Genome-wide detection of copy number variants in European autochthonous and commercial pig breeds by whole-genome sequencing of DNA pools identified breed-characterising copy number states.

In this study, we identified copy number variants (CNVs) in 19 European autochthonous pig breeds and in two commercial breeds (Italian Large White and Italian Duroc) that represent important genetic resources for this species. The genome of 725 pigs was sequenced using a breed-specific DNA pooling approach (30-35 animals per pool) obtaining an average depth per pool of 42×. This approach maximised CNV discovery as well as the related copy number states characterising, on average, the analysed breeds. By mining more than 17.5 billion reads, we identified a total of 9592 CNVs (~683 CNVs per breed) and 3710 CNV regions (CNVRs; 1.15% of the reference pig genome), with an average of 77 CNVRs per breed that were considered as private. A few CNVRs were analysed in more detail, together with other information derived from sequencing data. For example, the CNVR encompassing the KIT gene was associated with coat colour phenotypes in the analysed breeds, confirming the role of the multiple copies in determining breed-specific coat colours. The CNVR covering the MSRB3 gene was associated with ear size in most breeds. The CNVRs affecting the ELOVL6 and ZNF622 genes were private features observed in the Lithuanian Indigenous Wattle and in the Turopolje pig breeds respectively. Overall, the genome variability unravelled here can explain part of the genetic diversity among breeds and might contribute to explain their origin, history and adaptation to a variety of production systems.

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.

Effect of dietary energy level in finishing phase on performance, carcass and meat quality in immunocastrates and barrows in comparison with gilts and entire male pigs.

Immunocastration, a technique consisting of two vaccinations against gonadotropin-releasing hormone (GnRH), can be used as alternative to surgical castration of piglets. It reduces boar taint and allows higher economic and ecological efficiency compared to barrows. The feeding strategy of immunocastrates, however, can still be improved. After second vaccination, when immunisation becomes fully effective, feed intake of immunocastrates increases sharply. This study aimed to investigate whether energy intake of immunocastrates after second vaccination could be reduced by lowering the dietary energy level of the finishing phase, without negatively affecting animal performance and quality of pork production. We hypothesised that immunocastrates already reach their limits in voluntary feed intake after second vaccination, and therefore would not be able to compensate the lower dietary energy level, in contrast to barrows. Therefore, this study aimed to assess the effect of high-energy diet (HE, net energy (NE) = 10.2 MJ/kg) compared to low-energy diet (LE, NE = 8.8 MJ/kg) in barrows and immunocastrates and as a reference, gilts and entire male pigs on a standard high-energy diet were included. CP and standardised ileal digestible amino acid levels were similar in both diets. For each treatment, eight pen replicates of six pigs per pen were evaluated on performance, carcass quality, meat and fat quality, digestibility, economic and ecological sustainability, behaviour and effectiveness of immune response. No difference in feed intake of immunocastrates between LE and HE could be demonstrated. As a result, daily energy intake of immunocastrates was higher on HE compared to LE, which resulted in a higher daily gain on HE. Feed conversion ratio (FCR) of immunocastrates on HE did not differ significantly with FCR of entire males. Barrows did not show higher average daily gain on HE compared to LE. Nitrogen efficiency was better in HE compared to LE, without negative effects on digestibility, carcass quality, economic parameters, behaviour or immune response. Small positive effects on the palatability of the meat of immunocastrates on HE were observed, although consumers did not prefer one of both feeds. Immunocastration was successful in reducing sexual and aggressive behaviour as well as in lowering the prevalence of boar taint from 15% in EM to 0% in immunocastrates. However, in two out of 96 immunocastrates (one on HE and one on LE), the immunocastration was not fully effective. In conclusion, this study did not show advantages of feeding immunocastrates or barrows a low-energy diet.

The importance of pigs' castration strategy on carbon footprint of feed intake, nitrogen and phosphorus efficiency under different management conditions.

