Transcriptome annotation of 17 porcine tissues using nanopore sequencing technology.

The annotation of animal genomes plays an important role in elucidating molecular mechanisms behind the genetic control of economically important traits. Here, we employed long-read sequencing technology, Oxford Nanopore Technology, to annotate the pig transcriptome across 17 tissues from two Yorkshire littermate pigs. More than 9.8 million reads were obtained from a single flow cell, and 69 781 unique transcripts at 50 108 loci were identified. Of these transcripts, 16 255 were found to be novel isoforms, and 22 344 were found at loci that were novel and unannotated in the Ensembl (release 102) and NCBI (release 106) annotations. Novel transcripts were mostly expressed in cerebellum, followed by lung, liver, spleen, and hypothalamus. By comparing the unannotated transcripts to existing databases, there were 21 285 (95.3%) transcripts matched to the NT database (v5) and 13 676 (61.2%) matched to the NR database (v5). Moreover, there were 4324 (19.4%) transcripts matched to the SwissProt database (v5), corresponding to 11 356 proteins. Tissue-specific gene expression analyses showed that 9749 transcripts were highly tissue-specific, and cerebellum contained the most tissue-specific transcripts. As the same samples were used for the annotation of cis-regulatory elements in the pig genome, the transcriptome annotation generated by this study provides an additional and complementary annotation resource for the Functional Annotation of Animal Genomes effort to comprehensively annotate the pig genome.

Development of an Immunoassay for the Detection of Copper Residues in Pork Tissues.

The presence of high concentrations of copper (Cu) residues in pork is highly concerning and therefore, this study was designed to develop a high-throughput immunoassay for the detection of such residues in edible pork tissues. The Cu content in the pork samples after digestion with HNO3 and H2O2 was measured using a monoclonal antibody (mAb) against a Cu (II)-ethylenediaminetetraacetic acid (EDTA) complex. The resulting solution was neutralized using NaOH at pH 7 and the free metal ions in the solution were chelated with EDTA for the immunoassay detection. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed for Cu ion analysis. The half maximal inhibitory concentration of the mAb against Cu (II)-EDTA was 5.36 ng/mL, the linear detection range varied between 1.30 and 27.0 ng/mL, the limit of detection (LOD) was 0.43 µg/kg, and the limit of quantification (LOQ) was 1.42 µg/kg. The performances of the immunoassay were evaluated using fortified pig serum, liver, and pork samples and had a recovery rate of 94.53-102.24%. Importantly, the proposed immunoassay was compared with inductively coupled plasma mass spectroscopy (ICP-MS) to measure its performance. The detection correlation coefficients of the three types of samples (serum, pork, and liver) were 0.967, 0.976, and 0.983, respectively. Thirty pork samples and six pig liver samples were collected from local markets and Cu was detected with the proposed ic-ELISA. The Cu content was found to be 37.31~85.36 µg/kg in pork samples and 1.04-1.9 mg/kg in liver samples. Furthermore, we detected the Cu content in pigs with feed supplemented with tribasic copper chloride (TBCC) and copper sulfate (CS) (60, 110, and 210 mg/kg in feed). There was no significant difference in Cu accumulation in pork tissues between the TBCC and CS groups, while a remarkable Cu accumulation was found for the CS group in liver at 210 mg/kg, representing more than a two-fold higher level than seen in the TBCC group. Therefore, the proposed immunoassay was found to be robust and sensitive for the detection of Cu, providing a cost effective and practical tool for its detection in food and other complicated samples.

LC/MS/MS-based quantitation of pig and human S100A1 protein in cardiac tissues: Application to gene therapy.

The S100A1 protein is a target of interest for the treatment of heart failure as it has been previously reported to be depleted in failing cardiomyocytes. A gene therapy approach leading to increased expression levels of the protein directly in the heart could potentially lead to restoration of contractile function and improve overall cell survival. S100A1 is a relatively small soluble protein that is extremely well conserved across species with only a single amino acid difference between the sequences in human and pig, a commonly used pre-clinical model for evaluation of efficacy, biodistribution and safety for cardiac-directed gene therapy approaches. This high homology presents a bioanalytical challenge for the accurate detection and quantitation of both endogenous (pig) and exogenous (human) transduced S100A1 proteins post treatment using a human S100A1 gene therapy in pigs. Here we present a sensitive and selective LC-MS/MS approach that can easily differentiate and simultaneously quantitate both human and pig S100A1 proteins. Additionally, we report on a detailed profiling of S100A1 protein in various pig tissues, a comprehensive evaluation of S100A1 distribution in pig hearts and a comparison to S100A1 levels in human cardiac samples.

