Comparative evaluation of genomic inbreeding parameters in seven commercial and autochthonous pig breeds.

Single nucleotide polymorphism (SNP) genotyping tools, which can analyse thousands of SNPs covering the whole genome, have opened new opportunities to estimate the inbreeding level of animals directly using genome information. One of the most commonly used genomic inbreeding measures considers the proportion of the autosomal genome covered by runs of homozygosity (ROH), which are defined as continuous and uninterrupted chromosome portions showing homozygosity at all loci. In this study, we analysed the distribution of ROH in three commercial pig breeds (Italian Large White, n = 1968; Italian Duroc, n = 573; and Italian Landrace, n = 46) and four autochthonous breeds (Apulo-Calabrese, n = 90; Casertana, n = 90; Cinta Senese, n = 38; and Nero Siciliano, n = 48) raised in Italy, using SNP data generated from Illumina PorcineSNP60 BeadChip. We calculated ROH-based inbreeding coefficients (FROH) using ROH of different minimum length (1, 2, 4, 8, 16 Mbp) and compared them with several other genomic inbreeding coefficients (including the difference between observed and expected number of homozygous genotypes (FHOM)) and correlated all these genomic-based measures with the pedigree inbreeding coefficient (FPED) calculated for the pigs of some of these breeds. Autochthonous breeds had larger mean size of ROH than all three commercial breeds. FHOM was highly correlated (0.671 to 0.985) with FROH measures in all breeds. Apulo-Calabrese and Casertana had the highest FROH values considering all ROH minimum lengths (ranging from 0.273 to 0.189 and from 0.226 to 0.152, moving from ROH of minimum size of 1 Mbp (FROH1) to 16 Mbp (FROH16)), whereas the lowest FROH values were for Nero Siciliano (from 0.072 to 0.051) and Italian Large White (from 0.117 to 0.042). FROH decreased as the minimum length of ROH increased for all breeds. Italian Duroc had the highest correlations between all FROH measures and FPED (from 0.514 to 0.523) and between FHOM and FPED (0.485). Among all analysed breeds, Cinta Senese had the lowest correlation between FROH and FPED. This might be due to the imperfect measure of FPED, which, mainly in local breeds raised in extensive production systems, cannot consider a higher level of pedigree errors and a potential higher relatedness of the founder population. It appeared that ROH better captured inbreeding information in the analysed breeds and could complement pedigree-based inbreeding coefficients for the management of these genetic resources.

A machine learning approach for the identification of population-informative markers from high-throughput genotyping data: application to several pig breeds.

Single nucleotide polymorphisms (SNPs) able to describe population differences can be used for important applications in livestock, including breed assignment of individual animals, authentication of mono-breed products and parentage verification among several other applications. To identify the most discriminating SNPs among thousands of markers in the available commercial SNP chip tools, several methods have been used. Random forest (RF) is a machine learning technique that has been proposed for this purpose. In this study, we used RF to analyse PorcineSNP60 BeadChip array genotyping data obtained from a total of 2737 pigs of 7 Italian pig breeds (3 cosmopolitan-derived breeds: Italian Large White, Italian Duroc and Italian Landrace, and 4 autochthonous breeds: Apulo-Calabrese, Casertana, Cinta Senese and Nero Siciliano) to identify breed informative and reduced SNP panels using the mean decrease in the Gini Index and the Mean Decrease in Accuracy parameters with stability evaluation. Other reduced informative SNP panels were obtained using Delta, Fixation index and principal component analysis statistics, and their performances were compared with those obtained using the RF-defined panels using the RF classification method and its derived Out Of Bag rates and correct prediction proportions. Therefore, the performances of a total of six reduced panels were evaluated. The correct assignment of the animals to its breed was close to 100% for all tested approaches. Porcine chromosome 8 harboured the largest number of selected SNPs across all panels. Many SNPs were included in genomic regions in which previous studies identified signatures of selection or genes (e.g. ESR1, KITL and LCORL) that could contribute to explain, at least in part, phenotypically or economically relevant traits that might differentiate cosmopolitan and autochthonous pig breeds. Random forest used as preselection statistics highlighted informative SNPs that were not the same as those identified by other methods. This might be due to specific features of this machine learning methodology. It will be interesting to explore if the adaptation of RF methods for the identification of selection signature regions could be able to describe population-specific features that are not captured by other approaches.

Signatures of de-domestication in autochthonous pig breeds and of domestication in wild boar populations from MC1R and NR6A1 allele distribution.