The environmental impact of livestock production is under scrutiny nowadays and is being situated both on a global level and local level. On a global level, the global warming potential (GWP) of meat production is criticised. On a local level, the excretion of nitrogen (N) and phosphorus (P) is of concern as excess nutrients may lead to water eutrophication and soil acidification. The ERA-NET project SuSI evaluated if immunocastration may act as an ecologically, socially and economic more sustainable alternative compared to pork production with surgical castrates (SCs) and boars (BOs). Here, we report the environmental impact of immunocastrates (ICs) vs SC and BO, based on four trials carried out by European SuSi partners. More specifically, we aimed to compare IC with SC and BO in terms of GWP, N and P excretion per kg of pork production and test if this differed between experiments. There was an interaction between trial and sex category (PTrial × Sex category < 0.005) for all environmental sustainability parameters. Surgical castrates performed worse (higher carbon footprint of the feed intake, N and P excretion, lower N and P efficiency) compared to IC and BO, but the size of the effect was trial dependent. Immunocastrates scored intermediate, with mostly no significant differences from BO in most trials, but with significantly better values compared to SC. Over trials, the carbon footprint of the feed intake (land use change inclusive) per kg lean meat gain in the growing-finishing phase was 9-16% lower for IC vs SC and 9-22% lower for BO vs SC. Nitrogen efficiency of IC and BO was 7-10% and 9-14%, respectively, higher compared to SC. Phosphorus efficiency of IC and BO was higher than that of SC by 6-14% and 9-17%, respectively. Per kg of lean meat gain in the growing-finishing phase, IC excreted between 14 and 19% less N and between 14 and 24% less P than SC. For BO, it was between 14 and 27% less N and between 14 and 31% less P than SC. Differences between trials were larger than differences between castration strategies and trial design may have amplified the observed effects. Improving feed efficiency, adapting the feed to the needs of the animal (avoiding excess nutrients) and choosing low-impact ingredients are key for improving the environmental sustainability of pig production. Ending physical castration is another step to attain this goal.

The effect of immunocastration on adipose tissue deposition and composition in pigs.

Immunocastrated pigs (IC) exhibit intensive fat deposition after immunisation, but the underlying mechanisms of intensified fat metabolism and deposition are not yet fully understood. Moreover, there is also a lack of comparative studies performed on IC, entire males (EM) and surgical castrates (SC). The main objective of our research was, therefore, to characterise the adipose tissue from the quantitative, histo-morphological and biochemical perspectives in IC 5 weeks after their immunisation in comparison to EM and SC. Immunocastrated pigs had an intermediate position in carcass fatness traits between EM (the leanest) and SC (the fattest). The histo-morphological traits of the subcutaneous adipose tissue of IC were similar to those of SC and differed from those of EM; i.e., they exhibited larger adipocytes in the outer backfat and a larger lobulus surface area in both backfat layers than EM. Intensive fat tissue development in IC was corroborated with higher activities of lipogenic enzymes (i.e., fatty acid synthase, malic enzyme, glucose 6-phosphate dehydrogenase, citrate cleavage enzyme), which was especially pronounced in the subcutaneous adipose tissue of IC (1.5- to 2.7-fold higher activity than in EM or SC). The fatty acid composition of the backfat in IC was similar to that in EM pigs. Both IC and EM exhibited less saturated and more polyunsaturated fatty acids than SC. In contrast, the fatty acid composition of the intramuscular fat of longissimus dorsi muscle in IC pigs was more similar to SC than to EM (higher monounsaturated and lower polyunsaturated fatty acid content in IC and SC than EM). In this study, it was demonstrated that immunocastration notably influenced lipid metabolism. This was shown by increased quantity of lipid depots and with changes in adipose tissue cellularity compared to EM, with changes in the fatty acid composition of the intramuscular fat and enhanced lipogenic activity compared to both EM and SC. These results provide new insights into the specificity of adipose tissue development and deposition in IC compared to EM and SC.

Meat and fat quality of Krskopolje pigs reared in conventional and organic production systems.

