Exome Capture with Heterologous Enrichment in Pig (Sus scrofa)

The discovery of new protein-coding DNA variants related to carcass traits is very important for the Italian pig industry, which requires heavy pigs with higher thickness of subcutaneous fat for Protected Designation of Origin (PDO) productions. Exome capture techniques offer the opportunity to focus on the regions of DNA potentially related to the gene and protein expression. In this research a human commercial target enrichment kit was used to evaluate its performances for pig exome capture and for the identification of DNA variants suitable for comparative analysis. Two pools of 30 pigs each, crosses of Italian Duroc X Large White (DU) and Commercial hybrid X Large White (HY), were used and NGS libraries were prepared with the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). A total of 140.2 M and 162.5 M of raw reads were generated for DU and HY, respectively. Average coverage of all the exonic regions for Sus scrofa (ENSEMBL Sus_scrofa.Sscrofa10.2.73.gtf) was 89.33X for DU and 97.56X for HY; and 35% of aligned bases uniquely mapped to off-target regions. Comparison of sequencing data with the Sscrofa10.2 reference genome, after applying hard filtering criteria, revealed a total of 232,530 single nucleotide variants (SNVs) of which 20.6% mapped in exonic regions and 49.5% within intronic regions. The comparison of allele frequencies of 213 randomly selected SNVs from exome sequencing and the same SNVs analyzed with a Sequenom MassARRAY® system confirms that this “human-on-pig” approach offers new potentiality for the identification of DNA variants in protein-coding genes.     

家猪减数分裂粗线期二价体与有丝分裂中期染色体的比较研究

以性成熟公猪睾丸和外周血为材料,采用长低渗、高氯仿卡诺固定液固定和外周血细胞培养制备减数分裂粗线期二价体和有丝分裂中期染色体,通过对二价体和有丝分裂中期染色体分裂指数和长度的比较研究,发现二价体的分裂指数和长度分别是有丝分裂中期染色体的5倍和3．42倍（1．87～5．98）;同时以12号染色体为例,比较了二价体上的染色粒结构带与有丝分裂中期染色体G－带,表明染色粒结构带比中期染色体G－带纹丰富,而与早中期G－带带织吻合。     

Hemodynamic and histologic characterization of a swine (Sus scrofa domestica) model of chronic pulmonary arterial hypertension

The purpose of this work was to develop and characterize an aortopulmonary shunt model of chronic pulmonary hypertension in swine and provide sequential hemodynamic, angiographic, and histologic data by using an experimental endoarterial biopsy catheter. Nine Yucatan female microswine (Sus scrofa domestica) underwent surgical anastomosis of the left pulmonary artery to the descending aorta. Sequential hemodynamic, angiographic, and pulmonary vascular samples were obtained. Six pigs (mean weight, 22.4±5.3 kg; mean age, 7.3±2.7 mo at surgery) survived long-term (6 mo) and consistently developed marked pulmonary arterial hypertension. Angiography showed characteristic central pulmonary arterial enlargement and peripheral tortuosity and pruning. The biopsy catheter was safe and effective in obtaining pulmonary endoarterial samples for histologic studies, which showed neointimal and medial changes. Autopsy confirmed severe pulmonary vascular changes, including concentric obstructive neointimal and plexiform-like lesions. This swine model showed hemodynamic, angiographic, and histologic characteristics of chronic pulmonary arterial hypertension that mimicked the arterial pulmonary hypertension of systemic-to-pulmonary arterial shunts in humans. Experimental data obtained using this and other models and application of an in vivo endoarterial biopsy technique may aid in understanding mechanisms and developing therapies for experimental and human pulmonary arterial hypertension.     

Effects of Acute Dietary Iron Overload in Pigs (Sus scrofa) with Induced Type 2 Diabetes Mellitus