Autochthonous pig breeds are usually reared in extensive or semi-extensive production systems that might facilitate contact with wild boars and, thus, reciprocal genetic exchanges. In this study, we analysed variants in the melanocortin 1 receptor (MC1R) gene (which cause different coat colour phenotypes) and in the nuclear receptor subfamily 6 group A member 1 (NR6A1) gene (associated with increased vertebral number) in 712 pigs of 12 local pig breeds raised in Italy (Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano and Sarda) and south-eastern European countries (Krskopolje from Slovenia, Black Slavonian and Turopolje from Croatia, Mangalitsa and Moravka from Serbia and East Balkan Swine from Bulgaria) and compared the data with the genetic variability at these loci investigated in 229 wild boars from populations spread in the same macro-geographic areas. None of the autochthonous pig breeds or wild boar populations were fixed for one allele at both loci. Domestic and wild-type alleles at these two genes were present in both domestic and wild populations. Findings of the distribution of MC1R alleles might be useful for tracing back the complex genetic history of autochthonous breeds. Altogether, these results indirectly demonstrate that bidirectional introgression of wild and domestic alleles is derived and affected by the human and naturally driven evolutionary forces that are shaping the Sus scrofa genome: autochthonous breeds are experiencing a sort of 'de-domestication' process, and wild resources are challenged by a 'domestication' drift. Both need to be further investigated and managed.

The effects of different environmental conditions on thermoregulation and clinical and hematological variables in long-distance road-transported calves.

The aim of this study was to investigate the effects of long-distance road transport (19 h, from Poland to Italy) during 2 seasons (summer vs. winter) on clinical and hematological variables in calves. The environmental temperature range that could compromise the thermoregulation system (thermal stress) of the calves was tested. For the 7 Holstein calves in each transport, the BW and rectal temperature (RT) were measured, and blood samples were collected at the farm of origin, before loading at the transit center (T2), after unloading at the farm of destination (T3), and 1, 2, 3, and 4 d after arrival. The body temperature (BT) and heart rate (HR) were continuously monitored from T2 to T3. The data were statistically analyzed according to a mixed model that considered the fixed effects of transport (repeated measurements), season of journey, and their interaction. Within the observed temperature-humidity index (THI) range (30 to 80), effective thermoregulation allowed the calves to maintain their BT with small physiologic changes to prevent thermal stress, particularly in the summer. With no seasonal differences, the HR was greater at loading than unloading (120 vs. 115 beats per min; P = 0.012). As for the transport effect, the BW was less (P < 0.001) after unloading, and the RT was greater (P = 0.004). This effect was more marked in summer. The hematological variables indicated a moderate effect of transport on the hydration condition, reactive and muscular systems, and metabolism, although hematocrit (P = 0.004), erythrocytes, cortisol, NEFA, ß-hydroxybutyrate, lactate, creatine kinase, lactate dehydrogenase, and aspartate aminotransferase activity (P < 0.001), and total protein (P = 0.007) were greater after unloading. This was confirmed by a moderate decrease in total leukocytes (P = 0.031) and glucose concentration (P = 0.002). The changes in the clinical variables were similar for both seasons even though in the summer, hematocrit (P < 0.001), urea (P = 0.008), and total protein (P = 0.010) increased and glucose concentration (P = 0.038) decreased. In conclusion, the data did not show a pronounced effect attributable to the season of the journey. Long-distance road transport leads to notable changes in clinical and hematological variables at the end of the journey. However, these variables remained within their physiological ranges and returned to basal values within a few days after the journey.

Investigation on CAST, CAPN1 and CAPN3 porcine gene polymorphisms and expression in relation to post-mortem calpain activity in muscle and meat quality.

This study aimed to detect variability in CAST, CAPN1 and CAPN3 porcine genes and to investigate the effect of CAST and CAPN1 polymorphisms on the activity of native and autolyzed µ-calpain and m-calpain, measured from 1 to 72 h post-mortem in Longissimus dorsi (LD) muscle of 30 pigs. Effects of polymorphisms on meat quality parameter such as pH, color and drip loss were also evaluated. Samples carrying CAST EU137105:g.76,872AA genotype showed higher autolyzed µ-calpain activity 24 and 72 h post-mortem, as well as lower drip loss values. Expression of CAST, CAPN1 and CAPN3 was assessed in LD muscles divergent for shear force. Higher CAST and CAPN3 expression was found in LD with high shear force (P<0.2), confirming a direct role for calpastatin but not for calpain 3 in meat tenderization. In conclusion, CAST gene affected post-mortem activation time of calpain and drip loss.

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

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.

Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs.

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.

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.

Effect of journey time and environmental condition on bull behaviour and beef quality during road transport in northern Italy.