Data on production traits of the only Slovenian autochthonous pig breed, the Krskopolje pig, is very scarce. Krskopolje pigs are reared in conventional and organic production systems, which were compared in the present study. After weaning, 24 barrows were assigned within litter to either conventional (CON) or organic (ECO) rearing system. Group CON (n=12) was housed indoors in two pens (7.5 m2) with partly slatted floor. Group ECO (n=12) was held in a sty with sheltered area (concrete floor, bedded with straw, 16 m2) and outdoor paddock area (100 m2). The trial started when pigs had 68±8 kg BW and 157±6 days of age. Two diets were formulated with equivalent ingredients and composition. For ECO diet the ingredients used were ecological. Group ECO received a diet with 12.4 MJ metabolisable energy (ME) per kilogram and 12.9% CP and group CON a diet with 12.7 MJ ME/kg and 13.6% CP. Feed distribution was limited to 3.5 kg per pig daily. In line with the rules for organic production, ECO pigs were additionally given alfalfa hay ad libitum. After 73 days on trial, the pigs were slaughtered and carcass, meat and fat quality was evaluated. Meat quality traits (pH, colour, water holding capacity), fatty acid composition, lipid and protein oxidation, collagen content and solubility were analysed in longissimus lumborum (LL) muscle. Fatty acid composition, lipid oxidation, and vitamins A and E concentrations were determined in backfat. There were no significant differences in growth rate and carcass traits between ECO and CON pigs, however, ECO pigs tended (P<0.10) to have higher daily gain and lower dressing percentage, higher (P<0.001) pH 45 min and lower (P<0.01) pH 24 h postmortem, affecting (P<0.10) also water holding capacity and objective colour parameters (P<0.05) of LL muscle. There were no differences in intramuscular fat (IMF) content of LL muscle, however, IMF of ECO pigs had lower (P<0.05) proportion of saturated and higher (P<0.01) proportion of monounsaturated fatty acids accompanied by higher (P<0.001) values of thiobarbituric reactive substances (TBARS). In backfat, ECO pigs showed lower (P<0.05) vitamin E content, higher (P<0.001) TBARS, higher (P<0.01) degree of unsaturation (percentage of polyunsaturated fatty acids), and also higher (P<0.05) vitamin A concentration than CON pigs, which can be related to alfalfa hay supplementation of ECO pigs. In brief, organic rearing of Krskopolje pigs did not affect performances but had an effect on meat and fat quality.

Genomic diversity, linkage disequilibrium and selection signatures in European local pig breeds assessed with a high density SNP chip.

Genetic characterization of local breeds is essential to preserve their genomic variability, to advance conservation policies and to contribute to their promotion and sustainability. Genomic diversity of twenty European local pig breeds and a small sample of Spanish wild pigs was assessed using high density SNP chips. A total of 992 DNA samples were analyzed with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. Genotype data was employed to compute genetic diversity, population differentiation and structure, genetic distances, linkage disequilibrium and effective population size. Our results point out several breeds, such as Turopolje, Apulo Calabrese, Casertana, Mora Romagnola and Lithuanian indigenous wattle, having the lowest genetic diversity, supported by low heterozygosity and very small effective population size, demonstrating the need of enhanced conservation strategies. Principal components analysis showed the clustering of the individuals of the same breed, with few breeds being clearly isolated from the rest. Several breeds were partially overlapped, suggesting genetic closeness, which was particularly marked in the case of Iberian and Alentejana breeds. Spanish wild boar was also narrowly related to other western populations, in agreement with recurrent admixture between wild and domestic animals. We also searched across the genome for loci under diversifying selection based on FST outlier tests. Candidate genes that may underlie differences in adaptation to specific environments and productive systems and phenotypic traits were detected in potentially selected genomic regions.

Properties and aromatic profile of dry-fermented sausages produced from Krskopolje pigs reared under organic and conventional rearing regime.

