Swine Muscle Tissue Research Articles

Long-term longitudinal study on swine VML model

BackgroundVolumetric Muscle Loss (VML), resulting from severe trauma or surgical ablation, is a pathological condition preventing myofibers regeneration, since skeletal muscle owns the remarkable ability to restore tissue damage, but only when limited in size. The current surgical therapies employed in the treatment of this pathology, which particularly affects military personnel, do not yet provide satisfactory results. For this reason, more innovative approaches must be sought, specifically skeletal muscle tissue engineering seems to highlight promising results obtained from preclinical studies in VML mouse model. Despite the great results obtained in rodents, translation into human needs a comparable animal model in terms of size, in order to validate the efficacy of the tissue engineering approach reconstructing larger muscle mass (human-like). In this work we aim to demonstrate the validity of a porcine model, that has underwent a surgical ablation of a large muscle area, as a VML damage model.ResultsFor this purpose, morphological, ultrasound, histological and fluorescence analyses were carried out on the scar tissue formed following the surgical ablation of the peroneus tertius muscle of Sus scrofa domesticus commonly called mini-pig. In particular, the replenishment of the damaged area, the macrophage infiltration and the vascularization at different time-points were evaluated up to the harvesting of the scar upon six months.ConclusionHere we demonstrated that following VML damage, there is an extremely poor regenerative process in the swine muscle tissue, while the formation of fibrotic, scar tissue occurs. The analyses performed up to 180 days after the injury revealed the development of a stable, structured and cellularized tissue, provided with vessels and extracellular matrix acquiring the status of granulation tissue like in human.

A simplified sample pretreatment for the rapid determination of 22 β-agonist residues in swine muscle and liver tissues by ultra-high-performance liquid chromatography tandem mass spectrometry

A reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous identification and quantification of 22 β-agonist residues in swine muscle and liver tissues. A special focus was given to sample the pretreatment procedures and was crucial for an accurate and reliable analytical method. With regard to the high complexity of food matrices, the extraction solvent, time, and temperature, as well as clean-up cartridges, were optimized to improve the extraction efficiency and reduce matrix effects. The UHPLC-MS/MS method was further validated by determining the linearity (R2 ≥ 0.9900), sensitivity (limit of detection varying from 0.05 μg/kg to 0.8 μg/kg, limit of quantitation ranging from 0.2 μg/kg to 2.5 μg/kg), recovery (ranging from 76.4 to 111.7%) and precision (≤22.9%) in swine muscle and liver tissues. The UHPLC-MS/MS method was proven to be suitable for both screening and the confirmatory determination of 22 β-agonist residues in swine muscle and liver tissues.

A Convenient and Sensitive LC-MS/MS Method for Simultaneous Determination of Carbadox- and Olaquindox-Related Residues in Swine Muscle and Liver Tissues

This paper presents a convenient and sensitive LC-MS/MS method for the simultaneous determination of carbadox and olaquindox residues, including desoxyolaquindox (DOLQ), desoxycarbadox (DCBX), quinoxaline-2-carboxylic acid (QCA), 3-methyl-quinoxaline-2-carboxylic acid (MQCA), and the glycine conjugates of QCA and MQCA (namely, QCA-glycine and MQCA-glycine, resp.) in swine muscle and liver tissues. Tissue samples were extracted with 2% metaphosphoric acid in 20% methanol and cleaned up by solid-phase extraction (SPE) on a mixed-mode anion-exchange column (Oasis MAX). Analysis was performed on a C18 column by detection with mass spectrometry in the multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the six analytes were determined to be 0.01 μg·kg−1 to 0.25 μg·kg−1, and the limits of quantification (LOQs) were 0.02 μg·kg−1 to 0.5 μg·kg−1. The total recoveries of the six analytes in all tissues were higher than 79.1% with the RSD% less than 9.2%. The developed method can determine the real residue level of QCA and MQCA, whether they are present in free form or as glycine conjugates in tissues, together with the carcinogenic desoxy metabolites DCBX and DOLQ with high recovery. Therefore, this method was suitable for routine analysis of residue control programmes and the residue depletion study of CBX and OLQ on swine.

