Long-Term Effect of Maternal Antioxidant Supplementation on the Lipid Profile of the Progeny According to the Sow's Parity Number.

Pig feeding prior to the extensive fattening phase might affect the final lipid profile and product quality. This study evaluates how maternal supplementation with vitamin E (VITE) (100 mg/kg), hydroxytyrosol (HXT) (1.5 mg/kg), or combined administration (VE + HXT) affects the piglet's plasma and tissues' fatty acid profiles and lipid stability according to the sow's parity number (PN), as well as the possible changes to the lipid profile after extensive feeding. The sows' PN affected the total fatty acid profile of plasma, muscle, and liver of piglets, with lower Δ-9 and Δ-6 desaturase indices but higher Δ-5 in those from primiparous (P) than multiparous (M) sows. Dietary VITE was more effective at decreasing C16:0 and saturated fatty acids in the muscle of piglets born from M than P sows, and modified the liver phospholipids in a different way. Sows' supplementation with HXT increased C18:2n-6 in triglycerides and polyunsaturated fatty acids (PUFA) in muscle phospholipids. In the liver, HXT supplementation also increased free-PUFA and free-n-3 fatty acids. However, lipid oxidation of piglets' tissues was not affected by the antioxidant supplementation, and it was higher in the livers of piglets born from M sows. The fatty acid profile in the muscle of pigs after extensive feeding was not affected by the PN, but it was by the sows' antioxidant supplementation, with positive effects on quality by both compounds.

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.

Maternal Supplementation of Vitamin E or Its Combination with Hydroxytyrosol Increases the Gut Health and Short Chain Fatty Acids of Piglets at Weaning.

An adequate intestinal environment before weaning may contribute to diarrhea predisposition and piglet development. This study evaluates how the dietary supplementation of vitamin E (VE) (100 mg/kg), hydroxytyrosol (HXT) (1.5 mg/kg) or the combined administration (VE + HXT) given to Iberian sows from gestation affects the piglet's faecal characteristics, short chain fatty acids (SCFAs), fatty acid profile or intestinal morphology as indicators of gut health; and quantify the contribution of the oxidative status and colostrum/milk composition to the piglet's SCFAs content and intestinal health. Dietary VE increased isobutyric acid (iC4), butyric acid (C4), isovaleric acid (iC5), and ∑SCFAs, whereas HXT increased iC4 and tended to decrease ∑SCFAs of faeces. Piglets from HXT-supplemented sows also tended to have higher faecal C20:4n-6/C20:2 ratio C22:6 proportion and showed lower occludin gene expression in the duodenum. The combination of both antioxidants had a positive effect on iC4 and iC5 levels. Correlation analyses and regression equations indicate that faecal SCFAs were related to oxidative status (mainly plasma VE) and colostrum and milk composition (mainly C20:2, C20:3, C20:4 n-6). This study would confirm the superiority of VE over HXT supplementation to improve intestinal homeostasis, gut health, and, consequently piglet growth.

Different Effect of Vitamin E or Hydroxytyrosol Supplementation to Sow's Diet on Oxidative Status and Performances of Weaned Piglets.

Different feeding strategies are being applied to sows in order to obtain homogeneous piglets' weights and improved health status. This study evaluated how the dietary supplementation of vitamin E (VE) (100 mg/kg), hydroxytyrosol (HXT) (1.5 mg/kg) or the combined administration (VE + HXT) given to Iberian sows from day 85 of gestation affected the growth pattern of the piglets and their oxidative status; and quantified what these effects were due to. Dietary VE and HXT improved the oxidative status of sows and piglets. Both VE and HXT modified the growth pattern at birth and performances of the piglets in a different way according to the growing period. Piglets' performances were positively correlated with plasma VE and negatively with plasma malondialdehyde (MDA) of the sow. However, the highest variation in growth patterns was explained by the colostrum composition. Significant linear equations were observed between piglets' performances and colostrum saturated (SAT), n-7 monounsaturated fatty acids (C16:1n-7 and C18:1n-7) and different desaturases indices. This study would confirm that VE supplementation to the sow diet could be more adequate than HXT for the improved development during the first weeks of a piglet's life. The combined administration of both antioxidants would not produce additional positive effects compared to the individual supplementation.

