M. Colombo

An initial comparative map of copy number variations in the goat (Capra hircus) genome

BackgroundThe goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome.ResultsWe identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals.ConclusionsWe describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats.

A first comparative map of copy number variations in the sheep genome

We carried out a cross species cattle-sheep array comparative genome hybridization experiment to identify copy number variations (CNVs) in the sheep genome analysing ewes of Italian dairy or dual-purpose breeds (Bagnolese, Comisana, Laticauda, Massese, Sarda, and Valle del Belice) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. We identified 135 CNV regions (CNVRs; 24 reported in more than one animal) covering ~10.5 Mb of the virtual sheep genome referred to the bovine genome (0.398%) with a mean and a median equal to 77.6 and 55.9 kb, respectively. A comparative analysis between the identified sheep CNVRs and those reported in cattle and goat genomes indicated that overlaps between sheep and both other species CNVRs are highly significant (P<0.0001), suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Many sheep CNVRs include genes with important biological functions. Further studies are needed to evaluate their functional relevance.

Identification and association analysis of several hundred single nucleotide polymorphisms within candidate genes for back fat thickness in Italian Large White pigs using a selective genotyping approach1

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.

Investigation of candidate genes for glycolytic potential of porcine skeletal muscle: Association with meat quality and production traits in Italian Large White 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.

Assessment of the presence of chemosensing receptors based on bitter and fat taste in the gastrointestinal tract of young pig.

Knowledge on porcine bitter and fat taste receptors and on their expression in gastrointestinal tract of pigs is scarce. We searched for the presence of porcine homologous sequences for 13 human transcripts of bitter and fat taste receptors in ENSEMBL and National Center for Biotechnology Information databases. For taste 2 receptor (TAS2R) 8, alignment was not observed; for TAS2R13 and TAS2R46 the porcine predicted sequence aligned with several other human bitter genes. For 7 genes for bitter taste (TAS2R1, TAS2R3, TAS2R7, TAS2R9, TAS2R10, TAS2R16, and TAS2R38) and for 3 genes for fat taste (GPR40, GPR43, and GPR120), a full homology for exon sequences was found and primers were designed by Primer3. These 7 genes were amplified with real-time PCR and verified on agarose gel in 5 gastrointestinal segments of weaned pigs: oxyntic (ST1), pyloric (ST2), and cardiac to oxyntic transition mucosa (ST3), jejunum (JEJ), and colon (COL). Suitability of mRNA was verified by amplifying RPL4 and HMBS2 genes. Each bitter taste gene was detectable on agarose gel in at least 1 subject of all the gastrointestinal segments except for TAS2R3 and TAS2R38 that were never detected in ST1 and COL, respectively. The inspection of bitter taste genes amplification curve indicated that the expression was in general very low. GPR43 and GPR120 were present in all segments from all pigs. Expression was not detected for GPR40. Data also indicate that colon is the preeminent tract where fat detection by GPR120 takes place (P < 0.001). The presence of gene expression for several chemosensing receptors for bitter and fat taste in different compartments of the stomach confirms that this organ should be considered a player for the early detection of bolus composition.

Evaluation of post mortem stability of porcine skeletal muscle RNA

The objective of this study was to evaluate the effect of postmortem times on the quality of porcine skeletal muscle total RNA in order to consider the possibility to use postmortem material for gene expression analysis. Samples of Musculus semimembranosus were collected at 20min, 2h, 6h, 24h and 48h postmortem from the left legs of four commercial heavy pigs. Total RNA was analysed by agarose gel electrophoresis stained with ethidium bromide and by microfluidic capillary electrophoresis on an Agilent 2100 Bioanalyzer instrument obtaining 28S:18S rRNA peak ratios and RIN values. The average RIN values of the analysed samples were 7.45±0.13, 7.43±0.15, 7.45±0.10, 7.33±0.15 and 3.95±0.58 for the same postmortem times, respectively, indicating that RNA degradation was present at 48h postmortem. In a similar experiment, carried out by other authors on beef cattle muscle total RNA extracted at different postmortem times, RNA was stable up to 8days after death as indicated by intact 28S and 18S rRNA bands. Thus, differences among species or other environmental factors might affect the level of RNA degradation. In the porcine postmortem samples, qualitative assessment of GAPDH transcripts by PCR amplification of different cDNA fragments indicated that postmortem stages did not affect the possibility of analysing this housekeeping gene. Thus, postmortem porcine skeletal muscle can be an useful tissue to obtain gene expression based information.