Development of Chrysomya albiceps Wiedemann, 1819 (Diptera: calliphoridae) and Musca domestica Linnaeus, 1758 (Diptera: muscidae) reared together in pig tissues

Chysomya albiceps and Musca domestica are important for forensic entomology, and human and animal health. This study analyzed the effects of the coexistence of C. albiceps and M. domestica reared in four different assays in two pig tissues, brain and intestine: assay 1, interaction between the larvae of the same age; assay 2, interaction between larvae of C. albiceps 24 hours older than larvae of M. domestica; assay 3, interaction between larvae of M. domestica 24 hours older than the larvae of C. albiceps; assay 4, larvae of both species were reared together in flasks with a small supply of food. Weight of larvae, growth time and imago emergence frequency were studied. C. albiceps responded better than M. domestica under most conditions tested. Larvae of C. albiceps responded better in mixed cultures (together with M. domestica) than in pure cultures (larvae of the same species). In contrast, M. domestica responded better when reared in pure cultures. Both species presented shorter growth times when their larvae were reared in intestine tissue with larvae 24 hours younger than the larvae of the concurrent species, compared to their respective growth times in pure cultures. The results confirmed that trophic interactions are relevant to the successful colonization of carrion by C. albiceps. Coexistence of the two species may result in changes in values of their biological components. The results also help to shed light on the biology of the two species in carrion

Ochratoxin A determination in swine muscle and liver from French conventional or organic farming production systems.

Consumers generally considered organic products to be healthier and safer but data regarding the contamination of organic products are scarce. This study evaluated the impact of the farming system on the levels of ochratoxin A (OTA) in the tissues of French pigs (muscle and liver) reared following three different types of production (organic, Label Rouge and conventional). Because OTA is present at trace levels in animal products, a sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method using stable isotope dilution assay was developed and validated. OTA was detected or quantified (LOQ of 0.10 µg kg-1) in 67% (n = 47) of the 70 pig liver samples analysed, with concentrations ranging from <0.10 to 3.65 µg kg-1. The maximum concentration was found in a sample from organic production but there were no significant differences in the content of OTA between farming systems. OTA was above the LOQ in four out of 25 samples of the pork muscles. A good agreement was found between OTA levels in muscle and liver (liver concentration = 2.9 × OTA muscle concentration, r = 0.981).

Classification of trace elements in tissues from organic and conventional French pig production.

This study assesses the impact of the farming system on the levels of copper, zinc, arsenic, cadmium, lead and mercury in pig tissues from three types of production (Organic (n = 28), Label Rouge (n = 12) and Conventional (n = 30)) randomly sampled in different slaughterhouses. All the concentrations were below regulatory limits. In muscles, Cu, Zn and As were measured at slightly higher levels in organic samples but no differences between organic and Label Rouge was observed. Livers from conventional and Label Rouge pig farms exhibited higher Zn and Cd contents than the organic ones, probably due to different practice in zinc or phytase supplementation of fattening diets. Principal component analysis indicated a correlation between Cu and As concentrations in liver and carcass weight, and between Zn and Cd liver levels and lean meat percentage. The linear discriminant analysis succeeded in predicting the farming process on the basis of the lean meat percentage and the liver Cd level.

Quantification of piperazine in chicken and pig tissues by gas chromatography-electron ionization tandem mass spectrometry employing pre-column derivatization with acetic anhydride.

This paper describes a novel method that combines acetic anhydride derivatization with gas chromatography-electron ionization tandem mass spectrometry (GC-EI/MS/MS) for the sensitive and selective determination of piperazine in chicken and pig tissues. Samples were extracted using an accelerated solvent extraction (ASE) apparatus, purified by solid-phase extraction (SPE) and derivatized with acetic anhydride. This optimized method was validated according to the requirements defined by the European Union and the Food and Drug Administration. At the limit of quantification (LOQ) spiked levels of 50.0, 100.0, 500.0, 1000.0 and 2000.0µg/kg, the average recoveries of piperazine in chicken and pig tissues were 77.46-96.26%, with relative standard deviations (RSDs) of 1.55-6.64%. The intra-day RSDs were 1.39-5.92%, and the inter-day RSDs were 2.24-8.39%. The limits of detection (LODs) and the LOQs were 1.4-1.6µg/kg and 4.8-5.2µg/kg, respectively. The decision limits (CCα) were 102.02-105.17µg/kg, and the detection capabilities (CCß) were 104.03-109.09µg/kg. Finally, the new approach was verified for the quantitative determination of piperazine in 30 commercial chicken and pig tissues from local supermarkets.

Rapid analysis of ractopamine in pig tissues by dummy-template imprinted solid-phase extraction coupling with surface-enhanced Raman spectroscopy.

Ritodrine has similar skeleton structure to ractopamine and it was selected as the dummy-template molecule to synthesize the molecular imprinted polymers (MIPs). The MIPs exhibited better selectivity to ractopamine than to the dummy-template molecule: the imprint factor for ractopamine was 8.9, while 7.6 for ritodrine. The MIPs were used as sorbents in solid-phase extraction for selective enrichment of ractopamine, and some key parameters were optimized. After that, a rapid surface-enhanced Raman spectroscopy method was developed for analysis of ractopamine and isoxuprine in pig tissue samples. Under the optimal conditions, good linearity was achieved in the range of 20.0-200.0 µg/L for ractopamine and isoxsuprine at 842 cm(-1) and 993 cm(-1), respectively. The limits of detection were 3.1-4.3 µg/L, which were lower than the maximum allowed by U. S. Food and Drug Administration. The recoveries of ractopamine and isoxsuprine were 72.4-79.7% and 71.0-78.2% for the spiked pork and pig liver, respectively, while the relative standard deviations ranging from 7.4% to 13.0%. The results suggest that the proposed method is sensitive and selective, and it has good potential on the quantitative analysis of trace amounts of ß-agonists in complex samples.