Dietary Vitamin E and/or Hydroxytyrosol Supplementation to Sows during Late Pregnancy and Lactation Modifies the Lipid Composition of Colostrum and Milk.

Modifying the composition of a sow's milk could be a strategy to improve the intestinal health and growth of her piglet during the first weeks of life. This study evaluated how dietary supplementation of vitamin E (VE), hydroxytyrosol (HXT) or VE+HXT given to Iberian sows from late gestation affected the colostrum and milk composition, lipid stability and their relationship with the piglet's oxidative status. Colostrum from VE-supplemented sows had greater C18:1n-7 than non-supplemented sows, whereas HXT increased polyunsaturated (∑PUFAs), ∑n-6 and ∑n-3 fatty acids. In 7-day milk, the main effects were induced by VE supplementation that decreased ∑PUFAs, ∑n-6 and ∑n-3 and increased the Δ-6-desaturase activity. The VE+HXT supplementation resulted in lower desaturase capacity in 20-day milk. Positive correlations were observed between the estimated mean milk energy output and the desaturation capacity of sows. The lowest concentration of malondialdehyde (MDA) in milk was observed in VE-supplemented groups, whereas HXT supplementation increased oxidation. Milk lipid oxidation was negatively correlated with the sow's plasma oxidative status and to a great extent with the oxidative status of piglets after weaning. Maternal VE supplementation produced a more beneficial milk composition to improve the oxidative status of piglets, which could promote gut health and piglet growth during the first weeks, but more research is needed to clarify this.

Design of a low-density SNP panel for intramuscular fat content and fatty acid composition of backfat in free-range Iberian pigs.

Intramuscular fat (IMF) content and fatty acids (FA) profile determine important sensory and technological characteristics of the meat, being interesting traits to be included in breeding programs for Iberian pig. In a previous study, we analyzed the longissimus dorsi transcriptome of Iberian pigs with divergent breeding values for IMF identifying 63 and 12 functional candidate genes and long non-coding RNA being differentially expressed. The first objective of this work was to use the RNA-seq data generated in that study in order to design a low-density panel with 50 single-nucleotide polymorphisms (SNP). The second aim was to evaluate the effects of this SNP panel on IMF content and backfat FA profile in a closed commercial population of Iberian pigs. The IMF content and backfat FA composition were determined in 940 Iberian pigs. A total of 837 SNPs were identified in the selection of genes and lncRNA. Out of the 50 selected SNP, 11 were monomorphic in the population and 2 failed in their genotyping. Association analyses were carried out for the 37 SNP segregating in the population (minor allele frequency, MAF > 0.05). Regarding IMF content, a significant additive effect (q-value < 0.05) was observed for ADIPOQ:g.124646194T > G SNP, in which the G allele was linked to an increase of 7.59% (additive effect expressed as a percentage of the mean of the trait). For the backfat FA composition, we observed 24 SNP with significant associations. The strongest effects were detected for the ELOVL6:g.112186423A > G and FASN_rs331694510G > A SNP. The A allele of both SNPs were associated to a lower percentage of palmitic (C16:0) and palmitoleic (C16:1) acids and an increase in oleic acid (C18:1). In addition, the AMPD3:g.49043886C > G SNP had significant effects on C16:0, stearic (C18:0), C18:1 and on the sum of saturated and monounsaturated fatty acids. Finally, significant effects of the EGR2:66286345G > T SNP on C18:0 and C18:1 and other minor FA were observed. Our findings reveal ADIPOQ:g.124646194T > G, ELOVL6:g.112186423A > G, and FASN_rs331694510G > A may be useful in breeding programs for Iberian pigs including FA profile and IMF content as selection goals, provided that their potential effects on other traits of interest are controlled.

Fat infiltration of muscle and fatty acid profile of cover fat are essential traits in Iberian pig industry. However, their inclusion in genetic selection schemes can be tangled, since their determination is laborious and expensive. Molecular markers can be a useful alternative to improve these traits. In a previous study, we analyzed the longissimus dorsi transcriptome of Iberian pigs with divergent breeding values for intramuscular fat (IMF) identifying 63 and 12 functional candidate genes and long non-coding RNA being differentially expressed. From this information, we designed a low-density panel with 50 single-nucleotide polymorphisms, which we subsequently evaluated for IMF content and fatty acids (FA) backfat profile of 940 pigs. The present research identified five polymorphisms mapped to ADIPOQ, ELOVL6, FASN, AMPD3, and EGR2 genes that are associated to IMF content and backfat FA profile.

