Identification of candidate regulatory genes for intramuscular fatty acid composition in pigs by transcriptome analysis.

BACKGROUND: Intramuscular fat (IMF) content and its fatty acid (FA) composition are typically controlled by several genes, each with a small effect. In the current study, to pinpoint candidate genes and putative regulators involved in FA composition, we performed a multivariate integrative analysis between intramuscular FA and transcriptome profiles of porcine longissimus dorsi (LD) muscle. We also carried out a combination of network, regulatory impact factor (RIF), in silico prediction of putative target genes, and functional analyses to better support the biological relevance of our findings. RESULTS: For this purpose, we used LD RNA-Seq and intramuscular FA composition profiles of 129 Iberian × Duroc backcrossed pigs. We identified 378 correlated variables (13 FA and 365 genes), including six FA (C20:4n-6, C18:2n-6, C20:3n-6, C18:1n-9, C18:0, and C16:1n-7) that were among the most interconnected variables in the predicted network. The detected FA-correlated genes include genes involved in lipid and/or carbohydrate metabolism or in regulation of IMF deposition (e.g., ADIPOQ, CHUK, CYCS, CYP4B1, DLD, ELOVL6, FBP1, G0S2, GCLC, HMGCR, IDH3A, LEP, LGALS12, LPIN1, PLIN1, PNPLA8, PPP1R1B, SDR16C5, SFRP5, SOD3, SNW1, and TFRC), meat quality (GALNT15, GOT1, MDH1, NEU3, PDHA1, SDHD, and UNC93A), and transport (e.g., EXOC7 and SLC44A2). Functional analysis highlighted 54 over-represented gene ontology terms, including well-known biological processes and pathways that regulate lipid and carbohydrate metabolism. RIF analysis suggested a pivotal role for six transcription factors (CARHSP1, LBX1, MAFA, PAX7, SIX5, and TADA2A) as putative regulators of gene expression and intramuscular FA composition. Based on in silico prediction, we identified putative target genes for these six regulators. Among these, TADA2A and CARHSP1 had extreme RIF scores and present novel regulators in pigs. In addition, the expression of TADA2A correlated (either positively or negatively) with C20:4n-6, C18:2n-6, C20:3n-6, C18:1n-9, and that of CARHSP1 correlated (positively) with the C16:1n-7 lipokine. We also found that these two transcription factors share target genes that are involved in lipid metabolism (e.g., GOT1, PLIN1, and TFRC). CONCLUSIONS: This integrative analysis of muscle transcriptome and intramuscular FA profile revealed valuable information about key candidate genes and potential regulators for FA and lipid metabolism in pigs, among which some transcription factors are proposed to control gene expression and modulate FA composition differences.

Low n-6/n-3 Gestation and Lactation Diets Influence Early Performance, Muscle and Adipose Polyunsaturated Fatty Acid Content and Deposition, and Relative Abundance of Proteins in Suckling Piglets.

Elevated omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) ratios in swine diets can potentially impose a higher risk of inflammatory and metabolic diseases in swine. A low ratio between the two omega PUFAs has beneficial effects on sows' and piglets' production performance and immunity status. At present, there are few studies on how sow nutrition directly affects the protein and fat deposition in suckling piglets. Two groups of sows were fed diets with high or low n-6/n-3 polyunsaturated ratios of 13:1 (SOY) and 4:1 (LIN), respectively, during gestation and lactation. Longissimus dorsi muscle and adipose tissue from newborn piglets, nourished only with sow's milk, were subjected to fatty acid profiling by gas chromatography-mass spectrometry (GC-MS) and to proteomics assays based on nano-liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-HRMS). Fatty acid profiles on both muscle and adipose tissues resembled the magnitude of the differences between fatty acid across diets. Proteomic analysis revealed overabundance of 4 muscle and 11 adipose tissue proteins in SOY compared to LIN in both piglet tissues. The detected overabundance of haptoglobin, an acute-phase protein, and the stimulation of protein-coding genes and proteins related to the innate immune response and acute inflammatory response could be associated with the pro-inflammatory role of n-6 PUFAs.

Deciphering the regulation of porcine genes influencing growth, fatness and yield-related traits through genetical genomics.

