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Sea cucumbers are deposit feeding members of marine benthic communities. The over-exploitation of sea cucumber natural stocks, especially in the Mediterranean basin, is having negative impacts on the marine ecosystem. This concerns had led the Italian government to take legal actions to preserve these important marine resources. The aim of this study was to evaluate the level of genetic diversity and population structures within two sympatric Holothuria sea cucumber species, H. polii and H. tubulosa, across ten Mediterranean areas along Italy. A 323 bp portion of the COI mitochondrial gene was sequenced in a total of 441 holothurians (251 H. polii, 177 H. tubulosa and 13 from other species). Genetic diversity analyses and analysis of molecular variance (AMOVA) suggest that H. polii and H. tubulosa species are distinct, but within and among populations are homogeneous, indicating active gene flows across the Mediterranean areas investigated. H. polii showed a lower genetic diversity than H. tubulosa, probably related to differences in life history traits. Phylogenetic analyses showed a clear differentiation between the two species, even if six specimens morphologically assigned to a species clustered within the other species, indicating a possible occurrence of hybridization events. These data will be useful in implementing conservation actions for these holothurian genetic resources., (© 2024. The Author(s).)

The gilthead seabream ( Sparus aurata ) is a species of relevance for the Mediterranean aquaculture industry. Despite the advancement of genetic tools for the species, breeding programs still do not often include genomics. In this study, we designed a genomic strategy to identify signatures of selection and genomic regions of high differentiation among populations of farmed fish stocks. A comparative DNA pooling sequencing approach was applied to identify signatures of selection in gilthead seabream from the same hatchery and from different nuclei that had not been subjected to genetic selection. Identified genomic regions were further investigated to detect SNPs with predicted high impact. The analyses underlined major genomic differences in the proportion of fixed alleles among the investigated nuclei. Some of these differences highlighted genomic regions, including genes involved in general metabolism and development already detected in QTL for growth, size, skeletal deformity, and adaptation to variation of oxygen levels in other teleosts. The obtained results pointed out the need to control the genetic effect of breeding programs in this species to avoid the reduction of genetic variability within populations and the increase in inbreeding level that, in turn, might lead to an increased frequency of alleles with deleterious effects.

The aim of this research has been to assess the effect of the dietary protein level on piglet growth and post-weaning diarrhea (PWD) incidence. Piglet fecal microbiota and feces composition were also assessed. The experiment was carried out on 144 weaned piglets (Duroc × Large White; 72 piglets per treatment) and lasted from weaning (at 25 days of age) until the end of the post-weaning phase (at 95 days). Two dietary protein levels were compared: high (HP; 17.5% crude protein on average, during the experiment) and low (LP; 15.5% on average). Lower ( p < 0.01) average daily gain and feed conversion ratio were observed in LP piglets in the first growth phase. However, at the end of the post-weaning period, the growth parameters were not significantly different in the two diets. Diarrhea scores were lower in piglets fed LP diets than in piglets fed HP diets (28.6% of the total vs. 71.4% in the HP piglets). Fibrobacteres , Proteobacteria, and Spirochaetes were more abundant in the feces of the piglets fed LP diets. Feces nitrogen content was lower in piglets fed LP diets. In conclusion, low protein levels in the diet can reduce the incidence of PWD while only marginally affecting growth parameters.

CXCL8 (also known as IL-8) is a member of the CXC subfamily of chemokines that binds two of the seven transmembrane G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, to mediate and regulate leucocyte accumulation and activation at sites of inflammation. They are known to play a critical role in both disease susceptibility and infection outcome. The aim of this study was to investigate the entire sequences of CXCL8 and CXCR2 genes in thirty-one Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, and for somatic cell score (SCS). Five new single nucleotide polymorphisms (SNPs) were found in CXCR2 gene. The analysis of association indicated that one SNP in CXCL8 and two in CXCR2 influenced the considered traits. To evaluate the existence of functional haplotypic effects, combinations among the three genomic variants (SNP 1 in CXCL8 , SNP 6 and SNP 7 in CXCR2 ) were investigated. Four different haplotypic alleles were identified in the experimental population, one of which at a high frequency (61%). Bulls with Hap 4 (G-C-G at SNP 1, SNP 6, and SNP 7 respectively) had more favourable indexes for SCS ( P < 0.05). These results suggest that the SNPs in CXCL8 and CXCR2 may be potential genetic markers to improve udder health in the Simmental breed.

This study examined the environmental performance of mussel (Mytilus galloprovincialis) farming in the view of reduction of greenhouse gas emissions, through the Life Cycle Assessment (LCA) methodology. The LCA has been integrated with the evaluation of the carbon sequestration potential of the biocalcification process. Three case studies of mussel farming sited along the coastal area in the north Adriatic Sea, Italy, were analyzed. Two of them concerned mussels that do not require a depuration process (area Class A), and one inspected mussel production in the rearing area of Class B, which imposes a depuration phase after harvesting. This study examined all the relevant flows of materials and energy across the systems and explored the potential role of mussel biocalcification in stocking seawater carbon into the shells. Global Warming (GW) -related emissions amounted to 0.07-0.12 kg CO 2 eq for Class&#95;A case studies and to 0.53 kg CO 2 eq for Class&#95;B case study. Through biogenic calcification, 0.19-0.20 kg CO 2 kg -1 mussel is fixed in the shells, and 0.12 kg CO 2 kg -1 mussel is released. These flows resulted in a net sequestration of about 0.08 kg CO 2 kg -1 mussel. This study confirmed the good environmental performance of the mussel production in the farming systems analyzed. When considering greenhouse gasses emissions, the extent to which the seawater carbon fixed in the shell as calcium carbonate can be considered a carbon sink was discussed and substantiated by locally collected environmental data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cluster of differentiation 4 (CD4) is the accessory protein non-covalently bound to the T cell receptor that recognizes an invariant region of MHC class II on antigen presenting cells. Its cytoplasmic tail, physically associated with a protein tyrosine kinase, is important in the activation of helper/inducer T lymphocytes. In Bos taurus, CD4 gene is located on chromosome 5 from which two isoforms are transcribed, with a different number of amino acids due to splicing of exon 7 and variation in the reading frame. The aim of this study was to investigate the sequence of the entire CD4 gene in Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, as well as somatic cell score. The associations among genomic variants and indexes were analysed using the Allele and GLM procedures of SAS 9.4. The analysis indicated that only four of the thirty-one identified SNPs influenced the considered traits. Identified variants insist on coding zones and intronic sequences, where we revealed the presence of sites for transcription factors. To evaluate the existence of haplotypic effects, combinations among the four genomic variants (SNP 3, SNP 8, SNP 11 and SNP 19) were investigated. Six different haplotypic alleles were identified, but only four of them were frequent enough to allow for an evaluation of any haplotypic effect (at least six copies in the examined sample): Hap1, Hap2, Hap3 and Hap6. The analysis of associations between the selected haplotypes in the CD4 gene with milk related indexes showed that bulls with Hap2 (T-A-C-C) had better indexes for milk and protein yields (P < 0.05), whereas the presence of the Hap1 haplotype (A-G-A-T) caused a significant decrease of the index for protein yield (P < 0.05). Frequencies of the two alleles Hap1 and Hap2 (9 and 36% respectively) make them of interest for their possible inclusion in breeding schemes and support the hypothesis of considering this gene as a candidate for the improvement of milk-related traits in the Simmental breed.