Admixture and breed traceability in European indigenous pig breeds and wild boar using genome-wide SNP data.

Preserving diversity of indigenous pig (Sus scrofa) breeds is a key factor to (i) sustain the pork chain (both at local and global scales) including the production of high-quality branded products, (ii) enrich the animal biobanking and (iii) progress conservation policies. Single nucleotide polymorphism (SNP) chips offer the opportunity for whole-genome comparisons among individuals and breeds. Animals from twenty European local pigs breeds, reared in nine countries (Croatia: Black Slavonian, Turopolje; France: Basque, Gascon; Germany: Schwabisch-Hällisches Schwein; Italy: Apulo Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda; Lithuania: Indigenous Wattle, White Old Type; Portugal: Alentejana, Bísara; Serbia: Moravka, Swallow-Bellied Mangalitsa; Slovenia: Krskopolje pig; Spain: Iberian, Majorcan Black), and three commercial breeds (Duroc, Landrace and Large White) were sampled and genotyped with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. A dataset of 51 Wild Boars from nine countries was also added, summing up to 1186 pigs (~ 49 pigs/breed). The aim was to: (i) investigate individual admixture ancestries and (ii) assess breed traceability via discriminant analysis on principal components (DAPC). Albeit the mosaic of shared ancestries found for Nero Siciliano, Sarda and Moravka, admixture analysis indicated independent evolvement for the rest of the breeds. High prediction accuracy of DAPC mark SNP data as a reliable solution for the traceability of breed-specific pig products.

Understanding the role of myoglobin content in Iberian pigs fattened in an extensive system through analysis of the transcriptome profile.

Meat color is the first perceived sensory feature and one of the most important quality traits. Myoglobin is the main pigment in meat, giving meat its characteristic cherry-red color, highly appreciated by the consumers. In the current study, we used the RNA-seq technique to characterize the longissimus dorsi muscle transcriptome in two groups of Iberian pigs with divergent breeding values for myoglobin content. As a result, we identified 57 differentially expressed genes and transcripts (DEGs). Moreover, we have validated the RNA-seq expression of a set of genes by quantitative PCR (qPCR). Functional analyses revealed an enrichment of DEGs in biological processes related to oxidation (HBA1), lipid metabolism (ECH1, PLA2G10, PLD2), inflammation (CHST1, CD209, PLA2G10), and immune system (CD209, MX2, LGALS3, LGALS9). The upstream analysis showed a total of five transcriptional regulatory factors and eight master regulators that could moderate the expression of some DEGs, highlighting SPI1 and MAPK1, since they regulate the expression of DEGs involved in immune defense and inflammatory processes. Iberian pigs with high myoglobin content also showed higher expression of the HBA1 gene and both molecules, myoglobin and hemoglobin, have been described as having a protective effect against oxidative and inflammatory processes. Therefore, the HBA1 gene is a very promising candidate gene to harbor polymorphisms underlying myoglobin content, whereby further studies should be carried out for its potential use in an Iberian pig selection program.

Changes in Biceps femoris Transcriptome along Growth in Iberian Pigs Fed Different Energy Sources and Comparative Analysis with Duroc Breed.

This experiment was conducted to investigate the effects of developmental stage, breed, and diet energy source on the genome-wide expression, meat quality traits, and tissue composition of biceps femoris muscle in growing pure Iberian and Duroc pigs. The study comprised 59 Iberian (IB) and 19 Duroc (DU) animals, who started the treatment at an average live weight (LW) of 19.9 kg. The animals were kept under identical management conditions and fed two diets with different energy sources (6% high oleic sunflower oil or carbohydrates). Twenty-nine IB animals were slaughtered after seven days of treatment at an average LW of 24.1 kg, and 30 IB animals plus all the DU animals were slaughtered after 47 days at an average LW of 50.7 kg. The main factors affecting the muscle transcriptome were age, with 1832 differentially expressed genes (DEGs), and breed (1055 DEGs), while the effect of diet on the transcriptome was very small. The results indicated transcriptome changes along time in Iberian animals, being especially related to growth and tissue development, extracellular matrix (ECM) composition, and cytoskeleton organization, with DEGs affecting relevant functions and biological pathways, such as myogenesis. The breed also affected functions related to muscle development and cytoskeleton organization, as well as functions related to solute transport and lipid and carbohydrate metabolism. Taking into account the results of the two main comparisons (age and breed effects), we can postulate that the Iberian breed is more precocious than the Duroc breed, regarding myogenesis and muscle development, in the studied growing stage.

