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The European eel, a catadromous endangered species, displays plasticity in phenotypic traits and in trophic behaviour, features that can eventually affect growth. The aim of this study was to compare variation in carbon (?13C) and nitrogen (?15N) stable isotopes in muscle tissue of the adult eel life stages (i.e. yellow and silver eel), as function of growth. Eel samples were obtained from nine sites across the Mediterranean region, that included seven coastal lagoons of different size and ecological structure, and two freshwater habitats corresponding to one river and one lake. A large variability was found in both ?13C and ?15N values, suggesting that eels fed over a broad range of potential food sources. Eels from each location showed specific range values of ?13C, in most sites irrespective of stage, reflecting lagoon- or freshwater-specific carbon sources. A positive correlation was found between individual ?15N values and annual growth rates, suggesting that eels generally seem to feed on prey of higher ?15N values among the trophic resources available in each foraging habitat. This allows individuals to grow faster and to attain earlier the pre-spawning phase (i.e. the silver stage). A better understanding of eel trophic ecology across different life stages and different habitats is one of the prerequisites for the effective management of local stocks.

Extensive metabolic profiles of body fluids, obtained by nuclear magnetic resonance (NMR) spectroscopic or mass spectroscopic data, can provide information to deduce the effects of many different factors on biological processes. In particular, genetic factors can determine differences in physiological processes that are captured by metabolomics or metabonomics analyses. In this study we compared metabonomic profiles obtained from serum of two pig breeds (Italian Large White and Italian Duroc) with the aim to disentangle breed specific biological aspects. Twelve castrated males (6 Italian Large White and 6 Italian Duroc) were raised at the test station of the National Pig Breeders Association (ANAS) in the same conditions till they reached a live weight of about 160 kg. After a period of starvation, early in the morning pigs were transported all together at a commercial slaughterhouse, electrically stunned and jugulated within 10 minutes. Collected blood sample were processed after about 2 hours and serum was stored at -80°C till use. High Resolution Magic Angle Spinning-NMR serum spectra were recorded with a Bruker AVANCE spectrometer. Each sample was analysed in triplicate. Preliminary Principal Component Analysis of the obtained NMR spectra indicated that metabonomic analysis could be able to distinguish breed specific metabolic profiles. These results might represent useful information to disclose genetically determined regulatory pathways contributing to understand breed differences in biological processes indirectly linked with production traits.

Application of genomics (next generation sequencing and high throughput genotyping) and metabolomics technologies in farm animals are opening new opportunities for the identification of genetic factors affecting traits of economic relevance. In this study we combined several resources and data with the final aim to identify DNA polymorphisms and metabolites associated with production traits in Italian heavy pigs. Two genome wide association studies were carried out using a selective genotyping approach in Italian Large White pigs based on extreme and divergent estimated breeding values (EBVs) for average daily gain (ADG) and back fat thickness (BFT). Next generation sequencing was carried out using the Ion Torrent technology to identify single nucleotide polymorphisms (SNPs) from two reduced representation libraries constructed from pigs with extreme BFT EBVs. Metabolomics information was obtained using a mass spectrometry (MS/MS) analytical pipeline in a performance tested population. Integration of these data made it possible to identify markers (SNPs, copy number variation and metabolites) associated with ADG, BFT and several other correlated traits.

The development of high throughput genomics (next generation sequencing and high throughput genotyping) and metabolomics platforms has opened new perspectives for the identification of the genetics factors affecting traits of biological relevance in all species, including production traits in farm animals. In pigs, benefits derived from the recent sequencing of the pig genome can be fully exploited by combining advanced genomics and metabolomics approaches. In this work we integrated several resources, experiments and data with the final aim to identify markers (DNA polymorphisms and metabolites) associated with production traits in Italian Large White pigs. High throughput genotyping was carried out using the Illumina Porcine60SNP BeadChip array and customized Golden Gate system on extreme and divergent pigs for back fat thickness (BFT) estimated breeding values (EBVs) (300-560 animals) and average daily gain (ADG) EBVs (360 pigs), chosen among a population of about 12,000 performance tested pigs. Next generation sequencing was carried out using the Ion Torrent PGM machine to identify single nucleotide polymorphisms (SNPs) from two reduced representation libraries developed from pooled genomic DNA constructed from 50 pigs with most positive and 50 pigs with most negative BFT EBVs, respectively. A total of 7,510,918 reads were produced and 447,031 SNPs were called, using stringent criteria. Genome wide association studies made it possible to identify a quite large number of significant SNPs affecting BFT, ADG and correlated traits. In addition, several genome regions containing significant SNPs for BFT were enriched of SNPs identified from the Next Generation Sequencing experiment. Metabolomics information was obtained from 800 performance tested pigs using a mass spectrometry (MS/MS) analytical pipeline to measure 180 blood plasma metabolites. Estimated heritability and correlation among all these parameters and production traits indicated that a few metabolites could be useful predictors of EBVs for production traits. All these data will be used to develop a first systems biology platform to understand the fine biological mechanisms affecting production traits in pigs.