Krskopolje pig, the only Slovenian autochtonous breed is still relatively untapped, with almost no information available on the effect of rearing conditions or its aptitude for processing and meat product quality. In the present study, dry-fermented sausages were produced from Krskopolje pigs that were fed similar diets (with regard to ingredients, energy and protein content) but reared in either conventional (CON) or organic conditions (ECO). Sausage weight losses at the end of processing were recorded and their final pH, chemical composition (moisture, fat and protein content, proteolysis index, fat and protein oxidation, fatty acid and free fatty acid profile) were determined in addition to measurements of instrumental texture, sensory evaluation and volatile profile analysis. Compared with CON, ECO dry-fermented sausages had lower processing losses, lower pH, higher moisture, salt and thiobarbituric acid reactive substances content, less saturated fat and less free fatty acids. Volatile profile analysis showed that ECO dry-fermented sausages exhibited higher concentrations of compounds arising from lipid autooxidation and lower levels of volatile compounds resulting from microbial lipid ß-oxidation, amino acid degradation and spices. Panellists perceived ECO dry-fermented sausages as saltier and less colour intensive, while both rheological and sensory analysis indicated that ECO sausages had softer texture than CON. The present results indicate that rearing of Krskopolje pigs according to organic standards induced differences in meat and fat properties which affected the quality of dry-fermented sausages, most distinctly in terms of softer texture and different volatile profile.

Potential sensitivity of pork production situations aiming at high-quality products to the use of entire male pigs as an alternative to surgical castrates.

The perspective of a possible ban on surgical castration of male pigs in the EU is a real challenge for pork production systems aiming at (very) high-quality products. Information was collected from a total of 272 situations in 16 European countries, including 170 situations related to EU protected designations (Database of Origin & Registration (DOOR) database) and 102 other situations related to high-quality products or differentiated production systems, in order to evaluate their potential sensitivity to the use of entire male pigs along four dimensions: BT_Inc, likelihood of increased levels of boar taint compounds compared with conventional production of entire males; BT_Per, extent to which (some of) the associated pork product(s) are susceptible to perception of boar taint by consumers; FatQQ, likelihood that the quality of (some of) the related products is decreased due to the lower fat quantity and quality in entire males; Manag, increased likelihood of animal management and welfare problems compared with conventional production of entire males. Situations corresponding to EU protected designations (DOOR situations) were on average more sensitive to entire male production but 11% of the non-DOOR situations were highly potentially sensitive, whereas one-third of the DOOR situations had low potential sensitivity. In total, 37% of the situations where castration is not formally specified as mandatory exhibited high potential sensitivity to entire male production. Three main patterns of situations were identified via ascending hierarchical clustering. A first pattern including 31% of the DOOR situations and 74% of the other ones, had potentially no increased risk compared with conventional production of entire males. A second pattern including 28% of the DOOR situations and 16% of the other ones had a high, moderate and low potential sensitivity for FatQQ, BT_Inc and Manag, respectively. The third pattern including 41% of the DOOR situations and 11% of the other situations had high potential sensitivity for BT_Inc and FatQQ, associated with moderate to high sensitivity for Manag. The approach used to evaluate the sensitivity to entire male pig production from the limited information collected for this study has many limitations. More precise approaches using more specific information are needed to evaluate the actual sensitivity of individual situations to the use of entire male pigs. Still, the present study provides a first global insight on the capacity of European production systems aiming at high-quality products to use entire male pigs as an alternative to surgical castration.

Hydrolysable tannin fed to entire male pigs affects intestinal production, tissue deposition and hepatic clearance of skatole.