Genetical genomics approaches aim at identifying quantitative trait loci for molecular traits, also known as intermediate phenotypes, such as gene expression, that could link variation in genetic information to physiological traits. In the current study, an expression GWAS has been carried out on an experimental Iberian × Landrace backcross in order to identify the genomic regions regulating the gene expression of those genes whose expression is correlated with growth, fat deposition, and premium cut yield measures in pig. The analyses were conducted exploiting Porcine 60K SNP BeadChip genotypes and Porcine Expression Microarray data hybridized on mRNA from Longissimus dorsi muscle. In order to focus the analysis on productive traits and reduce the number of analyses, only those probesets whose expression showed significant correlation with at least one of the seven phenotypes of interest were selected for the eGWAS. A total of 63 eQTL regions were identified with effects on 36 different transcripts. Those eQTLs overlapping with phenotypic QTLs on SSC4, SSC9, SSC13, and SSC17 chromosomes previously detected in the same animal material were further analyzed. Moreover, candidate genes and SNPs were analyzed. Among the most promising results, a long non-coding RNA, ALDBSSCG0000001928, was identified, whose expression is correlated with premium cut yield. Association analysis and in silico sequence domain annotation support TXNRD3 polymorphisms as candidate to regulate ALDBSSCG0000001928 expression, which can be involved in the transcriptional regulation of surrounding genes, affecting productive and meat quality traits.

Epigenetic regulation of the ELOVL6 gene is associated with a major QTL effect on fatty acid composition in pigs.

BACKGROUND: In previous studies on an Iberian x Landrace cross, we have provided evidence that supported the porcine ELOVL6 gene as the major causative gene of the QTL on pig chromosome 8 for palmitic and palmitoleic acid contents in muscle and backfat. The single nucleotide polymorphism (SNP) ELOVL6:c.-533C > T located in the promoter region of ELOVL6 was found to be highly associated with ELOVL6 expression and, accordingly, with the percentages of palmitic and palmitoleic acids in longissimus dorsi and adipose tissue. The main goal of the current work was to further study the role of ELOVL6 on these traits by analyzing the regulation of the expression of ELOVL6 and the implication of ELOVL6 polymorphisms on meat quality traits in pigs. RESULTS: High-throughput sequencing of BAC clones that contain the porcine ELOVL6 gene coupled to RNAseq data re-analysis showed that two isoforms of this gene are expressed in liver and adipose tissue and that they differ in number of exons and 3'UTR length. Although several SNPs in the 3'UTR of ELOVL6 were associated with palmitic and palmitoleic acid contents, this association was lower than that previously observed with SNP ELOVL6:c.-533C > T. This SNP is in full linkage disequilibrium with SNP ELOVL6:c.-394G > A that was identified in the binding site for estrogen receptor alpha (ERα). Interestingly, the ELOVL6:c.-394G allele is associated with an increase in methylation levels of the ELOVL6 promoter and with a decrease of ELOVL6 expression. Therefore, ERα is clearly a good candidate to explain the regulation of ELOVL6 expression through dynamic epigenetic changes in the binding site of known regulators of ELOVL6 gene, such as SREBF1 and SP1. CONCLUSIONS: Our results strongly suggest the ELOVL6:c.-394G > A polymorphism as the causal mutation for the QTL on pig chromosome 8 that affects fatty acid composition in pigs.

From SNP co-association to RNA co-expression: novel insights into gene networks for intramuscular fatty acid composition in porcine.