In organic poultry production it is important to rear animals with a dynamic attitude to take advantage of outdoor areas. Farmers are reluctant to use such strains due to their lower productivity and older slaughtering age. However, fast growing lines grown in organic system often suffer poor health and welfare conditions. The kinetic metabolism of chickens is correlated with different types of muscle fiber: type I (in red muscles or oxidative) for prolonged and moderate movement and type II (in white muscles or glycogenic) for fast movements. Red muscle metabolism produces energy mainly by β-oxidation of Highly Unsaturated n-3 Fatty Acids (HUFA). Accordingly, kinetic activity causes higher consume of HUFA in red muscles than in white muscles, so the ratio between n-3 HUFA and their precursor C18:3 n-3 (ALA) is likely to be smaller in red than in white muscles. However, these ratios are highly affected by the environment. To reduce the effect of environmental variables, we propose an "Activity index" as the difference between n-3 HUFA/ALA in white and red muscle within the same bird. This index, measured after slaughtering, should represent the activity performed by the chicken during its life. Given that birds in good health had the possibility of moving, the "Activity index" would measure the activity actually performed by the animals. Should birds of a given strain show a higher activity level, this would be an indication of the suitability of that strain to outdoor systems. This work verified the application of this "Activity index" on 90 birds from 6 genetic strains with known kinetic behavior reared in an experimental farm. The "Activity index" was also tested on chicken strains collected form commercial organic farms. The results confirmed that strains recognized for higher kinetic attitude actually walked more and their behavior was clearly detected by the "Activity index" estimated from their muscles., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The gilthead seabream (Sparus aurata, Sparidae family) is commonly used for aquaculture. Despite its great economic value, several problems in its cultivation remain. One of the major concerns is the high frequency of morphological abnormalities occurring during the early developmental stages. Partial and/or total lack of operculum is the most frequent anomaly affecting the fish cranial region. The existence of genetic factors that can at least partially determine this defect has been hypothesized. In this work, two DNA pools of highly related fry, one composed of normal-looking (control) fish and the other lacking an operculum (case), were constructed and whole-genome resequencing data produced from the two were compared. The analysis revealed a 1 Mb region on chromosome 2 with higher heterozygosity in the lack of operculum DNA pool than in the control DNA pool, consistent with the enrichment, in the first DNA pool, of one or more haplotypes causing or predisposing to the defect together with other normal haplotypes. A window-based F ST analysis between the two DNA pools indicated that the same region had the highest divergence score. This region contained 2921 SNVs, 10 of which, with predicted high impacts (three splice donor and seven stop-gained variants), were detected in novel genes that are homologous to calcium-sensing receptor-like genes, probably involved in bone development. Other studies are needed to clarify the genetic mechanisms involved in predisposing fry to this deformity and then to identify associated markers that could be used in breeding programs to reduce the frequency of this defect in the broodstock., (© 2021 Stichting International Foundation for Animal Genetics.)

In the European rabbit (Oryctolagus cuniculus), a polytocous livestock species, the number of teats indirectly impacts the doe reproduction efficiency and, in turn, the sustainable production of rabbit meat. In this study, we carried out a genome-wide association study (GWAS) for the total number of teats in 247 Italian White does included in the Italian White rabbit breed selection program, by applying a selective genotyping approach. Does had either 8 (n = 121) or 10 teats (n = 126). All rabbits were genotyped with the Affymetrix Axiom OrcunSNP Array. Genomic data from the two extreme groups of rabbits were also analysed with the single-marker fixation index statistic and combined with the GWAS results. The GWAS identified 50 significant SNPs and the fixation index analysis identified a total of 20 SNPs that trespassed the 99.98th percentile threshold, 19 of which confirmed the GWAS results. The most significant SNP (P = 4.31 × 10 -11 ) was located on OCU1, close to the NUDT2 gene, a breast carcinoma cells proliferation promoter. Another significant SNP identified as candidate gene NR6A1, which is well known to play an important role in affecting the correlated number of vertebrae in pigs. Other significant markers were close to candidate genes involved in determining body length in mice. Markers associated with increased number of teats could be included in selection programmes to speed up the improvement for this trait in rabbit lines that need to increase maternal performances., (© 2021 Stichting International Foundation for Animal Genetics.)

New approaches are needed to improve the sustainability of feed production and utilization by ruminants. Promising approaches include increased use of buffaloes for more sustainable milk production, and arbuscular mycorrhizal fungi (AMF) to reduce crop production input needs. However, studies assessing the effect of crops grown in the presence of AMF on rumen microbial utilization are limited. Based on current knowledge, we hypothesized that maize grain grown on AMF-inoculated soil affected ruminal fermentation and microbiota, and that this effect differed between buffalo and cattle. A dietary cross-over study (four weeks per diet) was conducted using rumen-cannulated cattle ( n = 5) and buffalo ( n = 6) to assess the effect of maize grain (3.9% (w/v) of diet) grown on soil with or without AMF (15 kg/ha) on ruminal fermentation and microbiota. Production of maize on AMF-treated soil did not affect any of the assessed ruminal fermentation parameters, microbial concentrations, or prokaryotic community composition (using prokaryotic 16S rRNA gene sequence analysis). In contrast, host type had numerous effects. Protozoal counts, lactate, total VFA and isobutyrate, were significantly higher in buffaloes compared to cattle. Conversely, butyrate was significantly lower in buffaloes than in cattle. Host type explained 9.3% of the total variation in prokaryotic community composition, and relative abundance of nine amplicon sequence variants significantly differed between host types. These findings indicate that AMF treatment of maize crops has no detrimental impact on the value of the resulting maize grains as a ruminant feed, and provides additional insight into rumen-based differences between cattle and buffalo., (Copyright © 2020 Chiariotti, Edwards, Hermes, Catillo, Meo Zilio, Di Giovanni, Smidt and Buttazzoni.)

