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Vimentin (VIM) and desmin (DES) are muscle-specific proteins having crucial roles in maintaining the lateral organization and alignment of the sarcomeric structure during myofibrils’ regeneration. The present experiment was designed to ascertain the evolution of VIM and DES in Pectoralis major muscles (PM) of fast-growing (FG) and medium-growing (MG) meat-type chickens both at the protein and gene levels. MG broilers were considered as a control group whereas the evolution of VIM and DES over the growth period was evaluated in FG by collecting samples at different developmental stages (7, 14, 21, 28, 35, and 42 days). After performing a preliminary classification of the samples based on their histological features, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. Overall, the findings obtained at the protein level mirrored those related to their encoding genes, although a potential time lag required to observe the consequences of gene expression was evident. The two- and 3-fold higher level of the VIM-based heterodimer observed in FG at d 21 and d 28 in comparison with MG of the same age might be ascribed to the beginning and progressive development of the regenerative processes. This hypothesis is supported by IHC highlighting the presence of fibers to co-expressing VIM and DES. In addition, gene expression analyses suggested that, unlike VIM common sequence, VIM long isoform may not be directly implicated in muscle regeneration. As for DES content, the fluctuating trends observed for both the native protein and its heterodimer in FG might be ascribed to its importance for maintaining the structural organization of the regenerating fibers. Furthermore, the higher expression level of the DES gene in FG in comparison with MG further supported its potential application as a marker of muscle fibers’ regeneration. In conclusion, the findings of the present research seem to support the existence of a relationship between the occurrence of muscle regeneration and the growth rate of meat-type chickens and corroborate the potential use of VIM and DES as molecular markers of these cellular processes.

Growth-related abnormalities affecting modern chickens, known as White Striping (WS) and Wooden Breast (WB), have been deeply investigated in the last decade. Nevertheless, their precise etiology remains unclear. The present study aimed at providing new insights into the molecular mechanisms involved in their onset by identifying clusters of co-expressed genes (i.e., modules) and key loci associated with phenotypes highly related to the occurrence of these muscular disorders. The data obtained by a Weighted Gene Co-expression Network Analysis (WGCNA) were investigated to identify hub genes associated with the parameters breast width (W) and total crude protein content (PC) of Pectoralis major muscles (PM) previously harvested from 12 fast-growing broilers (6 normal vs. 6 affected by WS/WB). W and PC can be considered markers of the high breast yield of modern broilers and the impaired composition of abnormal fillets, respectively. Among the identified modules, the turquoise (r = -0.90, p < 0.0001) and yellow2 (r = 0.91, p < 0.0001) were those most significantly related to PC and W, and therefore respectively named “protein content” and “width” modules. Functional analysis of the width module evidenced genes involved in the ubiquitin-mediated proteolysis and inflammatory response. GTPase activator activity, PI3K-Akt signaling pathway, collagen catabolic process, and blood vessel development have been detected among the most significant functional categories of the protein content module. The most interconnected hub genes detected for the width module encode for proteins implicated in the adaptive responses to oxidative stress (i.e., THRAP3 and PRPF40A), and a member of the inhibitor of apoptosis family (i.e., BIRC2) involved in contrasting apoptotic events related to the endoplasmic reticulum (ER)-stress. The protein content module showed hub genes coding for different types of collagens (such as COL6A3 and COL5A2), along with MMP2 and SPARC, which are implicated in Collagen type IV catabolism and biosynthesis. Taken together, the present findings suggested that an ER stress condition may underly the inflammatory responses and apoptotic events taking place within affected PM muscles. Moreover, these results support the hypothesis of a role of the Collagen type IV in the cascade of events leading to the occurrence of WS/WB and identify novel actors probably involved in their onset.

Abstract In recent years, the poultry industry has experienced an increased incidence of myopathies affecting breasts of fast-growing broilers, such as White Striping (WS) and Wooden Breast (WB) defects. To explore the molecular mechanisms and genes involved in WS and WB onset, we decided to perform a Weighted Gene Co-expression Network Analysis (WGCNA) using the gene expression profile and meat quality parameters of Pectoralis major muscles analysed in our previous study. Among the 212 modules identified by WGCNA, the red, darkred, midnightblue and paleturquoise4 modules were chosen for subsequent analysis. Functional analysis evidenced pathways involved in extracellular matrix (ECM) organization, collagen metabolism, cellular signaling and unfolded protein response. The hub gene analysis showed several genes coding for ECM components as the most interconnected nodes in the gene network (e.g. COL4A1, COL4A2, LAMA2, LAMA4, FBLN5 and FBN1). In this regard, this study suggests that alterations in ECM composition could somehow activate the cascade of biological reactions that result in the growth-related myopathies onset, and the involvement of Collagen IV alterations in activating the endoplasmic reticulum (ER) stress response may be hypothesized. Therefore, our findings provide further and innovative knowledge concerning the molecular mechanisms related to the breast abnormalities occurrence in modern broilers.