Time-resolved study of the plasma produced from animal muscle tissue using a Nd:YAG laser

This paper reports studies on time-resolved laser induced breakdown spectroscopy (LIBS) of plasmas induced by Nd:YAG laser pulses on a sample lyophilized from swine muscle tissue.

Rapid multiresidue determination of pesticides in livestock muscle and liver tissue via modified QuEChERS sample preparation and LC-MS/MS

ABSTRACTMany multiresidue methods for the determination of pesticides in vegetables and fruits have been reported to date. However, few such methods have been employed to investigate pesticide residues in animal tissue. In this study, an LC-MS/MS multiresidue method coupled with modified QuEChERS extraction was developed and validated for the investigation of eight pesticide residues: prallethrin (PR), resmethrin (RMT), imidacloprid (IMC), diflubenzuron (DFB), cyromazine (CYR), etofenprox (EFP), dinotefuran (DNT) and phthalthrin (PTLT). This method involves initial extraction in a water/acetone system, the addition of salts and a subsequent extraction/partitioning step and, finally, a clean-up step utilising dispersive solid-phase extraction (SPE). The mean recoveries of seven of the pesticides (the exception being CYR) ranged between 74.7% and 113.5%, and the CVs of the livestock tissue – bovine, swine, and chicken muscle and liver tissue spiked at 10 ng g–1 (50 ng g–1 for RMT and DNT) and 100 ng g–1 – were < 13.8%. The recoveries of CYR in all muscle and liver spiked samples ranged from 56.9% to 78.3%, while those of RMT in swine liver were > 120%. Therefore, this method was considered as being unsuitable for the investigation of these samples. The limits of quantitation (LOQs) of seven of the investigated pesticides (the exception being swine liver) in the tissue samples ranged from 0.9 to 15.2 ng g–1. We therefore concluded that this LC-MS/MS multiresidue method is a valid and suitable for the investigation of seven pesticides in animal tissue, but it is unsuitable for the analysis of CYR in all animal tissues and RMT in swine liver tissue.

Differential microRNA expression profiling and target gene prediction in the muscle tissues of clenbuterol-fed Chinese miniature swine

ABSTRACTMicroRNAs (miRNAs) regulate gene expression by inhibiting the translation of target RNA. Clenbuterol (CLB) is a β-adrenergic agonist that has the ability to modify muscle characteristics by promoting protein synthesis and inhibiting the growth of fat. To determine the expression profile of miRNAs in the muscle of CLB-treated Chinese miniature swine and to predict their target genes, 13 microRNAs were detected and analyzed. We found that miR-133, miR-206, and miR-1 exhibited the highest expression, although no difference in expression levels was observed between the control and the treatment groups, whereas miR-29a and miR-29b were differentially expressed in the CLB-treated muscles. Furthermore, we analyzed the relationship between miR-29a/b expression, muscle growth, and candidate target genes, and determined that the expression of the four target genes (HnRNPF, SWI/SNF, TPM1, and LPL) was regulated by miR-29a and miR-29b and were functionally related to muscle development.

Plume Dynamics of Laser-Produced Swine Muscle Tissue Plasma.

We report on the plume dynamics of the plasma induced by laser ablation of a swine skeletal muscle tissue sample in different vacuum conditions. Pulses from a transversely excited atmospheric CO2 laser were focused onto a target sample and the induced plasma was allowed to expand in different air pressures. The expansion features were studied using fast photography of the overall visible emission by using a gated intensified charged coupled device. Free expansion and plume splitting were observed at different pressure levels. The expansion of the plasma plume front was analyzed using various expansion models and the velocity of the plume front was estimated. The effect of the number of accumulated laser shots on the crater volume at different ambient air pressures and an elemental analysis of the sample were performed using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) analysis. The surface morphology of the irradiated surface showed that increasing the pressure of the ambient gas decreased the ablated mass, or in other words it reduced significantly the laser-target coupling.