Flow cytometry is a powerful technology used in many fields of cell biology. It is also used as a routine method to count somatic cells in milk and to characterize bovine milk leukocytes. In this study, we used flow cytometry to simultaneously assess the viability, the percentage of the single subsets of leukocytes and to quantify the expression of CD11b, an immunological marker of cell activation status. Immunological markers were then related with on farm recorded parameters as milk electrical conductivity (MEC) and average milk flow rate (MFR). Composite milk samples were collected from 43 cows, nine of which had naturally infected udders and 34 of which had no infected udders. First, the milk samples were classified according to bacteriological test in positive and negative. The results showed that the negative samples to bacteriological test had: (i) significantly higher percentages of live lymphocytes; (ii) significantly lower percentages of CD11b + leukocytes; (iii) significantly lower MEC and higher MFR values. Then, samples were classified in three groups according to somatic cell count (SCC): Group A ( n = 15), samples with SCC ≤ 100,000 cells/mL, all negative to bacteriological analysis; Group B ( n = 11), samples with 100,000 < SCC < 300,000 cells/m, with four samples positive to bacteriological analysis; Group C ( n = 17), samples with SCC ≥ 300,000 cell/mL with five samples positive to bacteriological analysis. Multivariate discriminant analysis was used to verify which flow cytometry immunological markers and on farm recorded parameters could better discriminate among the different groups of SCCs. Linear discriminant analysis (LDA) indicated that 5 of the 10 parameters could best be used to reveal the differences between positive and negative samples among the considered groups of SCCs. Furthermore, the Canonical discriminant analysis (CDA) indicated that composite milk samples with different SCC and infection status were clearly separated from each other in a two-dimensional space. In conclusion, the study highlighted that: (1) the conventional flow cytometry analysis applied on milk samples is a useful tool to investigate immunological parameters as potential indicators of udder health; (2) the combined evaluation of live milk leukocytes and recorded farm parameters could be useful to assess udder health status in dairy cows. The results obtained from this pilot study on few data require new and larger trials to be confirmed.

The widespread use of genome-wide association studies resulted in the discovery of genomic regions associated with fatty acid (FA) composition in different porcine tissues, but little information exists about the genes involved in FA composition of meat obtained from heavy pigs selected for the production of Italian dry-cured hams. To this objective, we genotyped with a single nucleotide polymorphism (SNP) panel 795 Italian Large White heavy pigs to identify the markers and genomic regions associated with Semimembranosus muscle FA profile. Heritability estimates for intramuscular fat FA profile were of low-to-moderate magnitude, suggesting that these traits may be improved with genomic selection. On the whole, 45 SNPs were significantly associated with 14 FAs, and 4 of them (ALGA008109, ALGA0081097, CASI0010164 and SIRI0000267) were associated with more than 1 FA. The palmitoleic : palmitic and oleic : stearic ratios displayed the highest number of significant markers and the most significant associations (Bonferroni adjusted P < 5.00E-07). Of particular interest, the palmitoleic : palmitic ratio was strongly associated with markers located at 111 to 114 Mb on chromosome 14, in the same chromosomal region where Stearoyl-CoA desaturase Δ9 (SCD) gene is located. Several significant chromosomal regions were found; some of them harbour key genes playing pivotal roles in FA desaturation and elongation, such as SCD and some members of the Elongation of Very Long-Chain FA (ELOVL) gene family. The results suggest that the identification of causal mutations in these regions may provide a set of markers useful for selection schemes aimed at improving FA composition in pork products.

Semimembranosus muscle samples from 795 Large White heavy pigs were used to determine their intramuscular fatty acid composition and to estimate the heritability and the genetic correlations of these traits. Muscle fatty acids showed heritability estimates of low-to-moderate magnitude, ranging from 0.157 for total fatty acids to 0.237 for docosahexaenoic acid. Only small differences in heritability appeared among fatty acids based on their chain length, saturation and double bond position. Omega-6 polyunsaturated fatty acids showed positive genetic correlations with carcass lean % (0.563 ± 0.005) and loin thickness (0.438 ± 0.005) while being negatively related to backfat thickness measured both by calibre (-0.225 ± 0.008) and Fat-O-Meat'er (FOM) apparatus (-0.603 ± 0.004). Interestingly, the monounsaturated fatty acid class was not correlated with carcass measures and presented only a weak positive genetic correlation with intramuscular fat (0.145 ± 0.002). This result suggests that in heavy pig breeds monounsaturated fatty acids in muscle could be selected for without interfering with carcass traits., Competing Interests: Declaration of Competing Interest Roberta Davoli declares to be a member of Meat Science journal Editorial board. The other co-authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The gilthead seabream (Sparus aurata) is an important cultivated species in the Mediterranean area. A major problem for the gilthead seabream aquaculture sector derives from the high frequency of phenotypic abnormalities, including discolorations. In this study, we applied a whole-genome resequencing approach to identify a genomic region affecting a pigmentation defect that occurred in a cultivated S. aurata population. Two equimolar DNA pools were constructed using DNA extracted from 30 normally coloured and 21 non-pigmented fish collected among the offspring of the same broodstock nucleus. Whole-genome resequencing reads from the two DNA pools were aligned to the S. aurata draft genome and variant calling was performed. A whole-genome heterozygosity scan from single pool sequencing data highlighted a peak of reduced heterozygosity of approximately 5 Mbp on chromosome 6 in the non-pigmented pool that was not present in the normally coloured pool. The comparison of the non-pigmented with the normally coloured fish using a whole-genome F ST analysis detected three main regions within the coordinates previously detected with the heterozygosity analysis. The results support the presence of a major locus affecting this discoloration defect in this fish population. The results of this study have practical applications, including the possibility of eliminating this defect from the breeding stock, with direct economic advantages derived from the reduction of discarded fry. Other studies are needed to identify the candidate gene and the causative mutation, which could add information to understand the complex biology of fish pigmentation., (© 2020 Stichting International Foundation for Animal Genetics.)

Longevity is one of the most important traits determining dairy cow profitability. In the last decades dairy cows suffered a lowering in the age at culling. With the aim to identify the genes involved in longevity, dates of birth, yields, dates of calving during lifespan and culling dates were collected for 946 culled cows which had been genotyped with the Bovine High Density panel. Using the GenABEL package in R, genome-wide association analysis was performed on three potential traits of longevity: (1) 'days in production,' (2) 'days in herd,' (3) number of calvings over lifespan.' Five genome-wide significant single nucleotide polymorphisms (SNPs) associated with all three longevity traits were detected. Several consecutive SNPs identified on chromosomes 16 and 30 indicated the presence of two suggestive quantitative trait loci (QTL). The genes comprised in the QTL regions had biological functions related to fertility, reproductive disorders, heat stress and welfare of cows. These findings might contribute to improving breeding strategies to improve longevity.