BACKGROUND: Fatty acids (FA) play a critical role in energy homeostasis and metabolic diseases; in the context of livestock species, their profile also impacts on meat quality for healthy human consumption. Molecular pathways controlling lipid metabolism are highly interconnected and are not fully understood. Elucidating these molecular processes will aid technological development towards improvement of pork meat quality and increased knowledge of FA metabolism, underpinning metabolic diseases in humans. RESULTS: The results from genome-wide association studies (GWAS) across 15 phenotypes were subjected to an Association Weight Matrix (AWM) approach to predict a network of 1,096 genes related to intramuscular FA composition in pigs. To identify the key regulators of FA metabolism, we focused on the minimal set of transcription factors (TF) that the explored the majority of the network topology. Pathway and network analyses pointed towards a trio of TF as key regulators of FA metabolism: NCOA2, FHL2 and EP300. Promoter sequence analyses confirmed that these TF have binding sites for some well-know regulators of lipid and carbohydrate metabolism. For the first time in a non-model species, some of the co-associations observed at the genetic level were validated through co-expression at the transcriptomic level based on real-time PCR of 40 genes in adipose tissue, and a further 55 genes in liver. In particular, liver expression of NCOA2 and EP300 differed between pig breeds (Iberian and Landrace) extreme in terms of fat deposition. Highly clustered co-expression networks in both liver and adipose tissues were observed. EP300 and NCOA2 showed centrality parameters above average in the both networks. Over all genes, co-expression analyses confirmed 28.9% of the AWM predicted gene-gene interactions in liver and 33.0% in adipose tissue. The magnitude of this validation varied across genes, with up to 60.8% of the connections of NCOA2 in adipose tissue being validated via co-expression. CONCLUSIONS: Our results recapitulate the known transcriptional regulation of FA metabolism, predict gene interactions that can be experimentally validated, and suggest that genetic variants mapped to EP300, FHL2, and NCOA2 modulate lipid metabolism and control energy homeostasis in pigs.

New insight into the SSC8 genetic determination of fatty acid composition in pigs.

BACKGROUND: Fat content and fatty acid composition in swine are becoming increasingly studied because of their effect on sensory and nutritional quality of meat. A QTL (quantitative trait locus) for fatty acid composition in backfat was previously detected on porcine chromosome 8 (SSC8) in an Iberian x Landrace F2 intercross. More recently, a genome-wide association study detected the same genomic region for muscle fatty acid composition in an Iberian x Landrace backcross population. ELOVL6, a strong positional candidate gene for this QTL, contains a polymorphism in its promoter region (ELOVL6:c.-533C < T), which is associated with percentage of palmitic and palmitoleic acids in muscle and adipose tissues. Here, a combination of single-marker association and the haplotype-based approach was used to analyze backfat fatty acid composition in 470 animals of an Iberian x Landrace F2 intercross genotyped with 144 SNPs (single nucleotide polymorphisms) distributed along SSC8. RESULTS: Two trait-associated SNP regions were identified at 93 Mb and 119 Mb on SSC8. The strongest statistical signals of both regions were observed for palmitoleic acid (C16:1(n-7)) content and C18:0/C16:0 and C18:1(n-7)/C16:1(n-7) elongation ratios. MAML3 and SETD7 are positional candidate genes in the 93 Mb region and two novel microsatellites in MAML3 and nine SNPs in SETD7 were identified. No significant association for the MAML3 microsatellite genotypes was detected. The SETD7:c.700G > T SNP, although statistically significant, was not the strongest signal in this region. In addition, the expression of MAML3 and SETD7 in liver and adipose tissue varied among animals, but no association was detected with the polymorphisms in these genes. In the 119 Mb region, the ELOVL6:c.-533C > T polymorphism showed a strong association with percentage of palmitic and palmitoleic fatty acids and elongation ratios in backfat. CONCLUSIONS: Our results suggest that the polymorphisms studied in MAML3 and SETD7 are not the causal mutations for the QTL in the 93 Mb region. However, the results for ELOVL6 support the hypothesis that the ELOVL6:c.-533C > T polymorphism has a pleiotropic effect on backfat and intramuscular fatty acid composition and that it has a role in the determination of the QTL in the 119 Mb region.

Interrelation between gut microbiota, SCFA, and fatty acid composition in pigs.

The gut microbiota is a key player in the host metabolism. Some bacteria are able to ferment non-digestible compounds and produce short-chain fatty acids that the host can later transform and accumulate in tissue. In this study, we aimed to better understand the relationships between the microorganisms and the short-chain fatty acid composition of the rectal content, including the possible linkage with the fatty acid composition in backfat and muscle of the pig. We studied a Duroc × Iberian crossbred population, and we found significant correlations between different bacterial and archaeal genera and the fatty acid profile. The abundance of n-butyric acid in the rectal content was positively associated with Prevotella spp. and negatively associated with Akkermansia spp., while conversely, the abundance of acetic acid was negatively and positively associated with the levels of Prevotella spp. and Akkermansia spp., respectively. The most abundant genus, Rikenellaceae RC9 gut group, had a positive correlation with palmitic acid in muscle and negative correlations with stearic acid in backfat and oleic acid in muscle. These results suggest the possible role of Prevotella spp. and Akkermansia spp. as biomarkers for acetic and n-butyric acids, and the relationship of Rikenellaceae RC9 gut group with the lipid metabolism, building up the potential, although indirect, role of the microbiota in the modification of the backfat and muscle fatty acid composition of the host.IMPORTANCEThe vital role of the gut microbiota on its host metabolism makes it essential to know how its modulation is mirrored on the fatty acid composition of the host. Our findings suggest Prevotella spp. and Akkermansia spp. as potential biomarkers for the levels of beneficial short-chain fatty acids and the possible influence of Rikenellaceae RC9 gut group in the backfat and muscle fatty acid composition of the pig.

Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition.

BACKGROUND: In pigs, adipose tissue is one of the principal organs involved in the regulation of lipid metabolism. It is particularly involved in the overall fatty acid synthesis with consequences in other lipid-target organs such as muscles and the liver. With this in mind, we have used massive, parallel high-throughput sequencing technologies to characterize the porcine adipose tissue transcriptome architecture in six Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition (three per group). RESULTS: High-throughput RNA sequencing was used to generate a whole characterization of adipose tissue (backfat) transcriptome. A total of 4,130 putative unannotated protein-coding sequences were identified in the 20% of reads which mapped in intergenic regions. Furthermore, 36% of the unmapped reads were represented by interspersed repeats, SINEs being the most abundant elements. Differential expression analyses identified 396 candidate genes among divergent animals for intramuscular fatty acid composition. Sixty-two percent of these genes (247/396) presented higher expression in the group of pigs with higher content of intramuscular SFA and MUFA, while the remaining 149 showed higher expression in the group with higher content of PUFA. Pathway analysis related these genes to biological functions and canonical pathways controlling lipid and fatty acid metabolisms. In concordance with the phenotypic classification of animals, the major metabolic pathway differentially modulated between groups was de novo lipogenesis, the group with more PUFA being the one that showed lower expression of lipogenic genes. CONCLUSIONS: These results will help in the identification of genetic variants at loci that affect fatty acid composition traits. The implications of these results range from the improvement of porcine meat quality traits to the application of the pig as an animal model of human metabolic diseases.

Exon-intron split analysis reveals posttranscriptional regulatory signals induced by high and low n-6/n-3 polyunsaturated fatty acid ratio diets in piglets.

Polyunsaturated fatty acids (PUFA), such as omega-6 (n-6) and omega-3 (n-3), play a vital role in nutrient metabolism, inflammatory response, and gene regulation. microRNAs (miRNA), which can potentially degrade targeted messenger RNAs (mRNA) and/or inhibit their translation, might play a relevant role in PUFA-related changes in gene expression. Although differential expression analyses can provide a comprehensive picture of gene expression variation, they are unable to disentangle when in the mRNA life cycle the regulation of expression is taking place, including any putative functional miRNA-driven repression. To capture this, we used an exon-intron split analysis (EISA) approach to account for posttranscriptional changes in response to extreme values of n-6/n-3 PUFA ratio. Longissimus dorsi muscle samples of male and female piglets from sows fed with n-6/n-3 PUFA ratio of 13:1 (SOY) or 4:1 (LIN), were analyzed in a bidirectional contrast (LIN vs. SOY, SOY vs. LIN). Our results allowed the identification of genes showing strong posttranscriptional downregulation signals putatively targeted by significantly upregulated miRNA. Moreover, we identified genes primarily involved in the regulation of lipid-related metabolism and immune response, which may be associated with the pro- and anti-inflammatory functions of the n-6 and n-3 PUFA, respectively. EISA allowed us to uncover regulatory networks complementing canonical differential expression analyses, thus providing a more comprehensive view of muscle metabolic changes in response to PUFA concentration.

The relationship between dietary lipids, such as omega-6 and omega-3 polyunsaturated fatty acids (PUFA), and gene expression regulation was explored in piglet muscle. While these PUFA can influence nutrient metabolism and inflammatory response, small regulatory molecules called microRNAs (miRNA) can also influence the activity of genes. In this experiment, we used a computational approach dubbed exon­intron split analysis (EISA) to fully understand the role of miRNA in this context and how the genes and miRNA respond to changes in PUFA levels. Our findings demonstrated that some genes involved in lipid metabolism and immune response were affected by different PUFA concentrations and that EISA provides a more comprehensive view of how genes are regulated throughout their life cycle.