The addition of n-3 polyunsaturated fatty acids (n-3 PUFAs) to the swine diet increases their content in muscle cells, and the additional supplementation of antioxidants promotes their oxidative stability. However, to date, the functionality of these components within muscle tissue is not well understood. Using a published RNA-seq dataset and a selective workflow, the study aimed to find the differences in gene expression and investigate how differentially expressed genes (DEGs) were implicated in the cellular composition and metabolism of muscle tissue of 48 Italian Large White pigs under different dietary conditions. A functional enrichment analysis of DEGs, using Cytoscape, revealed that the diet enriched with extruded linseed and supplemented with vitamin E and selenium promoted a more rapid and massive immune system response because the overall function of muscle tissue was improved, while those enriched with extruded linseed and supplemented with grape skin and oregano extracts promoted the presence and oxidative stability of n-3 PUFAs, increasing the anti-inflammatory potential of the muscular tissue.

In this work, the Longissimus thoracis pig skeletal muscle was used as a model to investigate the impact of two different diets, supplemented with n-3 polyunsaturated fatty acids from extruded linseed (L) and polyphenols from grape skin and oregano extracts (L+P), on the lipidomic profile of meat. A standard diet for growing-finishing pigs (CTRL) was used as a control. Changes in lipids profile were investigated through an untargeted lipidomics and transcriptomics combined investigation. The lipidomics identified 1507 compounds, with 195 compounds fitting with the MS/MS spectra of LipidBlast database. When compared with the CTRL group, the L+P diet significantly increased 15 glycerophospholipids and 8 sphingolipids, while the L diet determined a marked up-accumulation of glycerolipids. According to the correlations outlined between discriminant lipids and genes, the L diet may act preventing adipogenesis and the related inflammation processes, while the L+P diet promoted the expression of genes involved in lipids' biosynthesis and adipogenic extracellular matrix formation and functioning.

The aim of this study was to investigate the expression of genes related to muscle growth, hypoxia and oxidative stress responses, a multi-substrate serine/threonine-protein kinase (AMPK) and AMPK-related kinases, carbohydrate metabolism, satellite cells activities and fibro- adipogenic progenitors (FAPs) in fast-growing (FG) (n = 30) and medium-growing (MG) chickens (n = 30). Pectoralis major muscles were collected at 7d, 14d, 21d, 28d, 35d and 42d of age. According to their macroscopic features, the samples from FG up to 21d of age were classified as unaffected, while all samples collected at an older age exhibited macroscopic features ascribable to white striping and/or wooden breast abnormalities. In contrast, MG samples did not show any feature associated to muscle disorders. The absolute transcript abundance of 33 target genes was examined by droplet digital polymerase chain reaction. The results showed differential gene expression profiles between FG and MG chickens at different ages. While most genes remained unchanged in MG chickens, the expression patterns of several genes in FG were significantly affected by age. Genes encoding alpha 1, alpha 2, beta 2 and gamma 3 isoforms of AMPK, as well as AMPK-related kinases, were identified as differentially expressed between the two strains. The results support the hypothesis of oxidative stress-induced muscle damage with metabolic alterations in FG chickens. An increased expression of ANXA2, DES, LITAF, MMP14, MYF5 and TGFB1 was observed in FG strain. The results suggest the occurrence of dysregulation of FAP proliferation and differentiation occurring during muscle repair. FAPs could play an important role in defining the proliferation of connective tissue (fibrosis) and deposition of intermuscular adipose tissue which represents distinctive traits of muscle abnormalities. Overall, these findings demonstrate that dysregulated molecular processes associated with myopathic lesions in chickens are strongly influenced by growth rate, and, to some extent, by age.