Epidemiological studies have reported an association between high iron (Fe) levels and elevated risk of developing type 2 diabetes mellitus (T2D). It is believed that the formation of Fe-catalyzed hydroxyl radicals may contribute to the development of diabetes. Our goal was to determine the effect of a diet with a high Fe content on type 2 diabetic pigs. Four groups of piglets were studied: (1) control group, basal diet; (2) Fe group, basal diet with 3,000 ppm ferrous sulfate; (3) diabetic group (streptozotocin-induced type 2 diabetes) with basal diet; (4) diabetic/Fe group, diabetic animals/3,000 ppm ferrous sulfate. For 2 months, biochemical and hematological parameters were evaluated. Tissue samples of liver and duodenum were obtained to determine mRNA relative abundance of DMT1, ferroportin (Fpn), ferritin (Fn), hepcidin (Hpc), and transferrin receptor by qRT-PCR. Fe group presented increased levels of hematological (erythrocytes, hematocrit, and hemoglobin) and iron parameters. Diabetic/Fe group showed similar behavior as Fe group but in lesser extent. The relative abundance of different genes in the four study groups yielded a different expression pattern. DMT1 showed a lower expression in the two iron groups compared with control and diabetic animals, and Hpc showed an increased on its expression in Fe and diabetic/Fe groups. Diabetic/Fe group presents greater expression of Fn and Fpn. These results suggest that there is an interaction between Fe nutrition, inflammation, and oxidative stress in the diabetes development.     

Detection of Antibodies Against Paracoccidioides brasiliensis in Free-Range Domestic Pigs (Sus scrofa)

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.     

Genetic diversity in wild (Sus scrofa scrofa) and domestic (Sus scrofa domestica) pigs and their hybrids based on polymorphism of a fragment of the D-loop region in the mitochondrial DNA

We examined the variation in mitochondrial DNA by sequencing the D-loop region in wild and domestic (large-white breed) pigs, in hybrids between domestic and wild pigs, and in Monteiro pigs. A D-loop fragment of approximately 330 bp was amplified by PCR. Sequencing of DNA amplicons identified haplotypes previously described as European and Asian types. Monteiro pigs and wild pigs had European haplotypes and domestic pigs had both European and Asian haplotypes.     

Genomic Diversity in Pig (Sus scrofa) and its Comparison with Human and other Livestock

We have reviewed the current pig (Sus scrofa) genomic diversity within and between sites and compared them with human and other livestock. The current Porcine 60K single nucleotide polymorphism (SNP) panel has an average SNP distance in a range of 30 - 40 kb. Most of genetic variation was distributed within populations, and only a small proportion of them existed between populations. The average heterozygosity was lower in pig than in human and other livestock. Genetic inbreeding coefficient (F(IS)), population differentiation (F(ST)), and Nei's genetic distance between populations were much larger in pig than in human and other livestock. Higher average genetic distance existed between European and Asian populations than between European or between Asian populations. Asian breeds harboured much larger variability and higher average heterozygosity than European breeds. The samples of wild boar that have been analyzed displayed more extensive genetic variation than domestic breeds. The average linkage disequilibrium (LD) in improved pig breeds extended to 1 - 3 cM, much larger than that in human (~ 30 kb) and cattle (~ 100 kb), but smaller than that in sheep (~ 10 cM). European breeds showed greater LD that decayed more slowly than Asian breeds. We briefly discuss some processes for maintaining genomic diversity in pig, including migration, introgression, selection, and drift. We conclude that, due to the long time of domestication, the pig possesses lower heterozygosity, higher F(IS), and larger LD compared with human and cattle. This implies that a smaller effective population size and less informative markers are needed in pig for genome wide association studies.     

Characterization of hepatic drug-metabolizing activities of Bama miniature pigs (Sus scrofa domestica): comparison with human enzyme analogs

We used various substrates and selective inhibitors of human cytochrome P450 (CYP) isozymes as probes to study the metabolism of liver microsomes from Chinese Bama miniature pigs. Nifedipine oxidation (NOD) and testosterone 6beta-hydroxylation (6beta-OHT) activities were similar between human liver microsomes and those from Bama miniature pigs. However, compared with those from humans, liver microsomes from Bama miniature pigs showed decreased phenacetin O-deethylation, coumarin 7-hydroxylation, and chlorzoxazone 6-hydroxylation activities, whereas dextromethorphan O-demethylation activity was increased. Ketoconazole selectively inhibited NOD and 6beta-OHT activities in microsomes from Bama pigs, and 8-methoxypsoralen and tranylcypromine inhibited coumarin 7-hydroxylation in pig microsomes. However, furafylline and quinidine failed to selectively inhibit phenacetin O-deethylation and dextromethorphan O-demethylation in microsomes from Bama pigs, whereas chlormethiazole more efficiently inhibited coumarin 7-hydroxylation activity than chlorzoxazone 6-hydroxylation in pig microsomes. Our results suggest that liver microsomes from Chinese Bama miniature pigs are similar to those from humans in regard to metabolism of nifedipine and testosterone (both are probe substrates for human CYP3A4). In addition, chemical inhibitors used as specific probes for human P450 enzymes did not always show the same selectivity toward corresponding enzyme activities in liver microsomes from Bama pigs. However, ketoconazole (a potent inhibitor of human CYP3A4) could be used as a selective inhibitor probe for the NOD and 6beta-OHT activities in liver microsomes from Chinese Bama miniature pigs.     