Time- and space-resolved spectroscopic characterization of laser-induced swine muscle tissue plasma

The spatial-temporal evolution of muscle tissue sample plasma induced by a high-power transversely excited atmospheric (TEA) CO2 pulsed laser at vacuum conditions (0.1–0.01Pa) has been investigated using high-resolution optical emission spectroscopy (OES) and imaging methods. The induced plasma shows mainly electronically excited neutral Na, K, C, Mg, H, Ca, N and O atoms, ionized C+, C2+, C3+, Mg+, Mg2+, N+, N2+, Ca+, O+ and O2+ species and molecular band systems of CN(B2Σ+–X2Σ+), C2(d3Πg–a3Πu), CH(B2Σ−–X2Π; A2Δ–X2Π), NH(A3Π–X3Σ−), OH(A2Σ+–X2 Σ+), and CaOH(B2Σ+–X2Σ+; A2Π–X2Σ+). Time-resolved two-dimensional emission spectroscopy is used to study the expanded distribution of different species ejected during ablation. Spatial and temporal variations of different atoms and ionic excited species are reported. Plasma parameters such as electron density and temperature were measured from the spatio-temporal analysis of different species. Average velocities of some plasma species were estimated.

Time and space-resolved spectroscopic and imaging study of a laser-produced swine muscle tissue plasma

SynopsisWe investigated the optical emission and imaging features of plasmas produced by a high-power transversely excited atmospheric CO2 laser pulses on a swine muscle tissue sample in different vacuum conditions. The analyzed plasma shows electronically excited neutral Na, K, C, Mg, H, Ca, N, and O atoms, ionized C+, C2+, C3+, Mg+, Mg2+, N+, N2+, Ca+, O+, and O2+ species and molecular band systems of CN, C2, CH, NH, OH and CaOH. Time-resolved 2D emission spectroscopy is employed to study the expanded distribution of several species ejected during ablation. The expansion of the plume front was analyzed using Shock wave and Drag models.

Purification of swine carbonic anhydrase isoenzymeIIIand measurement of its levels in tissues and plasma

The changes in the levels of carbonic anhydrase isozyme III (CA-III) in swine plasma and urine have not been previously determined or reported. CA-III is relatively specific to skeletal muscles, and should therefore be a useful diagnostic marker for muscle diseases. We isolated CA-III from swine muscle tissues and determined CA-III levels in the plasma and urine from both healthy and diseased pigs. The levels of CA-III in the tissues of female swine (age, 3months) and plasma of young swine (age, 1-5months) and adult female pigs (age, 2-3years) were determined using the ELISA system for swine CA-III. The mean (±SD) levels of CA-III in the skeletal muscles were 3.8±3.2mg/g (wet tissue), and in the plasma, 230±193ng/ml at 1month, 189±208ng/ml at 2months, 141±148ng/ml at 3months, 78±142ng/ml at 4months and 53±99ng/ml at 5months. The mean level of CA-III in the plasma samples from 2- to 3-year-old pigs was 18±60ng/ml. CA-III in the plasma samples was found to decrease from 1month until 3years of age (p<0.01). We performed far-western blotting to clarify the cause of the observed decrease in CA-III in plasma. Our results demonstrated that CA-III is bound to the transferrin and albumin. In addition, we determined that the levels of CA-III in plasma and urine samples were higher in diseased swine compared with the healthy pigs.

Evaluation of the keyhole technique applied to the proton resonance frequency method for magnetic resonance temperature imaging

To evaluate the temporal and spatial resolution of magnetic resonance (MR) temperature imaging when using the proton resonance frequency (PRF) method combined with the keyhole technique. Tissue-mimicking phantom and swine muscle tissue were microwave-heated by a coaxial slot antenna. For the sake of MR hardware safety, MR images were sequentially acquired after heating the subjects using a spoiled gradient (SPGR) pulse sequence. Reference raw (k-space) data were collected before heating the subjects. Keyhole temperature images were reconstructed from full k-space data synthesized by combining the peripheral phase-encoding part of the reference raw data and the center phase-encoding keyhole part of the time sequential raw data. Each keyhole image was analyzed with thermal error, and the signal-to-noise ratio (SNR) was compared with the self-reference (nonkeyhole) images according to the number of keyhole phase-encoding (keyhole-data size) portions. In applied keyhole temperature images, smaller keyhole-data sizes led to more temperature error increases, but the SNR did not decreased comparably. Additionally, keyhole images with a keyhole-data size of <16 had significantly different temperatures compared with fully phase-encoded self-reference images (P < 0.05). The keyhole technique combined with the PRF method improves temporal resolution and SNR in the measurement of the temperature in the deeper parts of body in real time.