Subcutaneous fat thickness and fatty acid composition (FAC) play an important role on seasoning loss and organoleptic characteristics of seasoned hams. Dry-cured ham industry prefers meats with low contents of polyunsaturated fatty acids (PUFA) because these negatively affect fat firmness and ham quality, whereas consumers require higher contents in those fatty acids (FA) for their positive effect on human health. A population of 950 Italian Large White pigs from the Italian National Sib Test Selection Programme was investigated with the aim to estimate heritabilities, genetic and phenotypic correlations of backfat FAC, Semimembranosus muscle intramuscular fat (IMF) content and other carcass traits. The pigs were reared in controlled environmental condition at the same central testing station and were slaughtered at reaching 150 kg live weight. Backfat samples were collected to analyze FAC by gas chromatography. Carcass traits showed heritability levels from 0.087 for estimated carcass lean percentage to 0.361 for hot carcass weight. Heritability values of FA classes were low-to-moderate, all in the range 0.245 for n-3 PUFA to 0.264 for monounsaturated FA (MUFA). Polyunsaturated fatty acids showed a significant genetic correlation with loin thickness (0.128), backfat thickness (-0.124 for backfat measured by Fat-O-Meat'er and -0.175 for backfat measured by calibre) and IMF (-0.102). Obviously, C18:2(n-6) shows similar genetic correlations with the same traits (0.211 with loin thickness, -0.206 with backfat measured by Fat-O-Meat'er, -0.291 with backfat measured by calibre and -0.171 with IMF). Monounsaturated FA, except with the backfat measured by calibre (0.068; P&lt;0.01), do not show genetic correlations with carcass characteristics, whereas a negative genetic correlation was found between MUFA and saturated FA (SFA; -0.339; P&lt;0.001). These results suggest that MUFA/SFA ratio could be increased without interfering with carcass traits. The level of genetic correlations between FA and carcass traits should be taken into account in dealing with the development of selection schemes addressed to modify carcass composition and/or backfat FAC.

Previous studies on the immune system of water buffalo (Bubalus bubalis) using cross-reactive monoclonal antibodies (mAbs) revealed significant similarities and differences to the bovine immune system. Herein, we extend these studies and document the pattern of expression of CD14, CD16, CD163 and CD172a on buffalo leukocytes using a set of cross-reactive mAbs that are known to recognize conserved epitopes within orthologous molecules in cattle, sheep and goats. Buffalo leukocytes were isolated and subjected to mAb labelling for flow cytometry. Single color flow cytometry confirmed mAbs recognition of buffalo orthologues of CD14, CD16, CD163 and CD172a, and revealed consistent patterns of expression similar to that reported in other ruminants. Multicolor flow cytometry revealed that buffalo CD14 + monocytes uniquely co-express CD16, CD163 and CD172a, whereas buffalo granulocytes co-express CD16 and CD172a. This study expands mAbs available to define and study the buffalo monocytes, and also extends information available on the unique features of the buffalo immune system., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protected designation of origin dry-cured hams are obtained from heavy pigs (slaughtered at about 160 kg of live weight). A specific breeding program designed to improve meat quality for this production has included as key traits the level of intermuscular fat between the leg muscles and ham weight loss during the seasoning period together with a balance between fat and lean cuts. In this study we carried out genome-wide association studies for seven traits used in the genetic merit of Italian Duroc heavy pigs, five related to meat and carcass quality traits (visible intermuscular fat, ham weight loss at first salting, backfat thickness, ham weight and lean cuts), and two related to performance and efficiency traits (average daily gain and feed : gain ratio). A total of 573 performance-tested pigs were genotyped with the Illumina PorcineSNP60 BeadChip and genome-wide association analyses were carried out using the Bayes B approach with the 1 Mb window option of GenSel and random residuals for each of the seven traits. Detected windows were supported by independent single nucleotide polymorphism analyses with a linear mixed model (LMM) approach on the same animals for the same traits. A total of 30 windows identifying different quantitative trait loci (QTL) were detected and among those, 27 were confirmed by LMM in one of these traits. Among the confirmed windows, three QTL were reported for visible intermuscular fat, seven for ham weight loss at first salting and five and four for backfat thickness and lean cut, respectively. A total of eight QTL were detected for the other production traits. No overlapping QTL were reported except for one window on porcine chromosome 10 between lean cuts and ham weight that contained the CACNB2 gene that has been already associated with loin marbling score in other Duroc pigs. Several regions contained genes that have been already associated with production traits in other pig breeds, including Duroc lines, related to fat deposition or muscle structure. This work reports, for the first time, genome-wide association study results for several traits in Italian Duroc heavy pigs. These results will be useful to dissect the genetic basis for dry-cured ham production traits that determine the total genetic merit index of Italian Duroc pigs.

Poly(ADP-ribosyl)ation (PAR) is a post-translational protein modification catalysed by enzyme member of the poly(ADP-ribose) polymerases (PARPs) family. The activation of several PARPs is triggered by DNA strand breakage and the main PARP enzyme involved in this process is PARP1. Besides its involvement in DNA repair, PARP1 is involved in several cellular processes including transcription, epigenetics, chromatin re-modelling as well as in the maintenance of genomic stability. Moreover, several studies in human and animal models showed PARP1 activation in various inflammatory disorders. The aims of the study were (1) to characterize PARP1 expression in bovine peripheral blood mononuclear cells (PBMC) and (2) to evaluate PAR levels as a potential inflammatory marker in cells isolated from blood and milk samples following different types of infection, including mastitis. Our results show that (i) bovine PBMC express PARP1; (ii) lymphocytes exhibit higher expression of PARP1 than monocytes; (iii) PARP1 and PAR levels were higher in circulating PBMCs of infected cows; (iv) PAR levels were higher in cells isolated from milk with higher Somatic Cell Counts (SCC > 100,000 cells/mL) than in cells from milk with low SCCs. In conclusion, these findings suggest that PARP1 is activated during mastitis, which may prove to be a useful biomarker of mastitis., (Copyright © 2018. Published by Elsevier B.V.)