Optical Coherence Tomography of Pulmonary Arterial Walls in Humans and Pigs (Sus scrofa domesticus)

Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by progressive elevation of the pulmonary pressures that, in the absence of therapy, results in chronic right-heart failure and premature death. The vascular pathology of PAH is characterized by progressive loss of small (diameter, less than 50 μm) peripheral pulmonary arteries along with abnormal medial thickening, neointimal formation, and intraluminal narrowing of the remaining pulmonary arteries. Vascular pathology correlates with disease severity, given that hemodynamic effects and disease outcomes are worse in patients with advanced compared with lower-grade lesions. Novel imaging tools are urgently needed that demonstrate the extent of vascular remodeling in PAH patients during diagnosis and treatment monitoring. Optical coherence tomography (OCT) is a catheter-based intravascular imaging technique used to obtain high-resolution 2D and 3D cross-sectional images of coronary arteries, thus revealing the extent of vascular wall pathology due to diseases such as atherosclerosis and in-stent restenosis; its utility as a diagnostic tool in the assessment of the pulmonary circulation is unknown. Here we show that OCT provides high-definition images that capture the morphology of pulmonary arterial walls in explanted human lungs and during pulmonary arterial catheterization of an adult pig. We conclude that OCT may facilitate the evaluation of patients with PAH by disclosing the degree of wall remodeling present in pulmonary vessels. Future studies are warranted to determine whether this information complements the hemodynamic and functional assessments routinely performed in PAH patients, facilitates treatment selection, and improves estimates of prognosis and outcome.Abbreviations: OCT, optical coherence tomography; PAC, pulmonary artery catheter; PAH, pulmonary arterial hypertensionPulmonary arterial hypertension (PAH) is a devastating disorder characterized by progressive elevation of pulmonary pressures that, when untreated, can lead to chronic right heart failure and death.¹⁴ The vascular pathology of PAH is characterized by neointimal formation, medial thickening, intravascular thrombi and, in severe cases, intravascular clusters of disorganized endothelial cells that give rise to tortuous endovascular channels.⁸ Most of the early vascular lesions are found in small (diameter, less than 50 μm) pulmonary arteries. However, as the disease advances, pulmonary arteries (diameter, 50 μm or larger) proximal to these lesions also display evidence of luminal narrowing and medial thickening.^7,8,15 Most patients with PAH are younger than those with chronic systemic vascular disorders (that is, coronary artery disease, peripheral vascular disease, systemic hypertension), whose vascular pathology involves mostly large to medium-sized arteries. However, both patient populations demonstrate various pathologic features, including vascular smooth-cell accumulation, neointimal formation, inflammation, luminal narrowing, and alterations in the composition of the extracellular matrix.^6,17The only definite way to diagnose PAH is through right heart catheterization to directly measure the pressure in the pulmonary circulation. Although pulmonary angiography during right heart catheterization cannot be used to diagnose PAH, it provides supportive evidence of PAH by demonstrating significant peripheral small vessel loss and luminal narrowing in the remaining central vessels. Angiography can help clinicians visualize pulmonary vessels in real time, but this diagnostic technique has important limitations. The use of ionized contrast can cause allergic reactions and may trigger acute renal failure due to contrast-induced nephropathy.²⁶ In addition, pulmonary angiography provides information regarding gross vessel appearance and small vessel perfusion but not about the state of vascular wall remodeling or the extent of luminal narrowing associated with PAH at any stage.^5,16 Therefore, imaging techniques are urgently needed that complement the hemodynamic information obtained via right heart catheterization with a safe and reproducible method to assess vascular wall pathology, thereby allowing clinicians to correlate the clinical evolution of PAH with the progression of vascular pathology.The last decade has seen tremendous progress in the development of intravascular imaging modalities that can identify patients at risk for developing complications related to systemic vascular disease and therefore prevent disease-related morbidity and mortality.⁴ One such modality is optical coherence tomography (OCT), an imaging technique that uses a thin (diameter, 1.0 mm) wire and near-infrared light to capture micrometer-resolution, 3D images from within optical scattering media (for example, biologic tissue).¹ Superior to other intravascular imaging techniques, OCT is frequently used in patients with coronary artery disease, where it provides high-resolution images of the coronary arterial wall that correlate highly with pathology seen in explanted vessels.^10,11,21 To date, several small studies have demonstrated the application of OCT to the evaluation of vascular remodeling in both idiopathic PAH and chronic thromboembolic PAH.^7,21 However, despite OCT''s obvious advantages in the characterization of vascular remodeling in discrete segments of the pulmonary circulation, whether OCT provides anatomic information across the length of the pulmonary artery has not been tested.Here, we report the capacity of OCT to obtain both longitudinal and cross-sectional images that provide accurate anatomic information on healthy pulmonary arteries in explanted human lungs and during the pulmonary arterial catheterization of a live adult pig (Sus scrofa domesticus).     

