Italian Duroc Research Articles

The porcine tribbles homolog 3 (TRIB3) gene: Identification of a missense mutation and association analysis with meat quality and production traits in Italian heavy pigs

TRIB3 plays an important role in energy metabolism. This work aimed to study the porcine tribbles homolog 3 (TRIB3) gene and to evaluate its association with meat quality and carcass traits in pigs. By sequencing a portion of the porcine TRIB3 gene two single nucleotide polymorphisms (SNPs) in the first coding exon (one synonymous SNP: c.132 T>C; and one missense mutation: c.146C>T, p.P49L) were identified. The two polymorphisms were in complete linkage disequilibrium. In silico analysis of the p.P49L mutation suggested that it could have functional effects. Association studies in four groups of pigs (651 animals in total) indicated that this gene marker was associated with back fat thickness in Italian Large White and Italian Duroc pigs in two different experimental designs (P<0.1 and P<0.05). This polymorphism tended to be associated with lactate content of the semimembranosus muscle (P<0.1). Among several other tissues, TRIB3 is expressed in fat and skeletal muscle.

The insulin-like growth factor 2 (IGF2) gene intron3-g.3072G&gt;A polymorphism is not the only Sus scrofa chromosome 2p mutation affecting meat production and carcass traits in pigs: Evidence from the effects of a cathepsin D (CTSD) gene polymorphism1

The objective of this study was to evaluate the effects of mutations in 2 genes [IGF2 and cathepsin D (CTSD)] that map on the telomeric end of the p arm of SSC2. In this region, an imprinted QTL affecting muscle mass and fat deposition was reported, and the IGF2 intron3-g.3072G>A substitution was identified as the causative mutation. In the same chromosome region, we assigned, by linkage mapping, the CTSD gene, a lysosomal proteinase, for which we previously identified an SNP in the 3'-untranslated region (AM933484, g.70G>A). We have already shown strong effects of this CTSD mutation on several production traits in Italian Large White pigs, suggesting a possible independent role of this marker in fatness and meat deposition in pigs. To evaluate this hypothesis, after having refined the map position of the CTSD gene by radiation hybrid mapping, we analyzed the IGF2 and the CTSD polymorphisms in 270 Italian Large White and 311 Italian Duroc pigs, for which EBV and random residuals from fixed models were calculated for several traits. Different association analyses were carried out to distinguish the effects of the 2 close markers. In the Italian Large White pigs, the results for IGF2 were highly significant for all traits when using either EBV or random residuals (e.g., using EBV: lean cuts, P = 2.2 x 10(-18); ADG, P = 2.6 x 10(-16); backfat thickness, P = 2.2 x 10(-9); feed:gain ratio, P = 2.3 x 10(-9); ham weight, P = 1.5 x 10(-6)). No effect was observed for meat quality traits. The IGF2 intron3-g.3072G>A mutation did not show any association in the Italian Duroc pigs, probably because of the small variability at this polymorphic site for this breed. However, a significant association was evident for the CTSD marker (P < 0.001) with EBV of all carcass and production traits in Italian Duroc pigs (lean content, ADG, backfat thickness, feed:gain ratio) after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A mutation were also confirmed in a subset of Italian Large White animals carrying the homozygous genotype IGF2 intron3-g.3072GG, and by haplotype analysis between the markers of the 2 considered genes in the complete data set. Overall, these results indicate that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition on SSC2p. Therefore, the CTSD g.70G>A polymorphism could be used to increase selection efficiency in marker-assisted selection programs that already use the IGF2 mutation. However, for practical applications, because the CTSD gene should not be imprinted (we obtained this information from expression analysis in adult skeletal muscle), the different modes of inheritance of the 2 genes have to be considered.

A single nucleotide polymorphism in the porcine cathepsin K (CTSK) gene is associated with back fat thickness and production traits in Italian Duroc pigs

Cathepsin K (CTSK) was selected as a candidate gene for fat deposition in pigs because recently, in human and mouse, it was shown that this lysosomal proteinase is an obesity marker. A single nucleotide polymorphism (SNP) was identified in intron 4 of the porcine CTSK gene (g.15G>A; FM209043). Allele frequencies of this polymorphism were analysed in seven pig breeds. Radiation hybrid mapping confirmed the localization of CTSK to porcine chromosome 4, close to the FAT1 QTL region. Three populations of pigs (one Italian Large White and two Italian Duroc groups of pigs) were selected for association analysis. In the Italian Large White breed the g.15G>A SNP was not informative. Association analysis including all Italian Duroc pigs showed that the CTSK marker was associated with back fat thickness and lean cuts (P < 0.01), and average daily gain and feed:gain ratio (P < 0.05) estimated breeding values.