Abstract Background Supplementing farm animals diet with functional ingredients may improve the nutritional quality of meat products. Diet composition has been also demonstrated to influence the gene expression with effect on biological processes and pathways. However, the knowledge on the effect of nutrients at the molecular level is scant. In particular, studies on the effects of antioxidants and polyphenols dietary supplementation have been investigated mainly in rodents, and only scarcely in farm animals so far. RNA-Seq with next-generation sequencing is increasingly the method of choice for studying changes in the transcriptome and it has been recently employed also in pig nutrigenomics studies to identify diet-induced changes in gene expression. The present study aimed to investigate the effect of diets enriched with functional ingredients (linseed, vitamin E and plant extracts) on the transcriptome of pig Longissimus thoracis to elucidate the role of these compounds in influencing genes involved in muscle physiology and metabolism compared to a standard diet. Results Eight hundred ninety-three significant differentially expressed genes (DEGs) (FDR adjusted P-value ≤ 0.05) were detected by RNA-Seq analysis in the three diet comparisons (D2-D1, D3-D1, D4-D1). The functional analysis of DEGs showed that the diet enriched with n-3 PUFA from linseed (D2) mostly downregulated genes in pathways and biological processes (BPs) related to muscle development, contraction, and glycogen metabolism compared to the standard diet. The diet supplemented with linseed and vitamin E/Selenium (D3) showed to mostly downregulate genes linked to oxidative phosphorylation. Only few genes involved in extracellular matrix (ECM) organization were upregulated by the D3. Finally, the comparison D4-D1 showed that the diet supplemented with linseed and plant extracts (D4) upregulated the majority of genes compared to D1 that were involved in a complex network of pathways and BPs all connected by hub genes. In particular, IGF2 was a hub gene connecting protein metabolism, ECM organization, immune system and lipid biosynthesis pathways. Conclusion The supplementation of pig diet with n-3 PUFA from linseed, antioxidants and plant-derived polyphenols can influence the expression of a relevant number of genes in Longissimus thoracis muscle that are involved in a variety of biochemical pathways linked to muscle function and metabolism.

Abstract Background Colostrum is the first secretion produced by mammary glands during the hours immediately preceding and succeeding parturition. This secretion differs from milk and represents an essential vehicle of passive immunity, prebiotic compounds and growth factors involved in intestinal development. Most of the literature concerning colostrum composition refers mainly to human and cow; and little is known about pig colostrum metabolome and how it varies between pig breeds and different farrowing parity. Thus, the aim of the present research is to provide new information about pig colostrum composition and the associations between metabolites, the sows’ breed and the survival and growth rates of their litters. Results Colostrum samples were gathered from 58 parturitions of sows belonging to three different breeds chosen for their importance in Italian heavy pig production: 31 Large White, 15 Landrace and 12 Duroc respectively. The defatted and ultrafiltered colostrum samples were analysed using 1H–NMR spectroscopy. Principal Components Analysis (PCA) was assessed on the obtained spectra. In addition, using a Stepwise Regression and a Linear Regression analyses the metabolites named after the signals assignment were tested for their associations with piglets’ performances. Twenty-five metabolites were identified, comprehending monosaccharides, disaccharides (such as lactose), organic acids (lactate, citrate, acetate and formate), nitrogenous organic acids (such as creatine) and other compounds, including nucleotides. PCA results evidence a clustering due to breed and season effects. Lactose was the main compound determining the assignment of the samples into different clusters according to the sow breed. Furthermore, some metabolites showed to be associated with piglets’ performance and survival traits: acetate and taurine were positively related to litter weight gain and piglets’ survival rate, respectively, while dimethylamine and cis-aconitate were linked to new-borns’ impaired ability to survive. Conclusions The results obtained suggest that colostrum composition is affected by breed, which, together with environmental conditions, may cause changes in colostrum metabolites content with possible consequences on piglets’ performances. Among the identified metabolites, acetate, taurine, dimethylamine and cis-aconitate showed consistent associations with piglets’ survival rate and litter weight gain, implying that these compounds may affect new-borns’ ability to survive.