Pig breeding selection schemes have been designed to maximize their effectiveness dealing with specific biological features of this species (e.g. short generation interval, large litter size, etc.) and with the limited reproduction technologies available (e.g. reduced number of semen doses per boar). The introduction of genomic selection in pig breeding could provide additional advantages to the current schemes, further improving their efficiencies and overcoming some limits of the already established selection strategies. Simulations studies can give preliminary information on the impacts and effects of genomic selection on pig breeding. A few studies have already simulated different scenarios for the introduction of genomic selection in North European pig breeding systems. However, the Italian heavy pig breeding industry has specific peculiarities that should be considered in a simulation analysis. To this aim, we used the software QMSim to simulate a structurated pig population with a genome marked with 60K bi-allelic single nucleotide polymorphisms. The population was selected over 25 generations based on BLUP breeding values for a trait with heritability of 0.40. Different genotyping strategies and methods of predicting genomic breeding values were considered and compared. The introduction of genomic evaluations might increase the accuracy levels both in young piglets and in selected reproducers and the choice of male candidate would be more accurate. Nevertheless the evaluation of the return on investment in the Italian heavy pig breeding industry would determine the implementation of genomic selection in practice

Genomic selection is becoming a routine application in dairy cattle breeding for the advantages it gives in terms of reduced generation interval, increased accuracy and increased selection intensity. Effectiveness and practical implementation of genomic selection on pig breeds is not clear yet due to biological and technically derived differences between pig and cattle breeding (e.g. much shorter generation interval, large litter size, reduced number of semen doses produced per boar in A.I.). However, a few preliminary North European simulation studies have reported that some advantage from genomic selection might also be expected in pigs. However, studies based on North European vision of pig breeding do not take into account the peculiarities of the Italian heavy pig breeding system. Italian pig selection is based on a sib-testing scheme in which 2 full sisters and 1 full castrated brother from the same litter of the candidate boar are performance tested. This scheme opens a wide range of different genotyping strategies to be evaluated. To this aim, a pig breeding population with a 18 chromosome genome marked with 60K bi-allelic single nucleotide polymorphisms and several segregating QTLs was simulated by using the software QMSim. The population was genetically selected over 25 generations based on BLUP estimated breeding values (EBV) for a trait with heritability of 0.40. Culling was based on EBV both in sires and dams. Different genotyping strategies as well as different methods of predicting genomic breeding values (GBLUP, Bayes or Single Step) were compared. Accuracies of genetic values were calculated as correlation with true breeding values. Considering the simulated scenarios, the introduction of genomic evaluations might increase accuracy levels both in young piglets and in selected reproducers. In particular, the choice of the male candidate would be more effective. Other simulations are under way to evaluate if genomic selection could actually give economic advantages to the Italian heavy pig breeding industry.

Combining different approaches (resequencing of portions of 54 obesity candidate genes, literature mining for pig markers associated with fat deposition or related traits in 77 genes, and in silico mining of porcine expressed sequence tags and other sequences available in databases), we identified and analyzed 736 SNP within candidate genes to identify markers associated with back fat thickness (BFT) in Italian Large White sows. Animals were chosen using a selective genotyping approach according to their EBV for BFT (276 with most negative and 279 with most positive EBV) within a population of similar to 12,000 pigs. Association analysis between the SNP and BFT has been carried out using the MAX test proposed for case-control studies. The designed assays were successful for 656 SNP: 370 were excluded (low call rate or minor allele frequency A polymorphism (P-nominal < 1.0E-50). The second most significant SNP was the MC4R c.1426A>G polymorphism (P-nominal = 8.0E-05). The third top SNP (P-nominal = 6.2E04) was the intronic TBC1D1 g.219G>A polymorphic site, in agreement with our previous results obtained in an independent study. The list of significant markers also included SNP in additional genes (ABHD16A, ABHD5, ACP2, ALMS1, APOA2, ATP1A2, CALR, COL14A1, CTSF, DARS, DECR1, ENPP1, ESR1, GH1, GHRL, GNMT, IKBKB, JAK3, MTTP, NFKBIA, NT5E, PLAT, PPARG, PPP2R5D, PRLR, RRAGD, RFC2, SDHD, SERPINF1, UBE2H, VCAM1, and WAT). Functional relationships between genes were obtained using the Ingenuity Pathway Analysis (IPA) Knowledge Base. The top scoring pathway included 19 genes with a P-nominal < 0.1, 2 of which (IKBKB and NFKBIA) are involved in the hypothalamic IKK beta/NF kappa B program that could represent a key axis to affect fat deposition traits in pigs. These results represent a starting point to plan marker-assisted selection in Italian Large White nuclei for BFT. Because of similarities between humans and pigs, this study might also provide useful clues to investigate genetic factors affecting human obesity.