Confirmed association between a single nucleotide polymorphism in the FTO gene and obesity-related traits in heavy pigs

We recently showed that a polymorphism in the fat mass and obesity associated (FTO) gene (AM931150: g.276T > G) is associated with fat deposition traits in pigs. To confirm this result, we genotyped this polymorphism in an Italian Duroc population made up by 313 performance tested pigs with known estimated breeding values (EBVs) for average daily gain, back fat thickness (BFT), feed:gain ratio, lean cuts (LC), and visible intermuscular fat (VIF, a measure of intermuscular fat in the hams). In addition, we genotyped 148 commercial heavy pigs for which several fat deposition traits and lean meat percentage were measured. The results of the association analyses confirmed the effect of the FTO mutation on obesity-related traits (VIF, BFT and LC) in the Italian Duroc pigs (P < 0.01) and in the commercial pigs (intramuscular fat content of different muscles, P < 0.05 or P < 0.10; lean meat content, P < 0.05; BFT, P < 0.05; intermuscular fat content in the hams, P < 0.05).

Study of porcine adiponectin ( ADIPOQ) gene and association of a missense mutation with EBVs for production and carcass traits in Italian Duroc heavy pigs

The adiponectin (adiponectin, C1Q and collagen domain containing) encoded by ADIPOQ is an adipokine that modulates several biological processes such as lipogenesis, gluconeogenesis, fatty acid oxidation, glucose uptake and insulin sensitivity. An objective of this study was to identify SNPs in the ADIPOQ gene both in cosmopolitan and local pig breeds reared in Italy and in Meishan samples. Resequencing of ADIPOQ gene regions in 16 pigs of 12 breeds revealed only two SNPs ( EU489740: g.1611G > A synonymous and EU489740: g.1735G > A missense mutations) already reported. The g.1735G > A polymorphism was genotyped by PCR-RFLP in 510 pigs from 11 breeds reared in Italy (Italian Large White, Italian Duroc, Italian Landrace, Belgian Landrace, Hampshire, Pietrain, Calabrese, Casertana, Cinta Senese, Mora Romagnola and Nero Siciliano) and from Chinese Meishan breed. The Italian local breeds were homozygous for the g.1735G allele while the Meishan samples resulted homozygous for the g.1735A allele. The polymorphism segregates in the cosmopolitan breeds, except for Hampshire, and the rarer g.1735A allele showed the highest frequency in Italian Duroc (0.12). Association analysis of g.1735G > A polymorphism with estimated breeding values (EBVs) for average daily gain (ADG), feed:gain ratio (FGR), weight of neck and loin (lean cuts, LC), weight of ham (H) and backfat thickness (BFT) were performed in Italian Duroc heavy pigs. The GA vs GG pigs showed higher least square means for ADG ( P = 0.003) and LC ( P = 0.036) EBVs and lower value for FGR EBV ( P = 0.033).

The porcine fat mass and obesity associated (FTO) gene is associated with fat deposition in Italian Duroc pigs

In humans, common variants in the fat mass and obesity associated (FTO) gene are associated with body mass index and obesity. Here we sequenced exon 4, parts of introns 3 and 4 and two portions of the 3'-untranslated region of the porcine FTO gene in a panel of nine pigs of different breeds and identified three SNPs. Allele frequencies of the g.276T>G (AM931150) mutation were studied in seven pig breeds. This mutation was used to linkage-map FTO to SSC6. Association analyses between the g.276T>G polymorphism and several traits [pH of semimembranosus muscle and estimated breeding values (EBV) for average daily gain, back fat thickness, lean cuts, ham weight and feed:gain ratio] were carried out in 257 sib-tested Italian Large White pigs. Only feed:gain ratio showed P<0.05. A selective genotyping approach was applied, analysing two extreme and divergent groups of Italian Large White pigs selected on the basis of back fat thickness EBV (50 with most positive and 50 with most negative values). Fisher's exact test (two-tailed) was not significant when comparing the allele frequencies of these two groups. The same approach was used in the Italian Duroc breed for which two extreme and divergent groups of animals were selected according to visible intermuscular fat EBV. Differences of allele frequencies between these two groups were highly significant (P<0.00001, P<0.001 and P<0.0001, considering all animals or only two- or three-generation unrelated animals respectively), indicating association between the analysed FTO marker and intermuscular fat deposition.