Influence of genetic background and dietary oleic acid on gut microbiota composition in Duroc and Iberian pigs.

BACKGROUND: Phenotypic variability for productive and meat quality traits has been largely studied in Iberian pigs, especially in genetic selection and nutritional experiments. Complex interactions among genetic background, diet composition and gut microbiota hinder the correct assessment of each factor's contribution on phenotypes. In order to disentangle these interactions, we evaluated changes in gut microbiota composition comparing 48 Iberian and Duroc pigs fed diets with different energy source (standard diet with carbohydrates vs sunflower oil-enriched diet with high oleic acid content). RESULTS: A higher richness was observed for Iberian pigs (p < 0.05) and compositional analysis was applied for beta-diversity, differential abundance and pairwise log-ratio analyses. We found significant differences in overall microbiota composition between breeds, and also between diets inside breeds, to a lesser extent. Differential abundance analysis revealed that Duroc animals have more proportion of Actinobacteria and Prevotella, while Iberian replace those microorganisms with other more variable taxa. According to dietary differences, high-oleic fed animals were richer in Prevotella. We also found microbial ratios capable of separating animals by breeds and diets, mostly related to Actinobacteria. CONCLUSION: This study reveals that both genetic background and diet composition might have a relevant impact in gut microbiota composition. The application of compositional data analysis has facilitated the identification of microorganisms and ratios as possibly related to metabolic changes due to genetic background and, to a lower extent, to dietary changes. This may lead to a relevant progress in the knowledge of interactions between pig genetics, environment and gut microbiota.

Muscle Transcriptome Analysis Reveals Molecular Pathways Related to Oxidative Phosphorylation, Antioxidant Defense, Fatness and Growth in Mangalitsa and Moravka Pigs.

This work was aimed at evaluating loin transcriptome and metabolic pathway differences between the two main Serbian local pig breeds with divergent characteristics regarding muscle growth and fatness, as well as exploring nutrigenomic effects of tannin supplementation in Mangalitsa (MA) pigs. The study comprised 24 Mangalitsa and 10 Moravka (MO) males, which were kept under identical management conditions. Mangalitsa animals were divided in two nutritional groups (n = 12) receiving a standard (control) or tannin-supplemented diet (1.5%; MAT). Moravka pigs were fed the standard mixture. All animals were slaughtered at a similar age; 120 kg of average live weight (LW) and loin tissue was used for RNA-seq analysis. Results showed 306 differentially expressed genes (DEGs) according to breed, enriched in genes involved in growth, lipid metabolism, protein metabolism and muscle development, such as PDK4, FABP4, MYOD1 and STAT3, as well as a relevant number of genes involved in mitochondrial respiratory activity (MT-NDs, NDUFAs among others). Oxidative phosphorylation was the most significantly affected pathway, activated in Mangalitsa muscle, revealing the basis of a different muscle metabolism. Also, many other relevant pathways were affected by breed and involved in oxidative stress response, fat accumulation and development of skeletal muscle. Results also allowed the identification of potential regulators and causal networks such as those controlled by FLCN, PPARGC1A or PRKAB1 with relevant regulatory roles on DEGs involved in mitochondrial and lipid metabolism, or IL3 and TRAF2 potentially controlling DEGs involved in muscle development. The Tannin effect on transcriptome was small, with only 23 DEGs, but included interesting ones involved in lipid deposition such as PPARGC1B. The results indicate a significant effect of the breed on muscle tissue gene expression, affecting relevant biological pathways and allowing the identification of strong regulatory candidate genes to underlie the gene expression and phenotypic differences between the compared groups.

Describing variability in pig genes involved in coronavirus infections for a One Health perspective in conservation of animal genetic resources.