Identifying miRNA-mRNA regulatory networks on extreme n-6/n-3 polyunsaturated fatty acid ratio expression profiles in porcine skeletal muscle.

Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids with antagonistic inflammatory functions that play vital roles in metabolic health and immune response. Current commercial swine diets tend to over-supplement with n-6 PUFAs, which may increase the likelihood of developing inflammatory diseases and affect the overall well-being of the animals. However, it is still poorly understood how n-6/n-3 PUFA ratios affect the porcine transcriptome expression and how messenger RNAs (mRNAs) and microRNAs (miRNAs) might regulate biological processes related to PUFA metabolism. On account of this, we selected a total of 20 Iberian × Duroc crossbred pigs with extreme values for n-6/n-3 FA ratio (10 high vs 10 low), and longissimus dorsi muscle samples were used to identify differentially expressed mRNAs and miRNAs. The observed differentially expressed mRNAs were associated to biological pathways related to muscle growth and immunomodulation, while the differentially expressed microRNAs (ssc-miR-30a-3p, ssc-miR-30e-3p, ssc-miR-15b and ssc-miR-7142-3p) were correlated to adipogenesis and immunity. Relevant miRNA-to-mRNA regulatory networks were also predicted (i.e., mir15b to ARRDC3; mir-7142-3p to METTL21C), and linked to lipolysis, obesity, myogenesis, and protein degradation. The n-6/n-3 PUFA ratio differences in pig skeletal muscle revealed genes, miRNAs and enriched pathways involved in lipid metabolism, cell proliferation and inflammation.

Global analysis of the association between pig muscle fatty acid composition and gene expression using RNA-Seq.

Fatty acids (FAs) play an essential role as mediators of cell signaling and signal transduction, affecting metabolic homeostasis and determining meat quality in pigs. However, FAs are transformed by the action of several genes, such as those encoding desaturases and elongases of FAs in lipogenic tissues. The aim of the current work was to identify candidate genes, biological processes, and pathways involved in the modulation of intramuscular FA profile from longissimus dorsi muscle. FA profile by gas chromatography of methyl esters and gene expression by RNA-Seq were determined in 129 Iberian × Duroc backcrossed pigs. An association analysis between the muscle transcriptome and its FA profile was performed, followed by a concordance and functional analysis. Overall, a list of well-known (e.g., PLIN1, LEP, ELOVL6, SC5D, NCOA2, ACSL1, MDH1, LPL, LGALS12, TFRC, GOT1, and FBP1) and novel (e.g., TRARG1, TANK, ENSSSCG00000011196, and ENSSSCG00000038429) candidate genes was identified, either in association with specific or several FA traits. Likewise, several of these genes belong to biological processes and pathways linked to energy, lipid, and carbohydrate metabolism, which seem determinants in the modulation of FA compositions. This study can contribute to elucidate the complex relationship between gene expression and FA profile in pig muscle.

Copy Number Variation on ABCC2-DNMBP Loci Affects the Diversity and Composition of the Fecal Microbiota in Pigs.

Genetic variation in the pig genome partially modulates the composition of porcine gut microbial communities. Previous studies have been focused on the association between single nucleotide polymorphisms (SNPs) and the gut microbiota, but little is known about the relationship between structural variants and fecal microbial traits. The main goal of this study was to explore the association between porcine genome copy number variants (CNVs) and the diversity and composition of pig fecal microbiota. For this purpose, we used whole-genome sequencing data to undertake a comprehensive identification of CNVs followed by a genome-wide association analysis between the estimated CNV status and the fecal bacterial diversity in a commercial Duroc pig population. A CNV predicted as gain (DUP) partially harboring ABCC2-DNMBP loci was associated with richness (P = 5.41 × 10-5, false discovery rate [FDR] = 0.022) and Shannon α-diversity (P = 1.42 × 10-4, FDR = 0.057). The in silico predicted gain of copies was validated by real-time quantitative PCR (qPCR), and its segregation, and positive association with the richness and Shannon α-diversity of the porcine fecal bacterial ecosystem was confirmed in an unrelated F1 (Duroc × Iberian) cross. Our results advise the relevance of considering the role of host-genome structural variants as potential modulators of microbial ecosystems and suggest the ABCC2-DNMBP CNV as a host-genetic factor for the modulation of the diversity and composition of the fecal microbiota in pigs. IMPORTANCE A better understanding of the environmental and host factors modulating gut microbiomes is a topic of greatest interest. Recent evidence suggests that genetic variation in the pig genome partially controls the composition of porcine gut microbiota. However, since previous studies have been focused on the association between single nucleotide polymorphisms and the fecal microbiota, little is known about the relationship between other sources of genetic variation, like the structural variants and microbial traits. Here, we identified, experimentally validated, and replicated in an independent population a positive link between the gain of copies of ABCC2-DNMBP loci and the diversity and composition of pig fecal microbiota. Our results advise the relevance of considering the role of host-genome structural variants as putative modulators of microbial ecosystems and open the possibility of implementing novel holobiont-based management strategies in breeding programs for the simultaneous improvement of microbial traits and host performance.