Several arthropod-borne diseases are now rising with increasing impact and risks for public health, due to environmental changes and resistance to pesticides currently marketed. In addition to community surveillance programs and a careful management of herds, a next-generation of effective products is urgently needed to control the spread of these diseases, with special reference to arboviral ones. Natural product research can afford alternative solutions. Recently, a re-emerging of bluetongue disease is ongoing in Italy. Bluetongue is a viral disease that affects ruminants and is spread through the bite of bloodsucking insects, especially Culicoides species. In this review, we focused on the importance of vector control programs for prevention or bluetongue outbreaks, outlining the lack of effective tools in the fight against Culicoides vectors. Then, we analyzed a field case study in Sardinia (Italy) concerning the utilization of the neem cake (Azadirachta indica), to control young instar populations of Culicoides biting midges, the vectors of bluetongue virus. Neem cake is a cheap and eco-friendly by-product obtained from the extraction of neem oil. Overall, we propose that the employ of neem extraction by-products as aqueous formulations in muddy sites close to livestock grazing areas may represent an effective tool in the fight against the spread of bluetongue virus in the Mediterranean areas., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study seeks to verify the feasibility of increasing twinning in a herd of the Italian autochtonous Maremmana breed. The data set included 1260 individuals born from 1963 to 2014, 527 males and 733 females, 402 of them calving at least once from 1983 through 2015. Breeding values for twinning were estimated by a single-trait linear animal model. However, since twinning is a dichotomous trait and the frequency of twins is far smaller than the frequency of single births, breeding values were also estimated by a single-trait animal threshold model. Heritability of twinning was 0.014±0.018 and 0.062±0.093 for the linear and the threshold models, respectively. Repeatability was 0.071±0.004 and 0.286± 0.012, respectively, for the two models. Genotyping with the Illumina BovineSNP54 BeadChip was performed for cows living on farm in 2012 (119 cows) and a genome-wide association analysis was performed on the corrected phenotype of all calving during the lifespan of each cow, using the GenABEL package in R and a three step GRAMMAR-GC approach. Genomic heritability, calculated from the genomic kinship matrix estimated through genomic marker data, was 0.29±0.021. The most significant detected single nucleotide polymorphisms (Hapmap22923-BTA-129564) was located in proximity of two genes, ARHGAP8 and TMEM200C, which might be potential functional candidates for twinning rate in cattle.

Intermuscular fat content in protected designations of origin dry-cured hams is a very important meat quality trait that affects the acceptability of the product by the consumers. An excess in intermuscular fat (defined as the level of fat deposition between leg muscles) is a defect that depreciates the final product. In this study we carried out a genome-wide association study for visible intermuscular fat (VIF) of hams in the Italian Large White pig breed. This trait was evaluated on the exposed muscles of green legs in 1122 performance-tested gilts by trained personnel, according to a classification scale useful for routine and cheap evaluation. All animals were genotyped with the Illumina PorcineSNP60 BeadChip. The genome-wide association study identified three QTL regions on porcine chromosome 1 (SSC1; accounting for ~79% of the SNPs below the 5.0E-04 threshold) and SSC2, two on SSC7 and one each on SSC3, SSC6, SSC9, SSC11, SSC13, SSC15, SSC16 and SSC17. The most significant SNP (ALGA0004143 on SSC1 at 77.3 Mb; P FDR  < 0.05), included in the largest QTL region which spanned about 6.8 Mb on SSC1, is located within the glutamate ionotropic receptor kainate type subunit 2 (GRIK2) gene. Functional annotation of all genes included in QTL regions for VIF suggested that intermuscular fat in the Italian Large White breed is a complex trait apparently influenced by complex biological mechanisms also involving obesity-related processes. These QTL target mainly chromosome regions different from those affecting subcutaneous and intramuscular fat deposition., (© 2017 Stichting International Foundation for Animal Genetics.)

Protected designation of origin dry-cured hams are the most important productions of the Italian heavy pig industry. Hams capable of minimal seasoning losses produce better quality dry-cured hams. Ham weight loss during the first 7 days in brine (first salting) is highly correlated with the total loss of weight up to the end of seasoning, and it has quite high heritability (0.30-0.61). For these reasons, ham weight loss at first salting has been included as a meat quality trait in the Italian heavy pig selection program. In this work, we carried out a genome-wide association study for this parameter in the Italian Large White pig breed by genotyping 1365 animals with the Illumina BeadChip PorcineSNP60 chip. A total of 44 single nucleotide polymorphisms (SNPs) had a P nominal value below 5.0E-04, five of which were below 5.0E-05 and one of them (ALGA0057985 on chromosome 10) was associated with this trait at a P Bonferroni threshold of 0.10. These SNPs identified a total of at least 29 putative QTLs that were located on most porcine autosomal chromosomes. This study provides genomic information that could be useful in dissecting this complex trait by identifying potential candidate genes whose function could contribute to understanding the biological mechanisms affecting meat quality for seasoning aptitude., (© 2016 Stichting International Foundation for Animal Genetics.)

Calcium, magnesium and phosphorus are essential electrolytes involved in a large number of biological processes. Imbalance of these minerals in blood may indicate clinically relevant conditions and are important in inferring acute or chronic pathologies in humans and animals. In this work, we carried out a genome-wide association study (GWAS) for the level of these three electrolytes in the serum of 843 performance-tested Italian Large White pigs. All pigs were genotyped with the Illumina PorcineSNP60 BeadChip, and GWAS was carried out using genome-wide efficient mixed-model association. For the level of Ca(2+) , eight single nucleotide polymorphisms (SNPs) were significant, considering a false discovery rate (FDR) < 0.05, and another eight were above the moderate association threshold (Pnominal value  < 5.00E-05). These SNPs are distributed in four porcine chromosomes (SSC): SSC8, SSC11, SSC12 and SSC13. In particular, a few putative different signals of association detected on SSC13 and one on SSC12 were in genes or close to genes involved in calcium metabolism (P2RY1, RAP2B, SLC9A9, C3orf58, TSC22D2, PLCH1 and CACNB1). Only one SNP (on SSC7) and six SNPs (on SSC2 and SSC7) showed moderate association with the level of magnesium and phosphorus respectively. The association signals for these two latter minerals might identify genes not known thus far for playing a role in their biological functions and regulations. In conclusion, our GWAS contributed to increased knowledge on the role that calcium, magnesium and phosphorus may play in the genetically determined physiological mechanisms affecting the natural variability of mineral levels in mammalian blood., (© 2016 Stichting International Foundation for Animal Genetics.)

Background: The climatic and cultural diversity of the Italian Peninsula triggered, over time, the development of a great variety of horse breeds, whose origin and history are still unclear. To clarify this issue, analyses on phenotypic traits and genealogical data were recently coupled with molecular screening., Methodology: To provide a comprehensive overview of the horse genetic variability in Italy, we produced and phylogenetically analyzed 407 mitochondrial DNA (mtDNA) control-region sequences from ten of the most important Italian riding horse and pony breeds: Bardigiano, Esperia, Giara, Lipizzan, Maremmano, Monterufolino, Murgese, Sarcidano, Sardinian Anglo-Arab, and Tolfetano. A collection of 36 Arabian horses was also evaluated to assess the genetic consequences of their common use for the improvement of some local breeds., Conclusions: In Italian horses, all previously described domestic mtDNA haplogroups were detected as well as a high haplotype diversity. These findings indicate that the ancestral local mares harbored an extensive genetic diversity. Moreover, the limited haplotype sharing (11%) with the Arabian horse reveals that its impact on the autochthonous mitochondrial gene pools during the final establishment of pure breeds was marginal, if any. The only significant signs of genetic structure and differentiation were detected in the geographically most isolated contexts (i.e. Monterufolino and Sardinian breeds). Such a geographic effect was also confirmed in a wider breed setting, where the Italian pool stands in an intermediate position together with most of the other Mediterranean stocks. However, some notable exceptions and peculiar genetic proximities lend genetic support to historical theories about the origin of specific Italian breeds.