Intramuscular fat content (IMF) is a complex trait influencing the technological and sensorial features of meat products and determining pork quality. Thus, we aimed at analyzing through RNA-sequencing the Semimembranosus muscle transcriptome of Italian Large White pigs to study the gene networks associated with IMF deposition. Two groups of samples were used; each one was composed of six unrelated pigs with extreme and divergent IMF content (0.67 ± 0.09% in low IMF vs. 6.81 ± 1.17% in high IMF groups) that were chosen from 950 purebred individuals. Paired-end RNA sequences were aligned to Sus scrofa genome assembly 11.1 and gene counts were analyzed using WGCNA and DeSeq2 packages in R environment. Interestingly, among the 58 differentially expressed genes (DEGs), several were related to primary cilia organelles (such as Lebercilin 5 gene), in addition to the genes involved in the regulation of cell differentiation, in the control of RNA-processing, and G-protein and ERK signaling pathways. Together with cilia-related genes, we also found in high IMF pigs an over-expression of the Fibroblast Growth Factor 2 (FGF2) gene, which in other animal species was found to be a regulator of ciliogenesis. Four WGCNA gene modules resulted significantly associated with IMF deposition: grey60 (P = 0.003), darkturquoise (P = 0.022), skyblue1 (P = 0.022), and lavenderblush3 (P = 0.030). The genes in the significant modules confirmed the results obtained for the DEGs, and the analysis with "cytoHubba" indicated genes controlling RNA splicing and cell differentiation as hub genes. Among the complex molecular processes affecting muscle fat depots, genes involved in primary cilia may have an important role, and the transcriptional reprogramming observed in high IMF pigs may be related to an FGF-related molecular cascade and to ciliogenesis, which in the literature have been associated with fibro-adipogenic precursor differentiation.

Desmin (DES) and Vimentin (VIM) exert an essential role in maintaining muscle cytoarchitecture and since are considered reliable markers for muscle regeneration, their expression has been extensively investigated in dystrophic muscles. Thus, exhibiting features similar to those of human dystrophic muscles, the present study aimed at assessing the distribution of VIM and DES proteins and the expression of the corresponding genes in Pectoralis major muscles affected by white striping (WS), wooden breast (WB), and spaghetti meat (SM) abnormalities as well as in those having macroscopically normal appearance (NORM). For this purpose, 20 Pectoralis major muscles (5/group) were collected from the same flock of fast-growing broilers to perform immunohistochemistry, immunoblotting and gene expression. Immunohistochemical analyses showed an increased number of fibers immunoreactive to both VIM and DES in WS and WB, while only a few immunoreactive fibers were observed in NORM. Concerning the protein level, if compared with NORM, a 55% increase in VIM content was found in WB affected cases (P < 0.05) thus suggesting the development of intense regenerative processes in an early-stage within these muscles. The significantly higher amount of DES (+53%) found in WS might be attributed to a progression of the regenerative processes that require its synthesis to preserve the structural organization of the developing fibers. On the other hand, significantly lower VIM and DES contents were found in SM. About gene expression, VIM mRNA levels gradually increased from the NORM to the SM group, with significantly higher gene expressions in WB and SM samples compared to the NORM group (P = 0.009 for WB vs. NORM and P = 0.004 for SM vs. NORM). Similarly, the expression of DES gene showed an increase from the NORM to WB group (P = 0.05). Overall, the findings of the present study suggest that intense regenerative processes take place in both WB and WS muscles although a different progression of regeneration might be hypothesized. On the other hand, the lack of correspondence between VIM gene expression and its protein product observed in SM suggests that VIM may also exert a role in the development of the SM phenotype.

To date, high-throughput technology such as RNA-sequencing has been successfully applied in livestock sciences to investigate molecular networks involved in complex traits, such as meat quality. Pork quality depends on several organoleptic, technological, and nutritional characteristics, and it is also influenced by the fatty acid (FA) composition of intramuscular fat (IMF). To explore the molecular networks associated with different IMF FA compositions, the Semimembranosus muscle (SM) from two groups of Italian Large White (ILW) heavy pigs divergent for SM IMF content was investigated using transcriptome analysis. After alignment and normalization, the obtained gene counts were used to perform the Weighted Correlation Network Analysis (WGCNA package in R environment). Palmitic and palmitoleic contents showed association with the same gene modules, comprising genes significantly enriched in autophagy, mitochondrial fusion, and mitochondrial activity. Among the key genes related to these FAs, we found TEAD4, a gene regulating mitochondrial activity that seems to be a promising candidate for further studies. On the other hand, the genes comprised in the modules associated with the IMF contents of oleic, n-6, and n-3 polyunsaturated FAs (PUFAs) were significantly enriched in Mitogen-Activated Protein Kinase (MAPK) signaling, in agreement with previous studies suggesting that several MAPK players may have a primary role in regulating lipid deposition. These results give an insight into the molecular cascade associated with different IMF FA composition in ILW heavy pigs. Further studies are needed to validate the results and confirm whether some of the identified key genes may be effective candidates for pork quality.