Liver transcriptome profile in pigs with extreme phenotypes of intramuscular fatty acid composition.

BACKGROUND: New advances in high-throughput technologies have allowed for the massive analysis of genomic data, providing new opportunities for the characterization of the transcriptome architectures. Recent studies in pigs have employed RNA-Seq to explore the transcriptome of different tissues in a reduced number of animals. The main goal of this study was the identification of differentially-expressed genes in the liver of Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition using RNA-Seq. RESULTS: The liver transcriptomes of two female groups (H and L) with phenotypically extreme intramuscular fatty acid composition were sequenced using RNA-Seq. A total of 146 and 180 unannotated protein-coding genes were identified in intergenic regions for the L and H groups, respectively. In addition, a range of 5.8 to 7.3% of repetitive elements was found, with SINEs being the most abundant elements. The expression in liver of 186 (L) and 270 (H) lncRNAs was also detected. The higher reproducibility of the RNA-Seq data was validated by RT-qPCR and porcine expression microarrays, therefore showing a strong correlation between RT-qPCR and RNA-Seq data (ranking from 0.79 to 0.96), as well as between microarrays and RNA-Seq (r=0.72). A differential expression analysis between H and L animals identified 55 genes differentially-expressed between groups. Pathways analysis revealed that these genes belong to biological functions, canonical pathways and three gene networks related to lipid and fatty acid metabolism. In concordance with the phenotypic classification, the pathways analysis inferred that linolenic and arachidonic acids metabolism was altered between extreme individuals. In addition, a connection was observed among the top three networks, hence suggesting that these genes are interconnected and play an important role in lipid and fatty acid metabolism. CONCLUSIONS: In the present study RNA-Seq was used as a tool to explore the liver transcriptome of pigs with extreme phenotypes for intramuscular fatty acid composition. The differential gene expression analysis showed potential gene networks which affect lipid and fatty acid metabolism. These results may help in the design of selection strategies to improve the sensorial and nutritional quality of pork meat.

Expression analysis of porcine miR-33a/b in liver, adipose tissue and muscle and its potential role in fatty acid metabolism.

mir-33a and mir-33b are co-transcribed with the SREBF2 and SREBF1 transcription factors, respectively. The main role of SREBF1 is the regulation of genes involved in fatty acid metabolism, while SREBF2 regulates genes participating in cholesterol biosynthesis and uptake. Our objective was to study the expression of both miR-33a and miR-33b, together with their host SREBF genes, in liver, adipose tissue and muscle to better understand the role of miR-33a/b in the lipid metabolism of pigs. In our study, the expression of miR-33a, miR-33b and SREBF2 in liver, adipose tissue, and muscle was studied in 42 BC1_LD (25% Iberian x 75% Landrace backcross) pigs by RT-qPCR. In addition, the expression of in-silico predicted target genes and fatty acid composition traits were correlated with the miR-33a/b expression. We observed different tissue expression patterns for both miRNAs. In adipose tissue and muscle a high correlation between miR-33a and miR-33b expression was found, whereas a lower correlation was observed in liver. The expression analysis of in-silico predicted target-lipid related genes showed negative correlations between miR-33b and CPT1A expression in liver. Conversely, positive correlations between miR-33a and PPARGC1A and USF1 gene expression in liver were observed. Lastly, positive and negative correlations between miR-33a/b expression and saturated fatty acid (SFA) and polyunsaturated fatty acid (PUFA) content, respectively, were identified. Overall, our results suggested that both miRNAs are differentially regulated and have distinct functions in liver, in contrast to muscle and adipose tissue. Furthermore, the correlations between miR-33a/b expression both with the expression of in-silico predicted target-lipid related genes and with fatty acid composition, opens new avenues to explore the role of miR33a/b in the regulation of lipid metabolism.