The effect of adding hydrolysable tannins to the diet of fattening boars was studied. Performance, reproductive organ weights, salivary gland morphology, boar taint compounds and skatole metabolism were evaluated. At 123 days of age and 52 ± 6 kg liveweight, 24 Landrace × Large White boars were assigned within a litter to four treatment groups: control (T0 fed mixture with 13.2 MJ/kg, 17.5% crude proteins) and three experimental diets for which the T0 diet was supplemented with 1%, 2% and 3% of hydrolysable tannin-rich extract (T1, T2 and T3, respectively). Pigs were kept individually with ad libitum access to feed and water and slaughtered at 193 days of age and 122 ± 10 kg liveweight. Adding hydrolysable tannins to the diet had no negative effect on growth performance at 1% and 2%, whereas the 3% inclusion reduced feed intake and resulted in an adaptive response of the salivary glands (particularly parotid gland hypertrophy). Relative to T0, fat tissue skatole concentration was increased in the T1 group, but was similar in T2 and T3. Across treatments tissue skatole concentrations were proportional to the activity of hepatic CYP450. The results indicate the potential of tannin supplementation to reduce boar taint although further investigations are needed in order to establishing optimal dosage.

Steroid hormones, boar taint compounds, and reproductive organs in pigs according to the delay between immunocastration and slaughter.

The producer of vaccine against GnRH recommends that immunocastrated pigs are to be slaughtered within 4 to 6 weeks after the second vaccination (V2). The objective of the study was to examine the effect of shorter or longer delay on steroid hormones, boar taint compounds, and morphologic and histologic traits of reproductive organs. Forty male pigs (individually housed and fed a commercial diet) were assigned within litter to four treatment groups, 10 pigs were left entire (EM27) and the others were vaccinated against GnRH (Improvac, Pfizer Animal Health) at the age of 12 and 19 weeks. Pigs were slaughtered at 21 (IC21), 24 (IC24), and 27 (IC27 and EM27) weeks of age. Two EM27 pigs died during the experiment, one IC21 pig was excluded because of illness, one IC27 pig was a nonresponder, and two pigs (IC24 and IC27) were hermaphrodites. To assess the effect on steroid hormones, blood was taken at 12, 15, 19, 21, and 24 weeks of age. Subcutaneous fat and reproductive organs were sampled after slaughter for determination of androstenone, skatole, morphologic, and histologic measurements. Immmunocastration interrupted the rise of estrogen and caused a substantial fall of testosterone in IC21, IC24, and IC27 pigs. As a result, androstenone and skatole levels were successfully reduced regardless of the time elapsed from V2. The weight of the reproductive organs was also drastically reduced, the shrinkage being proportional to the length of the interval between V2 and slaughter and was the most evident for vesicular glands, followed by bulbourethral glands, and testes. Corresponding changes were observed also on a histologic level with a progressive decrease in the size and number of Leydig cells, a diminishing immunoreactivity of 3ß-hydroxysteroid dehydrogenase/Δ-5-4 isomerase, and luteinizing hormone receptor, along with a shrinkage of tubuli seminiferi, atrophy of seminiferous epithelium, and a loss of germ cells, indicating a disruption in testicular spermatogenetic function. Regression of the glandular tissue with a decreasing amount of secreta was also observed for bulbourethral and vesicular glands. The investigated physiologic, morphologic, and histologic traits were progressive with the increasing delay to slaughter (clearly seen already 2 weeks after V2), though no signs of functional or morphological restoration was observed within 8 weeks after V2.

Factors in pig production that impact the quality of dry-cured ham: a review.

This study reviews the factors of pig production that impact the quality of dry-cured ham. When processing is standardized, the quality of the final dry-cured product is primarily determined by the quality of the meat before curing (green ham). This has been defined as the aptitude for seasoning and is determined by the green ham weight, adipose tissue quantity and quality, meat physico-chemical properties and the absence of visual defects. Various ante-mortem factors including pig age and weight, genetic type, diet, feeding strategy and slaughter conditions determine green ham properties such as the dynamics of water loss, salt intake and, as a consequence, proteolysis and lipolysis. Muscle conditions (pH, salt concentration, water content and availability, temperature) influence enzymatic activity and development of characteristic texture and flavor. Generally, hams of older and heavier pigs present better seasoning aptitude because of higher adiposity. Adiposity is also positively correlated with fat saturation, which is desired to avoid rancidity and oiliness. The fatty acid profile of tissue lipids can be manipulated by diet composition. Feeding strategy affects tissue accretion and protein turnover, thus directly impacting proteolysis. With respect to the impact of pig genotype on dry-cured ham quality, local breeds are generally considered more suitable for producing quality dry hams; however, the majority of dry-cured hams on the market today are from modern pig breeds raised in conventional systems, providing lean hams. The importance of all these factors of pig production is discussed and synthesized, with an emphasis on the main difficulties encountered in dry-cured ham production.

