muscle) or in relative (mg/g of fatty acid) terms, as well as for their ratio (C20:4/C18:2), were high (> 0.40), revealing that the C18:2 to C20:4 pathway is subjected to substantial genetic influence. Litter effects were not negligible, with values ranging from 8% to 15% of the phenotypic variance. The genetic correlations of C18:2 and C20:4 with IMF and BT were negative (− 0.75 to − 0.66, for IMF, and − 0.64 to − 0.36, for BT), if expressed in relative values, but almost null (− 0.04 to 0.07), if expressed in absolute values, except for C18:2 with IMF, which was highly positive (0.88). The ratio of C20:4 to C18:2 also displayed a stronger genetic correlation with IMF (− 0.59) than with BT (− 0.10).ConclusionsThe amount of C18:2 in muscle can be used as an IMF-specific biomarker. Selection for the absolute amount of C18:2 is expected to deliver a similar response outcome as selection for IMF at restrained BT. Further genetic analysis of the C18:2 metabolic pathway may provide new insights into differential fat deposition among adipose tissues and on candidate genes for molecular markers targeting specifically for one of them.

Data Mining and Validation of AMPK Pathway as a Novel Candidate Role Affecting Intramuscular Fat Content in Pigs.

Simple SummaryIntramuscular fat (IMF) is increasingly being recognized as a key meat trait in the modern pork industry. The aims of this research were to identify potential signaling pathways associated with IMF content in the longissimus dorsi (LD) muscle of different pig breeds and investigate the gene expression levels in the screened signaling pathways. Our results indicated that the AMPK signaling pathway may be related to IMF deposition in the LD muscle of pigs. The results of qRT-PCR analysis showed that the expression of ten key hub genes (AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKKβ, CPT1A, CPT1B, PGC-1α, CD36, and ACC1) differed between the LD muscle of Min and Large White pigs. The protein expression levels of AMPK, LKB1, CaMKK2, CPT1A, and ACC1 were similar to the genes expression patterns in the LD muscle of Large White pigs. The results of this study provide novel insights into the regulatory function of the AMPK signaling pathway in relation to the IMF content in the LD muscle of different pigs.Intramuscular fat (IMF) is an important economic trait for pork quality and a complex quantitative trait regulated by multiple genes. The objective of this work was to investigate the novel transcriptional effects of a multigene pathway on IMF deposition in the longissimus dorsi (LD) muscles of pigs. Potential signaling pathways were screened by mining data from three gene expression profiles in the Gene Expression Omnibus (GEO) database. We designed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) arrays for the candidate signaling pathways to verify the results in the LD muscles of two pig breeds with different IMF contents (Large White and Min). Western blot analysis was used to detect the expression levels of several candidate proteins. Our results showed that the AMPK signaling pathway was screened via bioinformatics analysis. Ten key hub genes of this signaling pathway (AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKKβ, CPT1A, CPT1B, PGC-1α, CD36, and ACC1) were differentially expressed between the Large White and Min pigs. Western blot analysis further confirmed that LKB1/CaMKK2-AMPK-ACC1-CPT1A axis dominates the activity of AMPK signaling pathway. Statistical analyses revealed that AMPK signaling pathway activity clearly varied among the two pig breeds. Based on these results, we concluded that the activation of the AMPK signaling pathway plays a positive role in reducing IMF deposition in pigs.

A Key Gene, PLIN1, Can Affect Porcine Intramuscular Fat Content Based on Transcriptome Analysis.

Intramuscular fat (IMF) content is an important indicator for meat quality evaluation. However, the key genes and molecular regulatory mechanisms affecting IMF deposition remain unclear. In the present study, we identified 75 differentially expressed genes (DEGs) between the higher (H) and lower (L) IMF content of pigs using transcriptome analysis, of which 27 were upregulated and 48 were downregulated. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEG perilipin-1 (PLIN1) was significantly enriched in the fat metabolism-related peroxisome proliferator-activated receptor (PPAR) signaling pathway. Furthermore, we determined the expression patterns and functional role of porcine PLIN1. Our results indicate that PLIN1 was highly expressed in porcine adipose tissue, and its expression level was significantly higher in the H IMF content group when compared with the L IMF content group, and expression was increased during adipocyte differentiation. Additionally, our results confirm that PLIN1 knockdown decreases the triglyceride (TG) level and lipid droplet (LD) size in porcine adipocytes. Overall, our data identify novel candidate genes affecting IMF content and provide new insight into PLIN1 in porcine IMF deposition and adipocyte differentiation.

Identification and Functional Analysis of Long Intergenic Non-coding RNAs Underlying Intramuscular Fat Content in Pigs.