Fat deposition is a crucial aspect of pig meat quality: a suitable marbling and the right balance between lean meat and fat in carcass are both essential features to produce high quality dry cured hams. Moreover fat content influences both organoleptic and nutritive characteristics of meat products and consumer acceptance. Differences among breeds in intramuscular fat content and backfat thickness have been reported but the molecular events underlying the different aptitude to fat deposition are poorly known. The analysis of the expression profile of lipogenic genes in muscle and fat tissues in different pig breeds can contribute to a better knowledge of fat deposition control in pig meat and can provide new insights into carcass adiposity. The objective of this study is to investigate the expression level of the key adipogenic genes ATP citrate lyase and Fatty acid synthase in Italian Duroc and Italian Large White pigs with different fat deposition capacities. The obtained results suggest a tissue-specific mechanisms regulating gene transcription of FASN and ACL in the analysed samples. In adipose tissue, differential expression between the analysed breeds was observed and ILW pigs showed the highest FASN transcriptional level. In skeletal muscle tissue the greatest mRNA abundance of both lipogenic genes in ID pigs could be consistent with the high intermuscular fat deposition aptitude of this breed.

We identified, combining different approaches (sequencing, literature mining, and in silico porcine expressed sequence tag mining), a few hundreds of single nucleotide polymorphisms (SNPs; n. = 667) in candidate genes for fat deposition traits and genotyped these markers in Italian Large White pigs using a selective genotyping approach based on estimated breeding value for back fat thickness. Significant association (P nominal value

The aim of this work was to study the association between polymorphisms in five genes (CTSL, EPOR, ESR2, GPX5 and STAT5B) and functional teat number (TN). At each locus we genotyped at least 940 Italian Large White (ITLW) sows. All SNPs were segregating in the analyzed pigs with minor allele frequency ranging from 0.18 (CTSL and EPOR) to 0.40 (ESR2). The genotyped sows were categorized in two classes based on their TN: one with TN=14 (59% of the sample) and one with TN>14 (41%). The comparison of allele frequencies of the markers between the two classes was statistically significant (P

Lipogenesis is a complex process mainly influenced by genes coding for enzymes involved in de novo fatty acids synthesis. Changes in mRNA abundance of these genes have been associated to obesity related traits but the genetic regulation of fat deposition is not completely known. The variability at DNA and mRNA level of FASN (Fatty Acid Sinthase), ACL (ATP Citrate Lyase) and ACACA (Acetyl CoA Carboxylase) genes, involved in key steps of fatty acids metabolism, was investigated in porcine skeletal muscle and fat tissues. We genotyped Italian Duroc (IDU) and Italian Large White (ILW) pigs both for the SNP T265C of FASN gene (Munoz et al 2003) and the SNP T2956C of ACL gene that we newly identified by in silico analisys and sequencing. We localized ACL gene on SSC12 (RH panel, LOD: 11.49 with SW943 marker) where FASN and ACACA genes map and where QTLs for fat related traits were detected. The mRNA level of the three genes was determined in skeletal muscle and fat tissues samples of ILW and IDU pigs. Differential expression between breeds (FASN P=0.057; ACL P=0.021; ACACA P=0.041) only in skeletal muscle tissue was observed and IDU pigs showed the higher level. Studies are in progress to investigate on relationship between DNA variation and gene expression. The transcription of FASN, ACL and ACACA genes was found to be regulated in both tissues and breeds suggesting that these genes can represent promising functional and positional candidates for fat deposition in meat and carcass.