Response to a transport time of 1 and 3 hours on the road carried out under different environmental conditions was evaluated on 60 pure-breed Charolaise bulls. Temperature and relative humidity from loading to slaughter were expressed as THI (Temperature-Humidity Index). Regardless of journey time or environmental conditions, the most common standing orientation was either perpendicular or parallel to the direction of travel. Diagonal orientation increased with THI class. Cortisol and glucose plasma concentrations increased because of transport but they were not influenced by journey time or THI class. The CK plasma concentration, instead, increased according to THI class. The incidence of carcass bruising was not affected by the journey time or by the environmental conditions. Journey time also had a negligible effect on beef quality while, meat lightness increased and water holding capacity slightly decreased depending on THI.

Study of candidate genes for glycolytic potential of porcine skeletal muscle: identification and analysis of mutations, linkage and physical mapping and association with meat quality traits in pigs.

Several genes (PRKAA2, PRKAB1, PRKAB2, PRKAG3, GAA, GYS1, PYGM, ALDOA, GPI, LDHA, PGAM2 and PKM2), chosen according to their role in the regulation of the energy balance and in the glycogen metabolism and glycolysis of the skeletal muscle, were studied. Eleven single nucleotide polymorphisms (SNPs) were identified in six of these genes (PRKAB1, GAA, PYGM, LDHA, PGAM2 and PKM2). Allele frequencies were analyzed in seven different pig breeds for these loci and for a polymorphism already described for GPI and for three polymorphic sites already reported at the PRKAG3 locus (T30N, G52S and I199V). Linkage mapping assigned PYGM and LDHA to porcine chromosome (SSC) 2, PKM2 to SSC7, GAA to SSC12, PRKAB1 to SSC14 and PGAM2 to SSC18. Physical mapping, obtained by somatic cell hybrid panel analysis, confirmed the linkage assignments of PRKAB1 and GAA and localized ALDOA, PRKAB2 and GYS1 to SSC3, SSC4 and SSC6, respectively. Pigs selected for the association study, for which several meat quality traits were measured, were first genotyped at the PRKAG3 R200Q polymorphic site (RN locus), in order to exclude carriers of the 200Q allele, and then were genotyped for all the mutations considered in this work. Significant associations (P < or = 0.001) were observed for the PRKAG3 T30N and G52S polymorphic sites with meat colour (L* at 24 h post mortem). PGAM2 and PKM2 were significantly associated (P = 0.01) with drip loss percentage and glycogen content at one hour post mortem, respectively.

Investigation of candidate genes for meat quality in dry-cured ham production: the porcine cathepsin B (CTSB) and cystatin B (CSTB) genes.

Excessive softness is a serious defect of dry cured hams which seems related to high activity of lysosomal cysteine proteinases, such as cathepsin B, in fresh pork muscles a few days after slaughtering. As it has been shown that cathepsin B activity has a moderate heritability in Italian Large White pigs we started a candidate gene approach to identify the gene(s) that affect(s) this parameter. Here, we studied two candidate genes: cathepsin B (CTSB) and cystatin B (CSTB). We amplified and sequenced porcine DNA fragments for these two genes that were used to identify polymorphisms by SSCP and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Four and two alleles were detected at the CTSB and CSTB loci, respectively. Sequencing of the CSTB alleles showed a missense mutation that changes a codon for aspartic acid into a codon for asparagine in exon 3 of the gene. Allele frequencies for the two loci differed among the pig breeds studied (Large White, Landrace, Duroc, Belgian Landrace, Hampshire, Piétrain, Meishan, Cinta Senese, Casertana, Calabrese and Nero di Sicilia). Linkage, somatic cell hybrid panel and radiation hybrid panel analyses assigned CTSB to porcine chromosome (Sscr) 14 and CSTB to Sscr 13. The markers identified at the CTSB and CSTB loci were used in association studies with several traits of economic importance including parameters that may indicate the suitability of pig meat to produce dry-cured hams. Significant associations were observed between CTSB and back-fat thickness and between CSTB and average daily gain. In this study, cathepsin B activity was not associated with the polymorphisms identified at the CTSB and CSTB loci.

Analysis of raw meat to predict proteolysis in Parma ham.

Four hundred and thirty-seven pigs (223 purebred Italian Large White, 97 Italian Landrace, and 117 Duroc), were studied to examine the effect of breed on meat quality and assess the possibility of relating proteolysis of dry-cured hams to raw meat quality. The Duroc pigs had intramuscular fat contents and water holding capacities (M. Semimembranosus) significantly higher than those of the Large White and Landrace. The latter had a significantly higher pH(24h) and cathepsin B activities significantly lower than the Duroc breed. The dry-cured hams (M. Biceps femoris) from the three breeds were significantly different in proximate composition, proteolysis and weight loss at the end of ageing. Data for green hams (including salt content) were used to compute a model to fit the proteolysis of the corresponding dry-cured hams. The variables included in the model (R(2)=0.53 and P<0.01) were cathepsin B activity of raw ham, pH(24h), weight loss after the first salting step, and the salt content of the dry-cured ham. The raw hams with the highest cathepsin B activities, the lowest pH(24h), and the highest weight loss after the first salting were those in which greatest proteolysis occured.