Differential Gene Expression in the Oxyntic and Pyloric Mucosa of the Young Pig

The stomach is often considered a single compartment, although morphological differences among specific areas are well known. Oxyntic mucosa (OXY) and pyloric mucosa (PYL, in other species called antral mucosa) are primarily equipped for acid secretion and gastrin production, respectively, while it is not yet clear how the remainder of genes expressed differs in these areas. Here, the differential gene expression between OXY and PYL mucosa was assessed in seven starter pigs. Total RNA expression was analyzed by whole genome Affymetrix Porcine Gene 1.1_ST array strips. Exploratory functional analysis of gene expression values was done by Gene Set Enrichment Analysis, comparing OXY and PYL. Normalized enrichment scores (NESs) were calculated for each gene (statistical significance defined when False Discovery Rate % <25 and P-values of NES<0.05). Expression values were selected for a set of 44 genes and the effect of point of gastric sample was tested by analysis of variance with the procedure for repeated measures. In OXY, HYDROGEN ION TRANSMEMBRANE TRANSPORTER ACTIVITY gene set was the most enriched set compared to PYL, including the two genes for H+/K+-ATPase. Pathways related to mitochondrial activity and feeding behavior were also enriched (primarily cholecystokinin receptors and ghrelin). Aquaporin 4 was the top-ranking gene. In PYL, two gene sets were enriched compared with OXY: LYMPHOCYTE ACTIVATION and LIPID RAFT, a gene set involved in cholesterol-rich microdomains of the plasma membrane. The single most differentially expressed genes were gastrin and secretoglobin 1A, member 1, presumably located in the epithelial line, to inactivate inflammatory mediators. Several genes related to mucosal integrity, immune response, detoxification and epithelium renewal were also enriched in PYL (P<0.05). The data indicate that there is significant differential gene expression between OXY of the young pig and PYL and further functional studies are needed to confirm their physiological importance.

The Olfactory Receptor OR51E1 Is Present along the Gastrointestinal Tract of Pigs, Co-Localizes with Enteroendocrine Cells and Is Modulated by Intestinal Microbiota

The relevance of the butyrate-sensing olfactory receptor OR51E1 for gastrointestinal (GIT) functioning has not been considered so far. We investigated in young pigs the distribution of OR51E1 along the GIT, its relation with some endocrine markers, its variation with age and after interventions affecting the gut environment and intestinal microbiota. Immuno-reactive cells for OR51E1 and chromogranin A (CgA) were counted in cardial (CA), fundic (FU), pyloric (PL) duodenal (DU), jejunal (JE), ileal (IL), cecal (CE), colonic (CO) and rectal (RE) mucosae. OR51E1 co-localization with serotonin (5HT) and peptide YY (PYY) were evaluated in PL and CO respectively. FU and PL tissues were also sampled from 84 piglets reared from sows receiving either or not oral antibiotics (amoxicillin) around parturition, and sacrificed at days 14, 21, 28 (weaning) and 42 of age. JE samples were also obtained from 12 caesarean-derived piglets that were orally associated with simple (SA) or complex (CA) microbiota in the postnatal phase, and of which on days 26–37 of age jejunal loops were perfused for 8 h with enterotoxigenic Escherichia coli F4 (ETEC), Lactobacillus amylovorus or saline (CTRL). Tissue densities of OR51E1+ cells were in decreasing order: PL=DU>FU=CA>JE=IL=CE=CO=RE. OR51E1+ cells showed an enteroendocrine nature containing gastrointestinal hormones such as PYY or 5HT. OR51E1 gene expression in PL and FU increased during and after the suckling period (p<0.05). It was marginally reduced in offspring from antibiotic-treated sows (tendency, p=0.073), vs. control. Jejunal OR51E1 gene expression was reduced in piglets early associated with SA, compared with CA, and in ETEC-perfused loops vs. CTRL (p<0.01). Our results indicate that OR51E1 is related to GIT enteroendocrine activity. Moreover age, pathogen challenge and dietary manipulations influencing the gastrointestinal luminal microenvironment significantly affect the OR51E1 gene expression in GIT tissues presumably in association with the release of microbial metabolites.

Comparison of three patterns of feed supplementation with live Saccharomyces cerevisiae yeast on postweaning diarrhea, health status, and blood metabolic profile of susceptible weaning pigs orally challenged with Escherichia coli F4ac.

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic F4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of CNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 10(10) cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 10(10) cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 10(11) cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 10(8) cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P = 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P = 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P = 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P = 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P = 0.05). Blood concentrations of dodecenoyl-L-carnitine (P < 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P = 0.01 and P< 0.01, respectively), and α-amino adipic acid (P < 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P = 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P < 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

Next Generation Semiconductor Based-Sequencing of a Nutrigenetics Target Gene (GPR120) and Association with Growth Rate in Italian Large White Pigs

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.