Skeletal muscle expression analysis of fat metabolism genes in pig

Fat content and backfat thickness are important polygenic parameters influencing meat quality and carcass traits in pigs. Up to now, there is a lack of knowledge on the expression level of the genes encoding for enzymes involved in fatty acid metabolism in porcine skeletal muscle. In the present study we analysed, by quantitative real time PCR, the expression of three genes, acetyl-CoA carboxylase (ACACA), ATP citrate lyase (ACL) and fatty acid synthase (FASN) in skeletal muscle tissue samples of Italian Large White and Italian Duroc pigs with divergent breeding values for backfat thickness or visible intramuscular fat. Significant differences of the expression level for ACACA gene (P=0.04) and for ACL gene (P=0.02) were observed between the two breeds, comparing the samples selected for backfat thickness trait. The expression analysis of FASN gene in the samples with different genotype at the SNP c.265C>T showed that the TT genotype presented the lowest values in both breeds. The differences observed between breeds should be further considered to investigate the putative involvement of these genes on fat deposition traits.

Use of pure glycerol in fattening heavy pigs

Eighty Italian Duroc × Italian Large White pigs (BW 42.6 ± 3.37 kg) were used to determine the effects of pure glycerol on growth performance and meat quality of heavy pigs. Pigs were divided into five groups receiving 0% (control), 5% or 10% during the growing and finishing phases (42.6–160 kg BW) (G+F5,G+F10) or 5% or 10% during the finishing period (100–160 kg BW) (FIN5,FIN10) of pure glycerol in substitution for maize meal (on a dry matter basis). The pigs were slaughtered at approximately 160 kg BW. The growth performance of pigs fed 5% glycerol did not differ from controls regardless of feeding duration, whereas those fed 10% glycerol showed reduced growth and poorer feed:gain ratio. Fat quality and meat suitability for raw ham curing were not affected by dietary treatment. Differences were not consistent enough to draw any conclusion about the effects of feeding glycerol on sensory characteristics.

Study of Fatty Acid Synthase and Adiponectin SNPs in the Italian Duroc breed

Fatty acid synthase (FASN) is a multifunctional enzyme that plays a central role in fatty acid biosynthesis catalysing the conversion of acetyl-CoA and malonyl-CoA into long-chain saturated fatty acids and has an important role in energy homeostasis. Pig FASN gene has been assigned to chromosome 12p1.5 and a T>C polymorphism in the fourth exon was found. Adiponectin (ADN) is a fat-derived hormone involved in insulin sensitivity, in lipid and glucose metabolism. In literature is reported that the gene was mapped on chromosome 13 at 53.6 cM, in a region containing QTL for intramuscolar fat (IMF). In this gene several SNPs were identified and one of these polymorphisms (a G>A missense mutation within the 60th codon) determining the Val-Ile substitution in the protein, has been previously reported.The aim of this work is to analyse the variability of polymorphisms of fatty acid synthase described by Munoz et al., 2003 (Anim. Genet. 34:234) and adiponectin genes, candidates for meat and carcass quality, in Italian Duroc pigs. Up to now researches on these functional genes in this pig breed have not been performed.In order to estimate allele frequencies, the FASN T>C polymorphism and the ADN G>A mutation were studied by PCR-RFLP in 134 and 257 Italian Duroc, respectively. At the FASN locus the allele frequency was 0.041 for T allele; at the ADN locus the frequency of the alleles A was 0.136. We investigated the effect of FASN and ADN polymorphisms on Estimated Breeding Values (EBVs) for single traits (average daily gain, feed to gain ratio, weight of lean cuts, ham weight, backfat thickness and visible intermuscular fat) and for selection indexes (Total Index and Terminal Index) estimated by ANAS. The analyses were performed using the GLM procedure of SAS System and a model with the genotype at FASN or ADN locus as fixed factor.At the FASN locus only few pigs carried the T allele; therefore, we tried to perform the analysis comparing the pigs with both genotypes T/T plus C/T versus those genotyped C/C at this locus. The results didn’t show any association of the polymorphism with the examined traits.The same model was used for the ADN locus and the G/A genotyped pigs versus the G/G ones showed a significant higher estimated mean value for Total Index (P= 0.0114).For both the analysed genes the obtained results should be verified on a larger sample even if the present findings could suggest a possible role of ADN on the traits included in Total Index in the Duroc breed.