GC–MS Method for Simultaneous Determination of Four Sedative Hypnotic Residues in Swine Tissues

A robust, reliable and sensitive method is described for the simultaneous determination of diazepam, methaqualone, chlorpromazine and promethazine in swine muscle and liver tissues by GC–MS. The analytes were extracted with ethyl acetate and further clean-up performed with MCX solid-phase extraction cartridges. LODs and LOQs in the two tissues ranged from 0.2 to 0.3 μg kg−1 and 0.5 to 1 μg kg−1, respectively. Mean recoveries for the analytes ranged between 87.6 and 106.8%.

Simultaneous Determination of Florfenicol and Its Metabolite Florfenicol Amine in Swine Muscle Tissue by a Heterologous Enzyme-Linked Immunosorbent Assay

A sensitive and heterologous enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) in swine muscle tissue was developed. FFA was conjugated to bovine serum albumin by a formaldehyde coupling method as an immunogen to immunize rabbits. FFA, thiamphenicol glycinate, and modified FF were conjugated to ovalbumin as coating antigens. The effect of different types of hapten heterology on the sensitivity and specificity of the ELISA was evaluated. Using FF glutaric anhydride ester as a coating hapten and antibody raised against modified FFA, an ELISA was developed that showed an IC50 value of 0.48 ng/mL. The antibody showed a cross-reactivity of 100% with FFA, 97% with FF, 6% with thiamphenicol, and a negligible value with chloramphenicol. From fortified swine muscle samples at levels of 4-320 ng/g, the average recoveries of FF and FFA ranged from 58.2 to 96.8%, with coefficients of variation less than 14%. Analysis of incurred samples by the ELISA gave similar results to those by a previously developed liquid chromatographic method. The ELISA could be used as a rapid method for the simultaneous determination of FF and FFA in swine muscle tissue.

Cloning and analysis of differentially expressed ESTs in swine muscle tissue

The obvious difference in muscle growth and meat quality traits exists between Chinese indigenous pig and exotic pigs. In order to study the reason of these phenotypic differences and search the potential gene related to growth and meat quality traits, silver-stained mRNA differential display technique was used to detect the difference with mRNA of loin-eye muscle tissue from maturity pigs of Lantang in Guangdong Province and Large Yorkshire. One of the newly discovered expressed sequence tag (ESTsp3) was analyzed by using bioinformatic technique. The results showed: (1) nearly 2000 cDNA fragments were detected with 30 primer pairs, and 6 differentially expressed ESTs in the loin-eye muscle tissues from the two breeds were isolated and obtained. The differential fragments were cloned and sequenced. The all sequences were recorded in the GenBank. (2) The 786 bp fragment of ESTsp3 was obtained with in silico elongation system, the ORF analysis revealed that it existed as an 83 aa complete open reading frame, and the elongation sequences were verified by RT-PCR. The analysis of in silico expression profile showed that ESTsp3 is expressed in various growth stages and in most tissues and organs, such as soft tissue, skin, skeletal muscle and kidney, but with variant expression quantity.

Screening of Specific Genes Expressed in the Swine Tissues and Development of a Functional cDNA Chip

To develop a functional cDNA chip, specific genes expressed in the tissues of swine Kagoshima Berkshire were screened. A total of 4,434 ESTs were obtained by constructing a cDNA library from total RNA isolated from the muscle and fat tissues, affirming their functions by investigating similarity of nucleotide sequences with the database at the NCBI. Among them, 1,230 ESTs were confirmed as novel genes, which, to date, have not been identified. Attaching the genes to a cDNA microarray slide revealed expression patterns of genes in muscle and fat according to the growth stages of swine. As specific genes expressed in the muscle tissues of swine with body weight of 30 kg, 60 genes including actin, myosin, tropomysin, transfer RNA-trp synthetase, Kel-like protein 23, KIAA0182 and COI, Foocen-m, etc were obtained. In addition, 18 novel genes were obtained. As specific genes expressed in fat tissues of swine with body weight of 30 kg, 47 genes including annexin II, Collagen, Fibronectin, Pleckstrin homology domain, serine protease, etc were obtained. 21 novel genes were also obtained. The genes specifically expressed in the muscle and fat tissues of swine affect contraction and relaxation of the muscle and the fat. However, studies on the expression mechanisms of the genes are insufficient. To reveal species of structural genes in swine muscle and fat tissue, interrelation studies in expression and function of genes by using the cDNA chip should be conducted. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 7 : 933-941)