Intramuscular fat (IMF) content is an important trait that can affect pork quality. Previous studies have identified many genes that can regulate IMF. Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in various biological processes. However, lincRNAs related to IMF in pig are largely unknown, and the mechanisms by which they regulate IMF are yet to be elucidated. Here we reconstructed 105,687 transcripts and identified 1,032 lincRNAs in pig longissimus dorsi muscle (LDM) of four stages with different IMF contents based on published RNA-seq. These lincRNAs show typical characteristics such as shorter length and lower expression compared with protein-coding genes. Combined with methylation data, we found that both the promoter and genebody methylation of lincRNAs can negatively regulate lincRNA expression. We found that lincRNAs exhibit high correlation with their protein-coding neighbors in expression. Co-expression network analysis resulted in eight stage-specific modules, gene ontology and pathway analysis of them suggested that some lincRNAs were involved in IMF-related processes, such as fatty acid metabolism and peroxisome proliferator-activated receptor signaling pathway. Furthermore, we identified hub lincRNAs and found six of them may play important roles in IMF development. This work detailed some lincRNAs which may affect of IMF development in pig, and facilitated future research on these lincRNAs and molecular assisted breeding for pig.

374 Mechanism of continuous high ambient temperature affecting meat quality of finishing pigs

This study investigated the effect of high ambient temperature on meat quality and its mechanism in finishing pigs. All pigs received the same wheat–soybean meal diet. Twenty-four Landrace × Large White pigs (60 kg BW) were assigned to 3 groups—those fed ad libitum at 22°C, those fed ad libitum at 35°C, and those fed at 22°C, but fed the amount consumed by those kept at 35°C—and the experiment lasted for 30 d. Feed intake, weight gain, and intramuscular fat content of pigs were reduced, both directly by high temperature and indirectly through reduced feed intake. Transcriptome analysis showed that downregulated genes caused by feed restriction were mainly involved in muscle contraction, muscle development, and muscle system process or differentiation and that upregulated genes were mainly involved in response to nutrient levels or extracellular stimulus. Downregulated genes caused by high temperature were mainly involved in muscle structure and development, energy, or catabolic metabolism, whereas upregulated genes were mainly involved in DNA or protein damage or recombination, cell cycle process or biogenesis, stress response, or immune response. The mechanisms by which high temperature and reduced feed intake affected meat quality were not completely consistent. Apart from its effects of reducing feed intake, a direct effect of high temperature on meat quality also was involved in negatively regulating cell cycle, stimulating protein, DNA damage and cell apoptosis, and heat stress response. Based on this work, microRNA profiles were analyzed and the integrated analysis of microRNA–mRNA expression levels highlighted an inverse correlation between microRNA and their putative target genes, which further verified the negative effects of high ambient temperatures on feed intake and BW gain in finishing pigs. The results in this study indicate that high temperature decreased energy metabolism and muscle tissue development and increased DNA damage or stress response through regulating microRNA and their targets genes.

Identification of genes related to intramuscular fat content of pigs using genome-wide association study.

ObjectiveThe aim of this study is to identify single nucleotide polymorphisms (SNPs) and genes related to pig IMF and estimate the heritability of intramuscular fat content (IMF).MethodsGenome-wide association study (GWAS) on 704 inbred Berkshires was performed for IMF. To consider the inbreeding among samples, associations of the SNPs with IMF were tested as random effects in a mixed linear model using the genetic relationship matrix by GEMMA. Significant genes were compared with reported pig IMF quantitative trait loci (QTL) regions and functional classification of the identified genes were also performed. Heritability of IMF was estimated by GCTA tool.ResultsTotal 365 SNPs were found to be significant from a cutoff of p-value <0.01 and the 365 significant SNPs were annotated across 120 genes. Twenty five genes were on pig IMF QTL regions. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator, forkhead box protein O1, ectodysplasin A receptor, ring finger protein 149, cluster of differentiation, tyrosine-protein phosphatase non-receptor type 1, SRY (sex determining region Y)-box 9 (SOX9), MYC proto-oncogene, and macrophage migration inhibitory factor were related to mitogen-activated protein kinase pathway, which regulates the differentiation to adipocytes. These genes and the genes mapped on QTLs could be the candidate genes affecting IMF. Heritability of IMF was estimated as 0.52, which was relatively high, suggesting that a considerable portion of the total variance of IMF is explained by the SNP information.ConclusionOur results can contribute to breeding pigs with better IMF and therefore, producing pork with better sensory qualities.

Identification of differentially expressed genes in longissimus muscle of pigs with high and low intramuscular fat content using RNA sequencing.

Intramuscular fat (IMF) content in pork is an important element of consumer preference and is positively correlated with meat quality, including tenderness and juiciness. With advances in RNA sequencing technologies, transcriptome-related differences can be associated with specific traits in animals. The objective of this study was to investigate differentially expressed genes (DEGs) closely related to IMF content in porcine longissimus muscle using RNA sequencing. A total of 107 Berkshire pigs were used for IMF content measurements, and significant differences between extremely high (H, n=3) and low (L, n=3) IMF content groups were found (P<0.0001). From multi-dimensional scaling analyses, it was observed that the relationships between H and L groups were similar to each other. Here, we identified a total of 134 genes that were differentially expressed between the groups (false discovery rate <0.05; fold change ≥2). Functional analyses with DEGs revealed that lipid metabolism (SCD and FASN) was one of the significant biological processes related to IMF content determination. In addition, we found that DEGs related to muscle regeneration (MYOG and VEGFA) and extracellular matrix (COL1A1, COL1A2, COL5A1, COL14A1 and COL15A1) were changed among individuals with extreme IMF contents. These results will aid in understanding the regulation of IMF content in pigs.