Analysis of skeletal muscle tissue expression profiles in pig to identify genes involved in meat quality traits: effect of stress conditions before slaughtering in different pig breeds

The knowledge of genes and molecular processes involved in the stress susceptibility and reaction could be important for the improvement of meat quality traits. The aim of the research was the identification by microarray analysis of skeletal muscle genes differentially expressed in pigs divergent for meat quality. The expression profiles were studied in subjects exposed or not exposed to a physical stress (a run in controlled conditions) before slaughtering. Semimembranosus muscle samples were collected at slaughtering and stored at -80°C before RNA extraction. These samples were obtained from 38 unrelated castrated male pigs belonging to 3 different breeds and reared up to 120 kg in the University of Bologna experimental farm: 10 Italian Duroc and 10 Italian Large White pigs with genotype CC at the RYR 1 1843 polymorphic site and18 Pietrain pigs (CC, CT and TT in the not stressed group and only CC and CT in the stressed group). Microarray analysis was performed using two platforms: a cDNA based microarray produce by the University of Aarhus (~27,000 porcine cDNAs) and the Operon/Qiagen pig 11k Oligo set (version 1.0) (10,665 70mers oligos). Total RNA extracted from the samples was reverse transcribed, labelled with fluorescent Alexa Fluor 555 dye and individually hybridized to both cDNA and Oligo microarrays. The labelled samples were hybridised together with a common reference sample consisting of Alexa Fluor 647 labelled total RNA extracted from skeletal muscle and heart tissue from a pig not related with those of the experiment. ScanArray Gx (Perkin Elmer) scanner and ScanArray Express software were used for image acquisition and data normalization. ANOVA has been performed without to include 3 Pietrain pigs not stressed with genotype CC at RYR 1. Using the Mixed procedure of SAS that consider fixed and random factors the adjustment for overall effects of array and dye across genes was performed. The residuals from this normalization process were used to test the effects of the factors included in the model on gene expression. Differences in the least square means were obtained and tested for significant differences by using a t-test. The false discovery rate was used to account for multiple testing. A false discovery rate of 5% was accepted. Preliminary results of the comparison between stressed and not stressed pigs obtained with the Oligo array showed that more than 20% of the genes upregulated in the group of stressed pigs belong to functional categories involved in regulation of cellular physiological processes. The first results from the comparison between treatments within breed seems to evidence differences among breeds in stress response and can provide indication on potentially interesting genes influencing pig meat quality.

Investigation of SNPs in the ATP1A2, CA3 and DECR1 genes mapped to porcine chromosome 4: analysis in groups of pigs divergent for meat production and quality traits

Three genes (ATPase, Na+/K+ transporting, α 2(+) polypeptide, ATP1A2; carbonic anhydrase III, CA3; 2,4-dienoyl CoA reductase 1, mitochondrial, DECR1), isolated from a porcine skeletal muscle cDNA library and mapped on porcine chromosome 4 (SSC4), were investigated. A new single nucleotide polymorphism (SNP) was identified in the 3’-untranslated region of the CA3 gene and used to genetically map this locus on SSC4 together with the ATP1A2 and DECR1 loci for which SNPs were already reported. Allele frequencies of the three loci were reported for 11 pig breeds (Italian Large White, Italian Landrace, Italian Duroc, Belgian Landrace, Hampshire, Piùtrain, Meishan, Cinta Senese, Casertana, Calabrese and Nero Siciliano). Radiation hybrid mapping of these genes confirmed the linkage mapping results as well as mapping information reported by other authors. Then, the SNPs identified in the ATP1A2, CA3 and DECR1 genes were genotyped in Italian Large White and Italian Duroc animal groups with extreme and divergent estimated breeding value for several production traits. For CA3 significant differences in allele frequencies (P< 0.05) were observed between the extreme groups of pigs for the lean cuts (Italian Large White) and visible intermuscular fat (Italian Duroc) traits. For DECR1, a significant difference in allele frequencies was observed only for the visible intermuscular fat trait. ATP1A2, which maps close to the FAT1 locus, did not show any significant difference. A very high linkage disequilibrium (D’= 0.967; P< 0.0001) was identified between CA3 and DECR1 in the Italian Duroc population. Further investigations are needed to evaluate the effect of CA3 and DECR1 on the considered traits.