Protected Designation of Origin (PDO) dry-cured hams production is greatly dependent on raw meat quality. This study was performed to identify genetic markers associated with the quality of dry-cured ham. Carcass traits of 229 heavy pigs belonging to three commercial genetic lines were registered (weight, EUROP classification). Phenotypic traits (Semimembranosus muscle ultimate pH, ham weight and lean meat content, adsorbed salt) of the corresponding thighs, undergone PDO ham process in three different plants, were measured, using a fast and non-invasive technology. Green ham weight and lean meat percentage influenced the estimated salt content and the weight loss during salting, even if the processing plant greatly affected the variability of the measured ham traits. The genomic data were obtained with the GeneSeek Genomic Profiler (GGP) 70k HD Porcine Array, using the slaughter day and the sex of the animals in the statistical analyses. The phenotypic traits were associated with the genotypes through GenAbel software. The results showed that 18 SNPs located on nine porcine chromosomes were found to be associated with nine phenotypic traits, mainly related to ham weight loss during salting. New associations were found between markers in the genes Neural Precursor Cell Expressed Developmentally Down-Regulated 9 (NEDD9, SSC7), T-Cell Lymphoma Invasion and Metastasis 2 (TIAM2, SSC1), and the ham quality traits. After validation, these SNPs may be useful to improve the quality of thighs for the production of PDO dry-cured hams.

Supplementing pig diets with n-3 polyunsaturated fatty acids (n-3 PUFA) may produce meat products with an increased n-3 fatty acid content, and the combined antioxidants addition could prevent lipid oxidation in the feed. However, to date, the effects of these bioactive compounds at the molecular level in porcine skeletal muscle are mostly unknown. This study aimed to analyse changes in the Longissimus thoracis transcriptome of 35 pigs fed three diets supplemented with: linseed (L); linseed, vitamin E and Selenium (LES) or linseed and plant-derived polyphenols (LPE). Pigs were reared from 80.8 ± 5.6 kg to 151.8 ± 9.9 kg. After slaughter, RNA-Seq was performed and 1182 differentially expressed genes (DEGs) were submitted to functional analysis. The L vs LES comparison did not show differences, while L vs LPE showed 1102 DEGs and LES vs LPE 80 DEGs. LPE compared to the other groups showed the highest number of up-regulated genes involved in preserving muscle metabolism and structure. Results enlighten that the combined supplementation of bioactive lipids (n-3 PUFA from linseed) with plant extracts as a source of polyphenols increases, compared to the only addition of linseed, the expression of genes involved in mRNA metabolic processes and transcriptional regulation, glucose uptake and, finally, in supporting muscle development and physiology. These results improve the knowledge of the biological effect of bioactive compounds in Longissimus thoracis muscle, and sustain the growing interest over their use in pig production.

Porcine fat traits depend mostly on the interaction between nutritional and genetic factors. However, the pathways and biological processes influenced by this interaction are still poorly known in pigs, although they can have a huge impact on meat quality traits. The present research provides new knowledge insight into the effect of four diets (D1 = standard diet; D2 = linseed supplementation; D3 = linseed, vitamin E and selenium supplementation; D4 = linseed and plant-derived polyphenols supplementation) on the expression of 24 candidate genes selected for their role in lipid and energy metabolism. The data indicated that 10 out of 24 genes were differentially expressed among diets, namely ACACA, ADIPOQ, ADIPOR1, CHREBP (MLXPL), ELOVL6, FASN, G6PD, PLIN2, RXRA and SCD. Results from the univariate analysis displayed an increased expression of ACACA, ADIPOQ, ADIPOR1, CHREBP, ELOVL6, FASN, PLIN2, RXRA and SCD in D4 compared to D2. Similarly, ACACA, ADIPOQ, ADIPOR1, ELOVL6 and SCD were highly expressed in D4 compared to D3, while no differences were observed in D2-D3 comparison. Moreover, an increased expression of G6PD and ELOVL6 genes in D4 compared to D1 was observed. Results from the multivariate analysis confirmed that D2 was not different from D3 and that ACACA, SCD and FASN expression made D4 different from D2 and D3. Comparing D4 and D1, the expression levels of ELOVL6 and ACACA were the most influenced. This research provides evidence that the addition of both n-3 PUFA and polyphenols, derived from linseed, grape-skin and oregano supplementation in the diets, stimulates the expression of genes involved in lipogenesis and in oxidative processes. Results evidenced a greater effect on gene expression of the diet added with both plant extracts and n-3 PUFA, resulting in an increased expression of genes coding for fatty acid synthesis, desaturation and elongation in pig Longissimus thoracis muscle.

Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms) were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP) approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese) and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred) breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

In cattle, the MC1R gene has been the subject of several studies with the aim to elucidate the biology of coat colour. Then, polymorphisms of this gene have been proposed as tools for breed identification and animal products authentication. As a first step to identify breed specific DNA markers that can be used for the traceability of mono-breed dairy cattle products we investigated, using PCR-RFLP and PCR-APLP protocols, the presence and distribution of some alleles at the MC1R locus in 18 cattle breeds for a total of 1360 animals. For each of seven breeds (Italian Holstein, Italian Brown, Italian Simmental, Rendena, Jersey, Reggiana and Modenese) a large number of animals (>70) was genotyped so the obtained results can be considered with more confidence. Allele ED was identified only in black pied cattle (Italian Holstein and Black Pied Valdostana). Allele E (this nomenclature includes all alleles except ED, E1 and e) was observed in Italian Brown, Rendena, Jersey, Modenese, Italian Simmental, Grigio Alpina, Piedmontese, Chianina, Romagnola, Marchigiana, Swedish Red and White and Danish Red. Allele E1 was identified in Italian Brown, Rendena, Grigio Alpina, Piedmontese, Swedish Red and White and Danish Red. The recessive allele e, known to cause red coat colour, was fixed in Reggiana and almost fixed in Italian Simmental. This allele was observed also in Italian Holstein, Italian Brown, Rendena, Jersey and Modenese albeit with low frequency. Moreover, this allele was detected in Valdostana, Pezzata Rossa d’Oropa, Piedmontese, Romagnola, Swedish Red and White, Danish Red, Charoleis and Salers. In the case of the Reggiana breed, which is fixed for allele e, the MC1R locus is highly informative with respect to breeds that carry other alleles or in which allele e is at very low frequency. In theory, using the MC1R locus it is possible to identify the presence of milk from some other breeds in Parmigiano Reggiano cheese labelled as exclusively from the Reggiana breed. This possibility was practically tested by setting up protocols to extract and analyse polymorphisms of the MC1R locus in several dairy products, including Parmigiano Reggiano cheese cured for 30 months. The lower detection limit was estimated to be 5% of non expected DNA. This test can represent a first deterrent against fraud and an important tool for the valorisation and authentication of Parmigiano Reggiano cheese obtained from only Reggiana milk.

A major QTL for milk production traits was reported in the middle of bovine chromosome 20 and, for its map position, the growth hormone receptor (GHR) gene was considered a strong positional and functional candidate gene. A missense mutation in exon VIII (F279Y amino acid substitution) showed highly significant effects mainly on milk composition (protein percentage and fat percentage) as well as on milk yield in several dairy cattle populations. As no information about the frequency of these two GHR alleles is available in any population, we studied their distribution in dairy and dual purpose cattle breeds reared in Italy. A total of 679 animals belonging to seven cattle breeds (Italian Holstein-Friesian, n=108; Italian Brown, n=104; Italian Simmental, n=104; Jersey, n=104; Reggiana, n=108; Modenese, n=66; Rendena, n=85) were sampled. A new PCR-RFLP protocol was designed to analyse this mutation inserting an artificial restriction site for enzyme SspI. In all investigated breeds, allele F was the most frequent and ranged from 0.947 (Italian Brown and Jersey) to 0.727 (Italian Holstein-Friesian). In Rendena, Italian Simmental, Reggiana and Modenese it was 0.824, 0.909, 0.921 and 0.924, respectively. For all breeds no significant deviation from the Hardy-Weinberg equilibrium was observed. Differences in allele frequencies were statistically significant between the Italian Holstein Friesian and Rendena against all other breeds. Due to the high frequency of the putative positive allele for milk protein percentage the use of the GHR F279Y marker in marker assisted selection plans should not have a great impact on this trait in the studied breeds.