Coronaviruses silently circulate in human and animal populations, causing mild to severe diseases. Therefore, livestock are important components of a "One Health" perspective aimed to control these viral infections. However, at present there is no example that considers pig genetic resources in this context. In this study, we investigated the variability of four genes (ACE2, ANPEP and DPP4 encoding for host receptors of the viral spike proteins and TMPRSS2 encoding for a host proteinase) in 23 European (19 autochthonous and three commercial breeds and one wild boar population) and two Asian Sus scrofa populations. A total of 2229 variants were identified in the four candidate genes: 26% of them were not previously described; 29 variants affected the protein sequence and might potentially interact with the infection mechanisms. The results coming from this work are a first step towards a "One Health" perspective that should consider conservation programs of pig genetic resources with twofold objectives: (i) genetic resources could be reservoirs of host gene variability useful to design selection programs to increase resistance to coronaviruses; (ii) the described variability in genes involved in coronavirus infections across many different pig populations might be part of a risk assessment including pig genetic resources.

Differences in the Loin Tenderness of Iberian Pigs Explained through Dissimilarities in Their Transcriptome Expression Profile.

Tenderness is one of the most important meat quality traits and it can be measured through shear force with the Warner-Bratzler test. In the current study, we use the RNA-seq technique to analyze the transcriptome of Longissimus dorsi (LD) muscle in two groups of Iberian pigs (Tough and Tender) divergent for shear force breeding values. We identified 200 annotated differentially expressed genes (DEGs) and 245 newly predicted isoforms. The RNAseq expression results of 10 genes were validated with quantitative PCR (qPCR). Functional analyses showed an enrichment of DE genes in biological processes related to proteolysis (CTSC, RHOD, MYH8, ACTC1, GADD45B, CASQ2, CHRNA9 and ANKRD1), skeletal muscle tissue development (ANKRD1, DMD, FOS and MSTN), lipid metabolism (FABP3 and PPARGC1A) and collagen metabolism (COL14A1). The upstream analysis revealed a total of 11 transcription regulatory factors that could regulate the expression of some DEGs. Among them, IGF1, VGLL3 and PPARG can be highlighted since they regulate the expression of genes involved in biological pathways that could affect tenderness. The experiment revealed a set of candidate genes and regulatory factors suggestive to search polymorphisms that could be incorporated in a breeding program for improving meat tenderness.

Genetic parameter estimation and gene association analyses for meat quality traits in open-air free-range Iberian pigs.

Meat quality of Iberian pigs is defined by the combination of their genetic characteristics and the particular production system. To carry out a genetic analysis of the main meat quality traits, we estimated their heritabilities, genetic correlations and the association effects of 32 selected SNPs of 12 candidate genes. A total of ten traits were measured in longissimus dorsi samples from 1,199 Iberian pigs fattened in the traditional free-range system: water holding capacity (thawing, cooking and centrifuge force water losses), instrumental colour (lightness L*, redness a* and yellowness b*), myoglobin content, shear force on cooked meat, and shear force and maximum compression force on dry-cured loin. Estimated heritability values were low to moderate (0.01 to 0.43) being the lowest for L* and the highest for cooking loss. Strong genetic correlations between water holding capacity traits (0.93 to 0.96) and between myoglobin content and a* (0.94) were observed. The association analyses revealed 19 SNPs significantly associated with different traits. Consistent and strong effects were observed between PRKAG3 SNPs (rs319678464G > C and rs330427832C > T), MYH3_rs81437544T > C, CASP3_rs319658214G > T and CTSL_rs332171512A > G and water losses. Also for CAPN1_rs81358667G > A and CASP3_rs319658214G > T and shear force. The SNPs mapping on PRKAG3 showed the highest effects on Minolta colour traits. Genotyping of these SNPs could be useful for the selection of Iberian young boars with similar estimated breeding values for productive traits.

Whole-genome sequencing of European autochthonous and commercial pig breeds allows the detection of signatures of selection for adaptation of genetic resources to different breeding and production systems.