The quality of meat products is strongly influenced by pH. This trait is influenced by environmental factors and is also under genetic control. Up to now only two major genes related to pig meat pH (RYR1 and PRKAG3) have been identified and QTL regions (QTLRs) influencing it are reported in at least 10 porcine chromosomes. With the aim to fine maps some of these regions and to locate genes responsible of the QTL effects, three relevant QTLRs (SSC1 60-80 cM, SSC2 55-66 cM, SSC3 42-60 cM) were chosen and SNPs were searched in silico in transcribed sequences. We analysed 1822 porcine UniGene clusters located in the three QTLRs and chosen comparing pig and homologous human chromosomes through the NCBI map viewer tool. The clusters with a minimum of four sequences were aligned with MegaBLAST for the in silico search of SNPs. We marked as putative SNP a mutation detected in at least two sequences to avoid inconsistent results. The presence of some in silico identified SNPs was confirmed by an HRM analysis testing a panel of Italian Large White pigs divergent for pig meat pH. On the whole we found more than 400 SNPs that we will genotype by the high-throughput Illumina GoldenGate system to perform an association study with the considered trait and to identify the most interesting positional candidate genes located in these regions.

In pigs, several studies have been focused on structural and regulatory muscle genes and their possible relationship with meat production trait as they may play key role in muscle mass accretion. In the present work, we investigated four skeletal muscle genes, calpain 1 (CAPN1), calpastatin (CAST), myopalladin (MYPN) and titin (TTN). These genes were mapped on porcine chromosomes 2p14-p17, 2q21-q24, 14q25-q29 and 15q23-q26, respectively, and located in regions where quantitative trait loci for carcass traits were found. The calpain protein (coded by CAPN1 gene and expressed in skeletal muscle and other tissues) is involved in the early post mortem protein degradation promoting the initial disruption and destabilization of the myofibrillar structures. Calpastatin is a specific inhibitor of ubiquitous calpains and its post mortem activity influences meat tenderization process. Myopalladin is a sarcomeric protein which has central roles in the organization and assembly of the Z-line and seems to affect regulatory mechanisms of muscle gene expression. TTN encodes a large abundant protein of striated muscle that is involved in muscle assembly and ultrastructure and it is also implicated in muscle elasticity. To evaluate the putative effects of these four genes on carcass quality traits, we analysed single nucleotide polymorphisms (SNP) identified in the 3’ untranslated regions (MYPN and TTN) and in introns (CAST and CAPN1) in 272 sib tested Italian Large White (ILW) pigs and 114 sib tested Italian Duroc (ID). General linear model procedure of the SAS package was used to search for association between the SNPs and the productive traits average daily gain (ADG), backfat thickness (BFT), lean cuts (LC), ham weight (HW), feed conversion ratio (FCR) for both breeds and visible intramuscular fat (VIF) only for the ID breed. Statistically significant results for the association analyses were found for CAST in the ILW breed, with ADG, LC and FCR (P < 0.05) and in the ID breed with ADG, LC, BFT, HW (P < 0.05). Moreover, in the ID breed, the MYPN gene was associated with all considered traits (P < 0.01). The results confirm that CAST and MYPN could be considered positional and biological candidate genes affecting carcass traits, as previously reported in literature.

In humans, mutations in the fat mass and obesity associated (FTO) gene are associated with the obesity phenotype in several populations. In pigs, we already identified three single nucleotide polymorphisms (SNPs) in the FTO gene that we genetically assigned to porcine chromosome 6 (SSC6), in an imprinted QTL region for fat deposition. Then, using a selective genotyping approach, we showed that an SNP in this gene (g.276T>G) was associated with visible intermuscular fat (VIF, a measure that resembles intermuscular fat content) in Italian Duroc pigs. Here, we refined the mapping of this gene to SSC6 using a radiation hybrid panel and evaluate the effect of the same mutation in two different pig samples. The first was constituted by 157 sib-tested Italian Duroc animals for which estimated breeding values (EBV) for average daily gain, feed:gain ratio, back-fat thickness, lean cuts, ham weight and VIF were calculated. The second group of pigs was constituted by 117 commercial hybrids measured for a total of 31 carcass and fatness traits. In the Italian Duroc pigs the FTO marker was associated only with VIF EBV (P = 0.027). In the second group of pigs, the FTO genotypes showed a tendency to be associated with several fat deposition traits. These results confirm that FTO is an important source of variability for intermuscular fat deposition in Italian Duroc pigs while the effects on fatness traits in commercial pig populations should be further investigated.