Long-term evaluation of a selective retrograde coronary venous perfusion model in pigs (Sus scrofa domestica)

The lack of suitable target vessels remains a challenge for aortocoronary bypass grafting in end-stage coronary heart disease. This study aimed to investigate the arterialization of cardiac veins as an alternative myocardial revascularization strategy in an experimental long-term model in pigs. Selective retrograde perfusion of a coronary vein (aorta to coronary vein bypass, retrobypass) before ligation of the ramus interventricularis paraconalis (equivalent to the left anterior descending artery in humans) was performed in 20 German Landrace pigs (Sus scrofa domestica). Retroperfusion of the left anterior descending vein was performed in 10 pigs (RP+) but not in the other 10 (RP-), and the vena cordis magna was ligated (L+) in 5 pigs in each of these groups but left open (L-) in the remaining animals. Hemodynamic performance (for example, cardiac output) was significantly better in the group that underwent selective retroperfusion with proximal ligation of vena cordis magna (RP+L+; 4.1 L/min) compared with the other groups (RP+L-, 2.5 L/min; RP-L+, 2.2 L/min; RP-L-, 1.9 L/min). Long-term survival was significantly better in RP+L+ pigs (112±16 d) than in all other groups. Histologic follow-up studies showed significantly less necrosis in the RP+L+ group compared with all other groups. Venous retroperfusion is an effective technique to achieve long-term survival after acute occlusion of the left anterior descending artery in a pig model. In this model, proximal ligation of vena cordis magna is essential.     

Effects of ketamine-xylazine and isoflurane on insulin sensitivity in dehydroepiandrosterone sulfate-treated minipigs (Sus scrofa domestica)

Isoflurane and ketamine-xylazine (KX) combinations are widely used veterinary anesthetics, KX being the particularly common agent for immobilizing swine. Results of previous studies indicate that KX and xylazine suppress insulin release. The steroid hormones, dehydroepiandrosterone (DHEA) and its sulfated form, dehydroepiandrosterone-sulfate (DHEAS), have variable effects on insulin sensitivity in animals. We evaluated the effect of DHEAS on plasma glucose and insulin concentrations in female Yucatan swine under KX and isoflurane anesthesia. A 2 x 2 factorial design was used. Twenty-four 17-week-old gilts were randomly assigned to receive vehicle (placebo) or DHEAS as part of an ongoing study. The KX was given intramuscularly to all animals prior to blood sample collection at weeks two and four. At week three, all animals received isoflurane by inhalation. During KX anesthesia, mean insulin concentration in DHEAS-treated and control groups approximated half the postisoflurane values (P < 0.001). While under isoflurane, the DHEAS group had significantly higher mean plasma insulin concentration and mean insulin-to-glucose ratio, compared with values for controls (P < 0.05). These findings are consistent with changes in insulin values following DHEAS treatment observed previously in nonanesthetized swine. The effect of DHEAS treatment was absent in animals under KX anesthesia. These results suggest that KX significantly decreases plasma insulin concentration and blunts DHEAS-associated insulin resistance in female minipigs.     

Methyl-beta-cyclodextrin: an alternative carrier for intravenous infusion of palmitate during tracer studies in swine (Sus scrofa domestica).