Copy number variation in the porcine genome inferred from a 60 k SNP BeadChip.

BACKGROUND: Recent studies in pigs have detected copy number variants (CNVs) using the Comparative Genomic Hybridization technique in arrays designed to cover specific porcine chromosomes. The goal of this study was to identify CNV regions (CNVRs) in swine species based on whole genome SNP genotyping chips. RESULTS: We used predictions from three different programs (cnvPartition, PennCNV and GADA) to analyze data from the Porcine SNP60 BeadChip. A total of 49 CNVRs were identified in 55 animals from an Iberian x Landrace cross (IBMAP) according to three criteria: detected in at least two animals, contained three or more consecutive SNPs and recalled by at least two programs. Mendelian inheritance of CNVRs was confirmed in animals belonging to several generations of the IBMAP cross. Subsequently, a segregation analysis of these CNVRs was performed in 372 additional animals from the IBMAP cross and its distribution was studied in 133 unrelated pig samples from different geographical origins. Five out of seven analyzed CNVRs were validated by real time quantitative PCR, some of which coincide with well known examples of CNVs conserved across mammalian species. CONCLUSIONS: Our results illustrate the usefulness of Porcine SNP60 BeadChip to detect CNVRs and show that structural variants can not be neglected when studying the genetic variability in this species.

Hypothalamic expression of porcine leptin receptor (LEPR), neuropeptide Y (NPY), and cocaine- and amphetamine-regulated transcript (CART) genes is influenced by LEPR genotype.

The leptin receptor (LEPR) is a key gene in the control of food intake and energy homeostasis. The sequence variant LEPR{NM_001024587.1}:c.1987C>T has been associated with growth, fatness, and body composition in several pig populations. The purpose of this work was to confirm the phenotypic effects of this SNP in two new experimental backcrosses involving Iberian, Landrace, and Duroc breeds, and to evaluate the quantitative effects of the SNP on the hypothalamic expression of LEPR and two other downstream genes. Results indicate significant additive effects of the SNP on body weight, back fat thickness, and hypothalamic LEPR gene expression in both populations. Allele T fixed in the Iberian breed is systematically associated with a higher growth and fat deposition and leads to an intense reduction of LEPR hypothalamic expression, providing new functional evidence that supports the causality of the analyzed SNP with respect to previously reported and newly observed phenotypic effects. Also, some effects of the LEPR genotype on neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) genes are detected, although they are conditioned by the breed. Finally, a change in mRNA structure and an increase in free energy is predicted for allele T, agreeing with a cis-acting functional effect on mRNA stability, which also supports the causality hypothesis. The lower expression of the LEPR gene in Iberian pigs fits with obesity by leptin resistance observed in this breed. A reduction in leptin signaling could thus be considered one of the determinants of the obese phenotype characteristic of Iberian breed.

Identification of strong candidate genes for backfat and intramuscular fatty acid composition in three crosses based on the Iberian pig.

Meat quality has an important genetic component and can be modified by the fatty acid (FA) composition and the amount of fat contained in adipose tissue and muscle. The present study aimed to find genomic regions associated with the FA composition in backfat and muscle (longissimus dorsi) in 439 pigs with three different genetic backgrounds but having the Iberian breed in common. Genome-wide association studies (GWAS) were performed between 38,424 single-nucleotide polymorphisms (SNPs) covering the pig genome and 60 phenotypic traits related to backfat and muscle FA composition. Nine significant associated regions were found in backfat on the Sus scrofa chromosomes (SSC): SSC1, SSC2, SSC4, SSC6, SSC8, SSC10, SSC12, and SSC16. For the intramuscular fat, six significant associated regions were identified on SSC4, SSC13, SSC14, and SSC17. A total of 52 candidate genes were proposed to explain the variation in backfat and muscle FA composition traits. GWAS were also reanalysed including SNPs on five candidate genes (ELOVL6, ELOVL7, FADS2, FASN, and SCD). Regions and molecular markers described in our study may be useful for meat quality selection of commercial pig breeds, although several polymorphisms were breed-specific, and further analysis would be needed to evaluate possible causal mutations.