In this study, we investigated at the genome-wide level if 20 years of artificial directional selection based on boar genetic evaluation obtained with a classical BLUP animal model shaped the genome of the Italian Large White pig breed. The most influential boars of this breed (n = 192), born from 1992 (the beginning of the selection program of this breed) to 2012, with an estimated breeding value reliability of >0.85, were genotyped with the Illumina Porcine SNP60 BeadChip. After grouping the boars in eight classes according to their year of birth, filtered single nucleotide polymorphisms (SNPs) were used to evaluate the effects of time on genotype frequency changes using multinomial logistic regression models. Of these markers, 493 had a PBonferroni  < 0.10. However, there was an increasing number of SNPs with a decreasing level of allele frequency changes over time, representing a continuous profile across the genome. The largest proportion of the 493 SNPs was on porcine chromosome (SSC) 7, SSC2, SSC8 and SSC18 for a total of 204 haploblocks. Functional annotations of genomic regions, including the 493 shifted SNPs, reported a few Gene Ontology terms that might underly the biological processes that contributed to increase performances of the pigs over the 20 years of the selection program. The obtained results indicated that the genome of the Italian Large White pigs was shaped by a directional selection program derived by the application of methodologies assuming the infinitesimal model that captured a continuous trend of allele frequency changes in the boar population., (© 2015 Stichting International Foundation for Animal Genetics.)

In this study, we investigated whether a selection programme based on boar genetic evaluation obtained with a classical BLUP animal model can change allele frequencies in a pig population. All Italian Large White boars born from 1992 to 2012 with estimated breeding value reliability >0.85 (n = 200) were selected among all boars of this breed. Boars were genotyped with markers in major genes (IGF2 intron3-g.3072G>A, MC4R p.D298N, VRTN PRE1 insertion, PRKAG3 p.I199V and FTO g.276T>G). Genotyping data were analysed grouping boars in eight classes according to their year of birth. To evaluate the influence of time on allele frequencies of the genotyped markers, multinomial logistic regression models were computed. Four of five polymorphic sites (IGF2, MC4R, VRTN and FTO) showed significant (p < 0.01) changes in allele frequencies over time due to a progressive and continuous increase of one allele (associated with higher lean meat content, higher average daily gain and favourable feed: gain ratio) and, consequently, decrease of the other one, following the directional selection of the selection programme of this pig breed. The retrospective analysis that was carried out in Italian Large White boars suggests that selection based on methodologies assuming the infinitesimal model is able to modify in a quite short period of time allele frequencies in major genes, increasing the frequency of alleles explaining a relevant (non-infinitesimal) fraction of the overall genetic variability for production traits., (© 2015 Blackwell Verlag GmbH.)

Genomic selection is becoming a common practise in dairy cattle, but only few works have studied its introduction in pig selection programs. Results described for this species are highly dependent on the considered traits and the specific population structure. This paper aims to simulate the impact of genomic selection in a pig population with a training cohort of performance-tested and slaughtered full sibs. This population is selected for performance, carcass and meat quality traits by full-sib testing of boars. Data were simulated using a forward-in-time simulation process that modeled around 60K single nucleotide polymorphisms and several quantitative trait loci distributed across the 18 porcine autosomes. Data were edited to obtain, for each cycle, 200 sires mated with 800 dams to produce 800 litters of 4 piglets each, two males and two females (needed for the sib test), for a total of 3200 newborns. At each cycle, a subset of 200 litters were sib tested, and 60 boars and 160 sows were selected to replace the same number of culled male and female parents. Simulated selection of boars based on performance test data of their full sibs (one castrated brother and two sisters per boar in 200 litters) lasted for 15 cycles. Genotyping and phenotyping of the three tested sibs (training population) and genotyping of the candidate boars (prediction population) were assumed. Breeding values were calculated for traits with two heritability levels (h 2=0.40, carcass traits, and h 2=0.10, meat quality parameters) on simulated pedigrees, phenotypes and genotypes. Genomic breeding values, estimated by various models (GBLUP from raw phenotype or using breeding values and single-step models), were compared with the classical BLUP Animal Model predictions in terms of predictive ability. Results obtained for traits with moderate heritability (h 2=0.40), similar to the heritability of traits commonly measured within a sib-testing program, did not show any benefit from the introduction of genomic selection. None of the considered genomic models provided improvements in prediction ability of pigs with no recorded phenotype. However, a few advantages were found for traits with low heritability (h 2=0.10). These heritability levels are characteristic for meat quality traits recorded after slaughtering or for reproduction or health traits, typically recorded on field and not in performance stations. Other scenarios of data recording and genotyping should be evaluated before considering the implementation of genomic selection in a pig-selection scheme based on sib testing of boars.

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3'-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.

Myopalladin (MYPN) and titin (TTN) play key roles in skeletal muscle structural organisation and for this reason they may be functional candidates for meat production traits in pigs. We analysed by PCR-RFLP two polymorphisms previously identified and described in the 3'UTR of MYPN and TTN genes in a group of Italian Large White (ILW) and Italian Duroc (ID) pigs with extreme divergent estimated breeding values for some production traits. Significant allele frequency differences were observed for lean cuts (LC) at the MYPN locus in ILW breed and for visible intermuscular fat (VIF) at MYPN and TTN loci in ID breed. Moreover, association analysis between the two genes and some production traits (average daily gain, LC, backfat thickness, ham weight, feed conversion ratio and VIF) was performed in a sample of 409 ILW and 381 ID pigs. On the whole, the results showed significant effects of both genes on some of the considered traits in ID breed, and for TTN gene in ILW animals. These results indicate that both genes can be considered as candidates for meat and carcass traits in ID pigs.