Fatty acid-free albumin has been the standard carrier for intravenous infusion of fatty acids to study in vivo lipid metabolism. However, subjects can have adverse reactions to infusion of albumin. We sought an alternative to albumin as a carrier for intravenous infusion of fatty acids, using the pig as a model. Cyclodextrins are naturally occurring water-soluble molecules that can serve as carriers for lipid-soluble compounds. 13C-palmitate was complexed to either 20% methyl-beta-cyclodextrin, 20% 2-hydroxypropyl-beta-cyclodextrin, or 5% porcine albumin (isotopic purity of infusates: 99.22+/-0.06%). 13C-palmitate-albumin was infused under fed conditions and 13C-palmitate-methyl-beta-cyclodextrin was infused under fasted and fed conditions in 50-kg pigs. Palmitate remained in solution at 4 degrees C in methyl-beta-cyclodextrin, but precipitated at 25-30 degrees C in 2-hydroxypropyl-beta-cyclodextrin. Pigs infused with 13C-palmitate-methyl-beta-cyclodextrin maintained normal body temperature and appetite; those infused with 13C-palmitate-albumin became anorexic and exhibited other negative side effects to albumin. Palmitate oxidation rates under fed conditions were similar using either 13C-palmitate-methyl-beta-cyclodextrin or 13C-palmitate-albumin complexes. Fasting increased 13C-palmitate-methyl-beta-cyclodextrin oxidation by approximately eight-fold. These data suggest that methyl-beta-cyclodextrin may be a suitable substitute for albumin in fatty acid metabolism studies in swine.     

Pre- and perinatal oxygen transport in mammals: the embryonic hemoglobins of the domestic pig (Sus scrofa domestica)

Pigs express two globin genes of beta-type, the epsilon- and chains during their embryonic stage. With the alpha- and epsilon-chains they form four embryonic hemoglobins. We describe here in detail the experimental procedures for sequencing the epsilon- and chains from the pig (Sus scrofa domestica) and we discuss the data with respect to their special functional properties. From the components Gower I and Heide I, we obtained all embryonic chains by chromatography on CM-Cellulose. The sequence of the tryptic peptides of the beta-type chains was established by automatic Edman-Begg degradation. They were aligned by comparison with the corresponding human chains. The epsilon-chains from man and pig differ in 20 positions, porcine and epsilon-chains only in 4 positions. Up to now these have only been found in pigs. A fetal hemoglobin (gamma-chains) was not observed. As a result of this work the sequences of all peptide chains of pig's hemoglobin are determined.     

Localization of ribosomal genes and nucleolar activity in lymphocytes of swine (Sus scrofa domestica) stimulated by phytohemagglutinins

The Pig chromosomes that contain rDNA sites displayed a polymorphism in the distribution of the genes among the nucleolar organizers located on pairs Nos. 8 and 10. Two, or more often three, active sites were observed in the chromosomes of lymphocytes stimulated with phytohemagglutinin. Only 5% of the metaphases showed a 4th small active site. At the onset of stimulation most cells contained one-two nucleoli; four nucleoli were never observed. After prolonged stimulation, the number of nuclei containing three nucleoli increased. A 4th small nucleolus appeared in a few cells, presumably formed by activation of the smallest rDNA site.     

Social Complexification and Pig (Sus scrofa) Husbandry in Ancient China: A Combined Geometric Morphometric and Isotopic Approach

Pigs have played a major role in the economic, social and symbolic systems of China since the Early Neolithic more than 8,000 years ago. However, the interaction between the history of pig domestication and transformations in Chinese society since then, have not been fully explored. In this paper, we investigated the co-evolution from the earliest farming communities through to the new political and economic models of state-like societies, up to the Chinese Empire, using 5,000 years of archaeological records from the Xiawanggang (XWG) and Xinzhai (XZ) sites (Henan Province). To trace the changes of pig populations against husbandry practices, we combined the geometric morphometric analysis of dental traits with a study of the stable carbon and nitrogen isotope ratios from bone collagen. The domestication process intensified during the Neolithic Yangshao, prompted by greater selective pressure and/or better herd control against wild introgression. After that, pig farming, in XWG, relied on local livestock and a gradual change of husbandry practices overtime. This was characterized by a gentle increase in millet foddering and animal protein intake, until a complete change over to household management during the Han dynasty. The only rupture in this steady trend of husbandry occurred during the Longshan period, with the appearance of small sized and idiosyncratic pigs with specific feeding practices (relying on millet and household scraps). From three exploratory hypothesis, we explored the possibility of anti-elite pig production in XWG during the Longshan period, as a means to resist incorporation into a new economic model promoting intensified domestic production. This exploratory hypothesis is the most suitable to our dataset; however, numerous areas need to be explored further in order to adequately document the role of pigs in the rise of China’s complex societies.