Identification of eQTLs associated with lipid metabolism in Longissimus dorsi muscle of pigs with different genetic backgrounds.

Intramuscular fat content and its fatty acid composition affect porcine meat quality and its nutritional value. The present work aimed to identify genomic variants regulating the expression in the porcine muscle (Longissimus dorsi) of 45 candidate genes for lipid metabolism and fatty acid composition in three experimental backcrosses based on the Iberian breed. Expression genome-wide association studies (eGWAS) were performed between the muscle gene expression values, measured by real-time quantitative PCR, and the genotypes of 38,426 SNPs distributed along all chromosomes. The eGWAS identified 186 eSNPs located in ten Sus scrofa regions and associated with the expression of ACSM5, ACSS2, ATF3, DGAT2, FOS and IGF2 (FDR < 0.05) genes. Two expression quantitative trait loci (eQTLs) for IGF2 and ACSM5 were classified as cis-acting eQTLs, suggesting a mutation in the same gene affecting its expression. Conversely, ten eQTLs showed trans-regulatory effects on gene expression. When the eGWAS was performed for each backcross independently, only three common trans-eQTL regions were observed, indicating different regulatory mechanisms or allelic frequencies among the breeds. In addition, hotspot regions regulating the expression of several genes were detected. Our results provide new data to better understand the functional regulatory mechanisms of lipid metabolism genes in muscle.

Phytogenic actives supplemented in hyperprolific sows: effects on maternal transfer of phytogenic compounds, colostrum and milk features, performance and antioxidant status of sows and their offspring, and piglet intestinal gene expression.

Phytogenic actives (PA) are plant-derived natural bioactive compounds that may promote livestock health and well-being, as well as improve growth performance and production efficiency. The current study aims to evaluate their effects on sows and their offspring. Eighty-one hyperprolific sows (up to parity 7) were assigned to 3 experimental treatments. Control sows were offered a nonsupplemented diet during gestation and lactation, and treated sows were fed the control diet supplemented with 1 g/kg of a blend of PA (BPA) in lactation (L) or during gestation and lactation (GL). An evaluation was made of placental and milk maternal transfer of these BPA and colostrum-milk features, sows and piglets antioxidant status, reproductive performance (litter size), body weight (BW) changes, weaning-estrus interval, and litter performance. Finally, piglet´s jejunum gene expression was measured. The BPA supplementation during gestation (GL) increased the number of piglets born alive (P = 0.020) and reduced (P < 0.05) the newborn piglets BW, while there were no differences among treatments on the suckling (day 20) and weaned (day 7) piglets BW (P > 0.05). Dietary phytogenic volatile compounds reached GL placental fluid, and milk of L and GL sows (P < 0.05). Moreover, colostrum protein in GL and milk fat content in L and GL were increased (P < 0.05). Milk of GL showed inhibitory activity against Bacillus subtilis and Staphylococcus aureus (P < 0.05). Antioxidant status of GL sows showed an enhanced (P < 0.05) of catalase (CAT) and total antioxidant capacity levels at early gestation (day 35), whereas higher levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes at late gestation (day 110). Likewise, GL newborn piglets showed higher CAT levels, whereas both CAT and SOD levels in suckling piglets, as well as CAT, SOD, and GSH-Px in weaned piglets, were increased in L and GL (P < 0.05). Jejunum messenger ribonucleic acid abundance of suckling piglets in L and GL groups showed overexpression of barrier function MUC2, digestive enzyme IDO, and immune response PPARGC-α, TNF-α, TGF-ß1, and IL-10 genes (P < 0.05). In conclusion, dietary BPA supplementation in hyperprolific sows increased the litter size (born alive) and improved the composition and bioactivity of colostrum and milk, besides, modified the antioxidant status of sows and their offspring, as well as the suckling piglets gut health gene expression. Several BPA volatile compounds were prenatal and postnatal maternally transferred (placental fluid and milk).