Nero Siciliano (NS; Sicilian Black) is a local pig breed reared on the island of Sicily mainly under extensive management.The breed is well adapted to marginal conditions and is appreciated for its reproductive performance, disease resistanceand production of tasty meat. For a genetic characterization of this breed we analyzed the allele frequencies of singlenucleotide polymorphisms (SNPs) in eight major or candidate genes (ryanodine receptor 1, RYR1; Na+, K+ ATPase subunitα 2, ATP1A2; myosin heavy chain 2B, MYH4; sarcolipin, SLN; cathepsin B, CTSB; cystatin B, CSTB; estrogen receptor,ESR; melanocortin receptor 1, MC1R) for performance and phenotypic traits. The animals that were sampled andanalyzed represent about 6-8% of the total NS pig population. PCR-RFLP or PCR-SSCP techniques were used to type theDNA markers in the selected loci. Exact test of Hardy-Weinberg equilibrium was computed for each locus, Fis statisticsand heterozygosity were calculated for each locus and over all loci. Allele frequencies obtained in NS breed were comparedto the frequencies already available in literature for the Large White, Landrace, Duroc, Belgian Landrace, Piétrain,Hampshire and Meishan breeds. For the ESR locus, as no information on the distribution of the two alleles were available,we typed a sample of unrelated pigs from the considered breeds.Even if only eight loci were studied in NS breed, important elements were obtained from the data. The 1843T (n) alleleat the RYR1 locus is present in NS breed, thus the molecular test to identify the carriers of this allele should be adoptedto avoid its spreading in the population. Moreover, other studies are needed to clarify the allelic structure of the MC1Rgene, which affects coat color, in order to evaluate if this gene could be used in genetic tests for the traceability of themeat products of this breed. Finally, the present work represents an attempt to evaluate data on mutations within majorand candidate genes with the final aim to provide information that could be useful for the conservation and valorizationof local farm animal genetic resources.

Six batches of four commercial hybrids of heavy pigs, reared for the production of Italian dry-cured hams, were identifiedfor having homogeneous feeding and farm conditions. For a total of 235 pigs, slaughtered in the same slaughterhouse,carcass traits and muscle composition were measured. The pigs were genotyped for single nucleotide polymorphisms(SNPs) of Na+, K+-ATPase subunit alpha 2 (ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide; ATP1A2), cystatinB (CSTB), mitochondrial 2,4-dienoyl-CoA reductase 1 (DECR1), leptin (LEP; 3 SNPs), melanocortin receptor 4(MC4R), melanocortin receptor 5 (MC5R), sarcolipin (SLN) and titin (TTN) genes. All genes showed biallelic polymorphismsand the alleles were differently distributed between the six batches. Pigs were subsequentely clustered in “lean”and “fat” using either carcass traits (lean percentage, backfat thickness, loin muscle thickness, ham weight and hamcover fat thickness: 100 lean and 135 fat) or meat composition data (dry matter, protein, fat and ash of Biceps femorisand Vastus lateralis and pH after 24 hours: 126 lean and 109 fat). The association of gene polymorphisms with leanessand fatness of pigs was thus investigated using a logistic regression. ATP1A2, LEP (HinfI polymorphism) and MC4R,together with sex and ham weight were, included in the model to screen lean and fat pigs classified according to carcasstraits data, yielding a correct classification of 71%. For the lean and fat pigs classified according to muscle composition,sex, CSTB, DECR1, MC5R and LEP (AciI/TaqI polymorphisms) were included in the regression analysis, that yielded a66% of pigs correctly classified.These preliminary results may indicate that some of the selected candidate genes could be associated to production traitsand are worth of further investigations.

Three genes (ATPase, Na+/K+transporting, α2(+) 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 ATP1A2and DECR1loci 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, Piétrain, 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, CA3and DECR1genes were genotyped in Italian Large White and Italian Duroc animal groups with extreme and divergent estimated breeding value for several production traits. For CA3significant 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 vis- ible intermuscular fat trait. ATP1A2, which maps close to the FAT1locus, did not show any significant dif- ference. A very high linkage disequilibrium (D’= 0.967; P< 0.0001) was identified between CA3and DECR1 in the Italian Duroc population. Further investigations are needed to evaluate the effect of CA3and DECR1 on the considered traits.

The knowledge of genes and molecular processes controlling stress reactions and involved in the genetic system determining resistance to stress in pigs could be important for the improvement of meat quality. This research aimed to compare the expression profiles of skeletal muscle between physically stressed and not stressed pigs of different breeds immediately before slaughter. DNA microarray analysis showed that different functional categories of genes are up-regulated in stressed compared to not stressed pigs and relevant differences among breeds were found.

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.