Background: The pH is an important parameter influencing technological quality of pig meat, a trait affected by environmental and genetic factors. Several quantitative trait loci associated to meat pH are described on PigQTL database but only two genes influencing this parameter have been so far detected: Ryanodine receptor 1 and Protein kinase, AMP-activated, gamma 3 non-catalytic subunit. To search for genes influencing meat pH we analyzed genomic regions with quantitative effect on this trait in order to detect SNPs to use for an association study., Results: The expressed sequences mapping on porcine chromosomes 1, 2, 3 in regions associated to pork pH were searched in silico to find SNPs. 356 out of 617 detected SNPs were used to genotype Italian Large White pigs and to perform an association analysis with meat pH values recorded in semimembranosus muscle at about 1 hour (pH1) and 24 hours (pHu) post mortem.The results of the analysis showed that 5 markers mapping on chromosomes 1 or 3 were associated with pH1 and 10 markers mapping on chromosomes 1 or 2 were associated with pHu. After False Discovery Rate correction only one SNP mapping on chromosome 2 was confirmed to be associated to pHu. This polymorphism was located in the 3'UTR of two partly overlapping genes, Deoxyhypusine synthase (DHPS) and WD repeat domain 83 (WDR83). The overlapping of the 3'UTRs allows the co-regulation of mRNAs stability by a cis-natural antisense transcript method of regulation. DHPS catalyzes the first step in hypusine formation, a unique amino acid formed by the posttranslational modification of the protein eukaryotic translation initiation factor 5A in a specific lysine residue. WDR83 has an important role in the modulation of a cascade of genes involved in cellular hypoxia defense by intensifying the glycolytic pathway and, theoretically, the meat pH value., Conclusions: The involvement of the SNP detected in the DHPS/WDR83 genes on meat pH phenotypic variability and their functional role are suggestive of molecular and biological processes related to glycolysis increase during post-mortem phase. This finding, after validation, can be applied to identify new biomarkers to be used to improve pig meat quality.

Association mapping of the central part of porcine chromosome 2 harboring QTLs for carcass and meat quality traits was performed with 17 gene-tagged SNPs located between 44.0 and 77.5 Mb on a physical map (Sscrofa10.2) in Italian Large White pigs. For the analyzed animals records of estimated breeding values for average daily gain, back fat thickness, lean cuts, ham weight, feed conversion ratio, pH1, pHu, CIE L*, CIE a*, CIE b* and drip loss were available. A significant QTL for fat deposition (adjusted P=0.0081) and pH1 (adjusted P=0.0972) to MYOD1 at position 44.4 Mb and a QTL for growth and meatiness (adjusted P=0.0238-0.0601) to UBL5 at position 68.9 Mb were mapped. These results from association mapping are much more accurate than those from linkage mapping and facilitate further search for position candidate genes and causative mutations needed for application of markers through marker assisted selection., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The proprotein convertase subtilisin/kexin type 1 (PCSK1) gene encodes the prohormone convertase 1/3 enzyme that processes prohormones into functional hormones that, in turn, regulate central and peripheral energy metabolism. Mutations in the human PCSK1 gene cause severe monogenic obesity or confer risk of obesity. We herein investigated the porcine PCSK1 gene with the aim of identifying polymorphisms associated with fat deposition and production traits in Italian heavy pigs. By re-sequencing about 5.1 kb of this gene in 21 pigs of different breeds, we discovered 14 polymorphisms that were organized in nine haplotypes, clearly distributed in two clades of putative European and Asian origin. Then we re-mapped this gene on porcine chromosome 2 and analysed its expression in several tissues including gastric oxyntic mucosa of weanling pigs in which PCSK1 processes the pre-pro-ghrelin into ghrelin, which in turn is involved in the control of feed intake and energy metabolism. Association analyses between PCSK1 single-nucleotide polymorphisms (SNPs) and production, carcass and several other traits were conducted on five groups of pigs from three different experimental designs, for a total of 1221 animals. Results indicated that the analysed SNPs were associated (P < 0.01 or P < 0.05) with several traits including backfat thickness and visible intermuscular fat in Italian Duroc (ID) and growth performances in Italian Large White (ILW) and in ILW × Italian Landrace pigs. However, the effects estimated in the ILW were opposite to the effects reported in the ID pigs. Suggestive association (P < 0.10) was observed with muscle cathepsin B activity, opening, if confirmed, potential applications to reduce the excessive softness defect of the green hams that is of particular concern for the processing industry. The results obtained supported the need to further investigate the PCSK1 gene to fully exploit the value of its variability and apply this information in pig breeding programmes.

Background: Carcass fatness is an important trait in most pig breeding programs. Following market requests, breeding plans for fresh pork consumption are usually designed to reduce carcass fat content and increase lean meat deposition. However, the Italian pig industry is mainly devoted to the production of Protected Designation of Origin dry cured hams: pigs are slaughtered at around 160 kg of live weight and the breeding goal aims at maintaining fat coverage, measured as backfat thickness to avoid excessive desiccation of the hams. This objective has shaped the genetic pool of Italian heavy pig breeds for a few decades. In this study we applied a selective genotyping approach within a population of ~ 12,000 performance tested Italian Large White pigs. Within this population, we selectively genotyped 304 pigs with extreme and divergent backfat thickness estimated breeding value by the Illumina PorcineSNP60 BeadChip and performed a genome wide association study to identify loci associated to this trait., Results: We identified 4 single nucleotide polymorphisms with P≤5.0E-07 and additional 119 ones with 5.0E-07

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 ≈ 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 <5%), whereas the remaining 286 in 212 genes were taken for subsequent analyses, among which 64 showed a P(nominal) value <0.1. To deal with the multiple testing problem in a candidate gene approach, we applied the proportion of false positives (PFP) method. Thirty-eight SNP were significant (P(PFP) < 0.20). The most significant SNP was the IGF2 intron3-g.3072G>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) = 6.2E-04) 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 IKKβ/NFκB 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.

The melanocortin-4 receptor (MC4R) gene codes for a G protein transmembrane receptor playing an important role in energy homeostasis control. In pig a single nucleotide polymorphism c.1426G>A has been identified and associated to average daily gain, feed intake and fatness traits but a lack of agreement on the effects of the gene on carcass traits in different breeds comes out from many studies. In the present study the c.1426G>A polymorphism is analysed in two Italian pig breeds, Large White and Duroc to study the association of the MC4R gene with some carcass traits. The results show that the c.1426G>A polymorphism affects daily gain, feed conversion ratio and ham weight in both breeds, lean cuts in the Italian Duroc and backfat thickness in the Italian Large White. The presence of MC4R mRNA transcript in different porcine tissues was analysed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We genotyped single nucleotide polymorphisms (SNPs) in 5 cathepsin or cystatin genes (cathepsin F, CTSF; cathepsin L, CTSL; cathepsin S, CTSS; cathepsin Z, CTSZ; cystatin B, CSTB) in two groups of Italian Duroc pigs: the first group (n. 100) was chosen using a selective genotyping approach with extreme estimated breeding value (EBV) for visible intermuscular fat (VIF); the second group (n. 218) was made of performance-tested Duroc pigs not selected by any criteria. CTSL marker showed a tendency towards association (P<0.10) with VIF (first group) and back fat thickness (BFT) and average daily gain (ADG; second group). In the second group, the CTSL polymorphism was associated with weight of lean cuts (LC; P<0.05). Additive effects for all mentioned traits in the second group was significant (P<0.05). The results we obtained in the Italian Duroc pigs confirmed the results and the direction of the effects already reported for the Italian Large White breed.