BACKGROUND: Natural and artificial directional selection in cosmopolitan and autochthonous pig breeds and wild boars have shaped their genomes and resulted in a reservoir of animal genetic diversity. Signatures of selection are the result of these selection events that have contributed to the adaptation of breeds to different environments and production systems. In this study, we analysed the genome variability of 19 European autochthonous pig breeds (Alentejana, Bísara, Majorcan Black, Basque, Gascon, Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda, Krskopolje pig, Black Slavonian, Turopolje, Moravka, Swallow-Bellied Mangalitsa, Schwäbisch-Hällisches Schwein, Lithuanian indigenous wattle and Lithuanian White old type) from nine countries, three European commercial breeds (Italian Large White, Italian Landrace and Italian Duroc), and European wild boars, by mining whole-genome sequencing data obtained by using a DNA-pool sequencing approach. Signatures of selection were identified by using a single-breed approach with two statistics [within-breed pooled heterozygosity (HP) and fixation index (FST)] and group-based FST approaches, which compare groups of breeds defined according to external traits and use/specialization/type. RESULTS: We detected more than 22 million single nucleotide polymorphisms (SNPs) across the 23 compared populations and identified 359 chromosome regions showing signatures of selection. These regions harbour genes that are already known or new genes that are under selection and relevant for the domestication process in this species, and that affect several morphological and physiological traits (e.g. coat colours and patterns, body size, number of vertebrae and teats, ear size and conformation, reproductive traits, growth and fat deposition traits). Wild boar related signatures of selection were detected across all the genome of several autochthonous breeds, which suggests that crossbreeding (accidental or deliberate) occurred with wild boars. CONCLUSIONS: Our findings provide a catalogue of genetic variants of many European pig populations and identify genome regions that can explain, at least in part, the phenotypic diversity of these genetic resources.

Breed, Diet, and Interaction Effects on Adipose Tissue Transcriptome in Iberian and Duroc Pigs Fed Different Energy Sources.

In this study, we analyzed the effects of breed, diet energy source, and their interaction on adipose tissue transcriptome in growing Iberian and Duroc pigs. The study comprised 29 Iberian and 19 Duroc males, which were kept under identical management conditions except the nutritional treatment. Two isoenergetic diets were used with 6% high oleic sunflower oil (HO) or carbohydrates (CH) as energy sources. All animals were slaughtered after 47 days of treatment at an average live weight of 51.2 kg. Twelve animals from each breed (six fed each diet) were employed for ham subcutaneous adipose tissue RNA-Seq analysis. The data analysis was performed using two different bioinformatic pipelines. We detected 837 and 1456 differentially expressed genes (DEGs) according to breed, depending on the pipeline. Due to the strong effect of breed on transcriptome, the effect of the diet was separately evaluated in the two breeds. We identified 207 and 57 DEGs depending on diet in Iberian and Duroc pigs, respectively. A joint analysis of both effects allowed the detection of some breed-diet interactions on transcriptome, which were inferred from RNA-Seq and quantitative PCR data. The functional analysis showed the enrichment of functions related to growth and tissue development, inflammatory response, immune cell trafficking, and carbohydrate and lipid metabolism, and allowed the identification of potential regulators. The results indicate different effects of diet on adipose tissue gene expression between breeds, affecting relevant biological pathways.

Association between the pig genome and its gut microbiota composition.

The gut microbiota has been evolving with its host along the time creating a symbiotic relationship. In this study, we assess the role of the host genome in the modulation of the microbiota composition in pigs. Gut microbiota compositions were estimated through sequencing the V3-V4 region of the 16S rRNA gene from rectal contents of 285 pigs. A total of 1,261 operational taxonomic units were obtained and grouped in 18 phyla and 101 genera. Firmicutes (45.36%) and Bacteroidetes (37.47%) were the two major phyla obtained, whereas at genus level Prevotella (7.03%) and Treponema (6.29%) were the most abundant. Pigs were also genotyped with a high-throughput method for 45,508 single nucleotide polymorphisms that covered the entire pig genome. Subsequently, genome-wide association studies were made among the genotypes of these pigs and their gut microbiota composition. A total of 52 single-nucleotide polymorphisms distributed in 17 regions along the pig genome were associated with the relative abundance of six genera; Akkermansia, CF231, Phascolarctobacterium, Prevotella, SMB53, and Streptococcus. Our results suggest 39 candidate genes that may be modulating the microbiota composition and manifest the association between host genome and gut microbiota in pigs.

Identification of Candidate Genes and Regulatory Factors Underlying Intramuscular Fat Content Through Longissimus Dorsi Transcriptome Analyses in Heavy Iberian Pigs.