Combined effects of pre-slaughter treatments and lairage time on carcass and meat quality in pigs of different halothane genotype.

The effect of two different lairage times on meat quality was investigated in pigs with different halothane genotype previously submitted to controlled pre-slaughter treatments. One hundred and ninety nine Italian heavy pigs were loaded by ramp or lift and transported unmixed for 1 h to the abattoir at a stocking density of either <0.4 or >0.6 m(2) per 100 kg pigs. After unloading, an equal number of animals within each previous treatment was held in lairage for 2 h or overnight (22 h) before slaughter. Carcass and meat quality and incidence of skin damage were evaluated. Longer lairage did not negatively affect carcass traits and reduced the incidence of PSE meat without increased DFD occurrence. It also seriously increased the amount of skin damage due to fighting, resulting in long fasting. Pre-slaughter treatments and halothane genotype showed a negligible effect on the response to the resting time.

Influence of loading method and stocking density during transport on meat and dry-cured ham quality in pigs with different halothane genotypes.

The effect of loading method and stocking density in transit on meat and dry-cured ham quality was investigated in pigs with different halothane genotypes. A total of 507 Italian heavy pigs, supplied by two farms, were loaded by ramp or lift and transported unmixed for 35-55 min to the abattoir at a stocking density of either <0.4 or >0.6m(2) per 100 kg pigs. After overnight lairage in separate pens with free access to water, the pigs were slaughtered. Halothane genotype was assessed post mortem. Four hundred and thirty-nine pigs had a homozygous dominant (NN) genotype and 68 pigs were heterozygous (Nn). Carcass skin damage, meat quality traits and ham curing parameters were evaluated. Loading method and stocking density showed a negligible effect on meat and dry-cured ham quality while the predominant factor affecting these was the halothane genotype. Nn pigs produced meat with a faster rate of pH fall and lower water holding capacity as well as ham with higher weight losses in salting and greater incidence of defects in the dry-cured product. There were insignificant interactions between halothane genotype and loading method or stocking density. Overall, irrespective of pre-slaughter treatment, the Nn pigs were less suitable for the production of high quality products such as dry-cured ham.

Fibre optic probe measurements in landrace pigs of different halothane phenotypes.

Fibre Optic Probe (FOP) measurements were made in the longissimus dorsi muscle of 70 halothane-positive (HP) and 70 halothane negative (HN) pigs of a synthetic Landrace strain at 45 min (FOP(1)), 3 h (FOP(3)) and 17h (FOP(17)) post mortem. Measurements of pH, rigor and temperature were also made in the slaughterline. Carcasses were sampled (longissimus dorsi, 2-4 lumbar vertebrae) at 18h post mortem and meat quality objectively assessed. The incidence of slight PSE (visual score) was 49% in HP and 10% in HN pigs, while the incidence of serious PSE was 20% and 4%, respectively. Yet, the percentage drip loss during 48 h storage in HN/PSE pigs was higher than in HP/PSE pigs (10·2 vs 7·5%), which was associated with a lower ultimate pH (5·25 vs 5·44). Therefore, it is suggested that the remaining PSE problem in HN Landrace pigs, although less in frequency, might be more serious. In the whole population, of all FOP measurements taken correlations with colour (Hunter L(∗)-value) and percentage drip loss were highest for the FOP(17) measurements (r = 0·82 and 0·69, respectively). FOP(1) was better related than pH(1) to colour, while the relationships with drip loss were of a similar magnitude. Differences in FOP values between HP and HN pigs were larger for FOP(1) (148 vs 108) than for FOP(3) (148 vs 125) or FOP(17) (153 vs 130). The relationship between FOP1 and ultimate meat colour (L(∗)-value) appeared to be higher in HP (0·60) than in HN (0·48) pigs, while for the relationship between FOP(1) and percentage drip loss the reverse was true (0·48 and 0·60, respectively). The effect of halothane phenotype on the relationships found, may partially explain the different results of reflectance measurements reported in the literature as to their predictive value for ultimate meat quality. The results suggest that the measurement of reflectance in the slaughterline offers opportunities for identifying (potential) PSE carcasses. The large genetic influence of halothane phenotype on this parameter, may eventually justify its use as a base for payment for meat quality to the producer.