Reaction Products of Sulfamethazine and Sodium Nitrite in Swine Muscle Tissues

Sulfamethazine (SMZ) is an effective animal drug and is extensively used to treat acute and chronic bacteria infections and to enhance growth in swine. Residual SMZ in animal muscle tissues could react with sodium nitrite during further processing. This study investigated reaction products of SMZ and sodium nitrite in different reaction matrices including in vitro conditions, spiked and incurred in swine muscle tissues. Results from the in vitro study indicated that sodium nitrite and SMZ reacted and produced yellow precipitates under acid conditions. Three major reaction products were detected in the precipitates with molecular weights of 503, 556, and 567, respectively. The compound with molecular mass 567 could be l, 3-di-(4-[N-(4, 6-dimethyl-2-pyrimidinyl)] sulphamoyl-phenyl)-triazen. Three major compounds were detected in the supernatant of the reaction mixture, with molecular weights 307, 279, and 263, respectively. Those could be 4-diazohydroxy-N-(4, 6-dimethyl-2-pyrimidinyl) benzene-sulfonamide, 4-hydroxy-N-(4, 6-dimethyl-2-pyrimidinyl) benzene-sulfonamide, and desaminosulfamethazine, respectively. Only the molecular weight 279 and the molecular weight 263 compounds were detected in the reaction supernatant of the SMZ-spiked swine muscle tissues. Only the molecular weight 263 compound was detected in the reaction supernatant of the SMZ-incurred swine muscle tissues. Possible mechanisms for deriving these compounds were proposed.

Comparison of a Modified Digestion Assay with Trichinoscopy for the Detection of Trichinella Larvae in Pork

A pepsin-HCl digestion assay and two compressorium techniques (trichinoscopy) for the identification of swine muscle tissue containing low levels of Trichinella larvae were compared as part of the test validation process for quality assurance purposes. Compressoria read with a stereomicroscope detected more larvae (P < 0.0001, n = 57) and more tissues (P =0.0047, n = 57) than did compressoria read with a projection microscope (trichinoscope). The digestion assay evaluated was 3.2 times as likely as the best compressorium technique to identify a positive tissue when these procedures were used to test 1 g of infected muscle (P < 0.001; 95% confidence interval for the odds ratio, 2.0 to 5.4; n = 161 and n = 189, respectively). Detection by trichinoscopy improved as the number of larvae in tissues increased to >2 larvae per g, but trichinoscopy was less sensitive than the digestion assay regardless of the tissue larval load. These data indicate that the quality controlled digestion assay used in this study is more sensitive than trichinoscopic techniques in the detection of tissues containing low levels of Trichinella larvae.

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC–MS–MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2- n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1–22 μg/kg. The limits of detection were below 6 μg/kg and the limits of quantification for most benzimidazoles were below 10 μg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.

Comparison of the determination of four sulphonamides and their N 4-acetyl metabolites in swine muscle tissue using liquid chromatography with ultraviolet and mass spectral detection

A discharge-assisted LC—MS method has been developed and validated for the analysis of four sulphonamides (sulphathiazole, sulphadiazine, sulphamerazine and sulphadimidine) and their N 4-acetyl metabolites in the muscle of swine treated with Polysulpha-Complex, which contains all four drugs. The clean-up procedure developed involved chloroform—acetone extraction followed by Sep-Pak silica solid-phase extraction. In parallel a LC-UV method was validated using the same clean-up procedure. Blank tissue was fortified at levels between 20 and 100 μg/kg. [ 13C]sulphadimidine was used as internal standard. The samples were analysed with thermospray LC—MS. The [M + H] + ion was the major ion in all cases and was employed for single-ion monitoring. The limits of detection (LOD) were below 25 μg/kg and the limits of quantification (LOQ) for most sulphonamides were ca. 100 μg/kg. Incurred muscle tissues were measured by both LC methods and the concentrations of the sulphonamides were found to be similar. However, the LC—MS procedure is more suitable for confirmatory analysis due to its specificity.