One of the most important determinants of meat quality is the intramuscular fat (IMF) content. The development of high-throughput techniques as RNA-seq allows identifying gene pathways and networks with a differential expression (DE) between groups of animals divergent for a particular trait. The Iberian pig is characterized by having an excellent meat quality and a high content of intramuscular fat. The objectives of the present study were to analyze the longissimus dorsi transcriptome of purebred Iberian pigs divergent for their IMF breeding value to identify differential expressed genes and regulatory factors affecting gene expression. RNA-seq allowed identifying ∼10,000 of the 25,878 annotated genes in the analyzed samples. In addition to this, 42.46% of the identified transcripts corresponded to newly predicted isoforms. Differential expression analyses revealed a total of 221 DE annotated genes and 116 DE new isoforms. Functional analyses identified an enrichment of overexpressed genes involved in lipid metabolism (FASN, SCD, ELOVL6, DGAT2, PLIN1, CIDEC, and ADIPOQ) in animals with a higher content of IMF and an enrichment of overexpressed genes related with myogenesis and adipogenesis (EGR1, EGR2, EGR3, JUNB, FOSB, and SEMA4D) in the animals with a lower content of IMF. In addition to this, potential regulatory elements of these DE genes were identified. Co-expression networks analyses revealed six long non-coding RNAs (lncRNAs) (ALDBSSCG0000002079, ALDBSSCG0000002093, ALDBSSCG0000003455, ALDBSSCG0000004244, ALDBSSCG0000005525, and ALDBSSCG0000006849) co-expressed with SEMA4D and FOSB genes and one (ALDBSSCG0000004790) with SCD, ELOVL6, DGAT2, PLIN1, and CIDEC. Analyses of the regulatory impact factors (RIFs) revealed 301 transcriptionally regulatory factors involved in expression differences, with five of them involved in adipogenesis (ARID5B, CREB1, VDR, ATF6, and SP1) and other three taking part of myogenesis and development of skeletal muscle (ATF3, KLF11, and MYF6). The results obtained provide relevant insights about the genetic mechanisms underlying IMF content in purebred Iberian pigs and a set of candidate genes and regulatory factors for further identification of polymorphisms susceptible of being incorporated in a selection program.

Diversity across major and candidate genes in European local pig breeds.

The aim of this work was to analyse the distribution of causal and candidate mutations associated to relevant productive traits in twenty local European pig breeds. Also, the potential of the SNP panel employed for elucidating the genetic structure and relationships among breeds was evaluated. Most relevant genes and mutations associated with pig morphological, productive, meat quality, reproductive and disease resistance traits were prioritized and analyzed in a maximum of 47 blood samples from each of the breeds (Alentejana, Apulo-Calabrese, Basque, Bísara, Majorcan Black, Black Slavonian (Crna slavonska), Casertana, Cinta Senese, Gascon, Iberian, Krskopolje (Krskopoljski), Lithuanian indigenous wattle, Lithuanian White Old Type, Mora Romagnola, Moravka, Nero Siciliano, Sarda, Schwäbisch-Hällisches Schwein (Swabian Hall pig), Swallow-Bellied Mangalitsa and Turopolje). We successfully analyzed allelic variation in 39 polymorphisms, located in 33 candidate genes. Results provide relevant information regarding genetic diversity and segregation of SNPs associated to production and quality traits. Coat color and morphological trait-genes that show low level of segregation, and fixed SNPs may be useful for traceability. On the other hand, we detected SNPs which may be useful for association studies as well as breeding programs. For instance, we observed predominance of alleles that might be unfavorable for disease resistance and boar taint in most breeds and segregation of many alleles involved in meat quality, fatness and growth traits. Overall, these findings provide a detailed catalogue of segregating candidate SNPs in 20 European local pig breeds that may be useful for traceability purposes, for association studies and for breeding schemes. Population genetic analyses based on these candidate genes are able to uncover some clues regarding the hidden genetic substructure of these populations, as the extreme genetic closeness between Iberian and Alentejana breeds and an uneven admixture of the breeds studied. The results are in agreement with available knowledge regarding breed history and management, although largest panels of neutral markers should be employed to get a deeper understanding of the population's structure and relationships.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N2, room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.