In pigs, susceptibility to enterotoxigenic Escherichia coli (ETEC) K88 strains (locus F4bcR) is determined by a dominant allele, with the recessive allele determining resistance. The susceptible allele also appeared to be associated with higher growth rate even with discordant results. A single nucleotide polymorphism (SNP) in exon 7 of the mucin 4 (MUC4) gene (DQ848681:g.8227C>G), shown to be in close linkage disequilibrium with the F4bcR locus, has been used as marker to identify susceptible pigs, substituting invasive villous adhesion tests. We herein analyzed this SNP in Italian local breeds and applied a selective genotyping approach in Italian Large White, Italian Landrace, and Italian Duroc comparing allele frequency distribution in groups of pigs with extreme estimated breeding values (EBV) for average daily gain (ADG) and backfat thickness (BFT) to evaluate if this marker is associated with these traits. Allele G (associated with susceptibility to ETEC) was associated with higher ADG and BFT in Italian Large White (P=6.66E-04 and P=0.012, respectively) and higher ADG in Italian Landrace (P=7.23E-12). This polymorphism was poorly informative in Italian Duroc. Antagonistic associations of the MUC4 g.8227C>G alleles on susceptibility to ETEC and growth performances evidence the complexity of applying marker assisted selection in pig breeding.

PLIN2 (perilipin 2) is a cytosolic protein that promotes the formation and stabilization of the intracellular lipid droplets, organelles involved in the storage of lipid depots. Porcine PLIN2 gene represents a biological and positional candidate for fat deposition, a polygenic trait that affects carcass and meat quality. The aim of the present study was to screen PLIN2 gene for polymorphisms, to evaluate the association with carcass quality traits, and to investigate the gene expression in skeletal muscle. Six new single nucleotide polymorphisms (SNP) were detected by sequencing 32 samples from five pig breeds (Italian Large White, Italian Duroc, Italian Landrace, Belgian Landrace, Pietrain). Two SNP localized in introns, two in the 3'-untranslated region (UTR), and two missense SNP were found in exons. A 3'-UTR mutation (GU461317:g.98G>A), genotyped in 290 Italian Duroc pigs by High Resolution Melting, resulted significantly associated (P < 0.01) with average daily gain, feed conversion ratio, lean cuts and hams weight estimated breeding values. PLIN2 gene expression analysis in skeletal muscle of Italian Large White and Italian Duroc pigs divergent for backfat thickness and visible intermuscular fat showed a trend of higher expression level in pigs with higher intermuscular fat. These results suggest that PLIN2 can be a marker for carcass quality in pigs. Further investigation at both gene and protein level could elucidate its role on fat deposition.

TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1] is a Rab-GTPase-activating related protein implicated in regulating the trafficking of glucose transporter 4 (GLUT4 or SLC2A4) storage vesicles to the cell surface in response to insulin and AMPK-activating stimuli in skeletal muscle. Mutations in the human and mouse TBC1D1 genes confer risk of obesity or leanness. We identified five single nucleotide polymorphisms (SNPs) in the porcine TBC1D1 gene. One of them (FN677935:g.219G>A) was genotyped either by high resolution melting and PCR-RFLP analyses to study allele frequencies in a few pig breeds and evaluate association with meat production and carcass traits in five groups of sib-tested pigs of Italian Large White and Italian Duroc breeds. The g.219G>A SNP was associated (P < 0.05) with ham weight, back fat thickness and lean cuts content in Italian Large White and with visible intermuscular fat in Italian Duroc 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., (Copyright © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.)

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.

The aim of this work was to identify and analyse DNA markers in two cathepsin genes, cathepsin L (CTSL) and cathepsin S (CTSS), and study their association with meat quality parameters (including cathepsin activity of post mortem muscle) and several production traits in Italian Large White pigs. In silico analysis of the porcine CTSL and CTSS genes identified two new single nucleotide polymorphisms (SNPs), one located in exon 5 (CTSL) and one positioned in the 5'-untranslated region (CTSS). Allele frequencies of these two SNPs were evaluated in 7 different pig breeds. In addition, radiation hybrid mapping localized the CTSS gene on porcine chromosome 4, in an important QTL region for meat and fat deposition. Association analysis with meat quality traits, carried out in 268 Italian Large White pigs, indicated association of the CTSL polymorphism with average daily gain (ADG), weight of lean cuts (LC) and back fat thickness (BFT) estimated breeding values (P<0.05). Results for LC and BFT were also confirmed using a selective genotyping approach in other Italian Large White pigs (P<0.01). In the 268 Italian Large White pigs, the CTSS polymorphism was associated with feed:gain ratio and ADG (P<0.05). No association was observed between the analysed markers and meat quality parameters (pH(1), pH(u), lactate, glycogen, glycolytic potential and cathepsin activity, all measured on Musculus semimembranosus). Overall these results, together with those previously reported for other genes of this family, suggest that cathepsin genes play a role in defining economically important traits in pigs., (Copyright 2010 Elsevier Ltd. All rights reserved.)

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.

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).

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.

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, several porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cystatin B) were investigated. Single nucleotide polymorphisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand conformation polymorphism analysis. Sequencing and PCR-RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Piétrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously reported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested population (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glycogen, lactate, and glycolytic potential of semimembranosus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ratio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lysosomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs.

The objective of this study was to investigate the association of DNA markers in candidate genes for glycolytic potential on meat quality parameters (pH(1), pH(u), glycogen and lactate content and glycolytic potential of semimembranosus muscle) and estimated breeding values (EBVs) for average daily gain, lean cuts, back fat thickness, ham weight, and feed:gain ratio in 272 Italian Large White pigs. Three mutations in the PRKAG3 gene (T30N, G52S and I199V) were investigated as well as single nucleotide polymorphisms in two other skeletal muscle genes (PGAM2 and PKM2) involved in the glycolytic pathway. Association analysis with the PRKAG3 markers showed significant results (P<0.05) only for pH(1) (I199V, with significant additive effect) and lactate content (T30N), confirming, at least in part, the effects of this gene on meat quality traits. Significant association (P<0.05) was also observed for PGAM2 and ham weight EBV with significant additive and dominance effects. PKM2 was associated with average daily gain, lean cuts (P<0.001), back fat thickness and feed:gain ratio (P<0.05), with significant additive and/or dominance effects on these traits. PKM2 encodes for a key enzyme of the muscle glycolytic pathway and maps on porcine chromosome 7 where other studies have reported important QTL for the same traits. These data might suggest an important function of this gene in the mechanisms that produce the observed effects. The results will be important to evaluate the inclusion of some of these DNA polymorphisms in marker assisted selection programs.