PRKAG3 and CAST genetic polymorphisms and quality traits of dry-cured hams--III. Associations in Slovenian dry-cured ham Kraski prsut and their dependence on processing.

The aim of the present study, the third in a series of three papers, is to show the effects of PRKAG3 and CAST gene polymorphisms on the quality traits of the Slovenian dry-cured ham "Kraski prsut" and their interaction with ham producers. Significant interaction of polymorphisms with producer in the case of salt content, lipid oxidation (PRKAG3 Ile199Val), proteolysis index (CAST Arg249Lys) and pastiness (CAST Ser638Arg) indicated that genotype manifestation was reliant on the manufacturing practice. PRKAG3 Ile199Val polymorphism affected several physico-chemical, rheological and sensory traits. The Ile/Ile genotype yielded less salty and softer hams, indicating beneficial effects on dry-cured ham quality. The effect of CAST polymorphisms was less pronounced, although the observed associations with pastiness, proteolysis index and several free amino acid concentrations indicate its possible influence on proteolysis, with haplotype CAST 249Arg/638Ser being associated with a higher degree of proteolysis.

Effect of feed restriction on hormones, performance, carcass traits, and meat quality in immunocastrated pigs.

The aim of this study was to assess the effect of feed restriction applied to immunocastrated pigs in the period after the second vaccination (V2) against GnRH on hormonal status, performance, carcass traits, and meat quality. Immunocastrated pigs (IC) were compared with entire males (EM) and surgical castrates fed ad libitum. Pigs (Large White × Landrace) × Pietrain were either left entire or surgically castrated within 1 wk after birth (SC, n = 22). At 83 d of age, the entire males were further allotted to treatment groups (individual housing) of ad libitum fed EM (n = 24), ad libitum fed IC (IC-L, n = 21), or restrictively fed IC (IC-R, n = 21). At that time, the first vaccination (V1) was applied to IC-L and IC-R pigs. One week after V2 (age 130 d), feed restriction (≈ 80% of the ad libitum feed intake of SC pigs) was applied to IC-R pigs. The experiment ended 5 wk after V2, when pigs were 165 d old. Immunocastration successfully reduced boar taint compounds and size of reproductive organs. At 130 d, serum leptin concentrations were similar in all groups, whereas IGF-I concentration was less in SC (P ≤ 0.002) than in the other groups. Three weeks after V2, leptin concentrations of both IC groups were in between EM (least) and SC (greatest). The reverse was observed for IGF-I. Feed restriction had no effect on leptin or IGF-I concentrations in IC pigs. In the period V1 to V2, performance differed mainly between EM and SC, whereas both IC groups had feed intake and feed conversion ratio similar to EM and intermediate daily BW gain, not differing from either EM or SC. After V2, IC-L pigs increased their feed intake to the concentrations of SC, with faster growth compared with the other 3 groups (P < 0.05) and fatter carcasses compared with EM pigs (P = 0.007). Similar performance and carcass leanness were observed for IC-R and EM pigs. During preslaughter handling more carcass lesions were noted in EM and IC-R than in IC-L or SC pigs (P < 0.002). Neither immunocastration nor feed restriction had any effect on meat quality, but EM had greater drip loss, less intramuscular fat, and decreased tenderness than SC pigs. In conclusion, restricting feed intake can increase production efficiency but also aggressiveness of IC pigs.