Search

Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H2O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method's LODs ranged between 2.8 ng g−1 (6-phenyl-2-thiouracil) and 4.1 ng g−1 (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results. [ABSTRACT FROM AUTHOR]

Τhyreostats (TSs) are veterinary drugs used in livestock farming for fattening. Their administration is banned in the European Union since 1981, and their monitoring for food quality and safety control requires sensitive and confirmatory methods. The present study describes the development and validation of a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of 2-thiouracil (TU), 6-methyl-2-thiouracil (MTU), 6-propyl-2-thiouracil (PTU), 6-phenyl-2-thiouracil (PhTU), tapazole (TAP), and 2-mercaptobenzimidazole (MBI) in bovine muscle tissues. Investigation of the retention mechanism of the six analytes on the selected amide-based stationary phase showed that hydrophilic partition was the dominant interaction. The sample preparation included extraction with ACN/H2O (80/20), followed by dispersive solid-phase extraction (d-SPE) with C18 sorbent and hexane partitioning. The method was validated according to European guidelines using internal standards, including isotopically labelled ones. The method’s LODs ranged between 2.8 ng g−1 (6-phenyl-2-thiouracil) and 4.1 ng g−1 (2-thiouracil). Application of the proposed method to 48 bovine tissue samples showed non-detectable results.

Background: Vascular prosthetic graft infections are rare but associated with high morbidity and mortality. Treatment involves removal of the infected graft requiring arteriotomy closure. Previously this was performed with autologous graft, but bovine tissue has increasingly been used. The objective of this paper is to review the community hospital experience with bovine tissue repair in an infected vascular field.Materials and Methods: A retrospective review of all cases performed by a single surgeon in a community hospital for infected prosthetic grafts was completed. Sixteen cases were included where bovine tissue was used for repair. Presentation, location of graft, and causative organism were reviewed, and outcomes including reoperation and mortality were recorded.Results: Of the 16 patients, 15 (94%) had positive cultures of the graft. Methicillin-Resistant Staph Aureus was the most commonly isolated organism (50%). There were 3 unplanned reoperations including a revision from below to above knee amputation, drainage of a hematoma, and a wound debridement within the first year. Over the 1 year follow up period, 3 patients died for a mortality of 19%. There were no reinfections during follow-up.Discussion: Prosthetic graft infection is a rare but serious vascular surgery complication. The causative organism has shifted in the last few years to become increasingly drug resistant. Treatment requires excision, and bovine tissue has been demonstrated to provide a safe and durable method of repair. [ABSTRACT FROM AUTHOR]

Purpose: To compare the depth and volume of cavities produced by ablation on bovine bone tissue using different Er:YAG laser parameters. Materials and Methods: A bovine femur was cut in blocks and wet polished with 600-grit sandpaper. Different energies per pulse (100 mJ, 300 mJ, and 500 mJ) and different frequencies (5 Hz, 10 Hz, and 15 Hz) were used to create microcavities within a 10-s irradiation time. The samples were analyzed under an optical microscope (40X) and SEM for a better definition of the cavities and their features. The ablated volume was calculated and statistically analyzed with Kruskal-Wallis and Dunn tests. Results: The ablation rate in many groups showed significant differences up to 5%, where the energy per pulse was the only significant variable. As energy per pulse increased, so did the ablation rate. Morphological analysis showed differences in the homogeneity of the microcavities. Conclusions: Er:YAG laser is able to promote cold interaction, suggesting a precise tool for bone ablation. [ABSTRACT FROM AUTHOR]

Although the post-mortem diagnosis of bovine tuberculosis is mainly achieved through microbiological culture, the development of other techniques to detect Mycobacterium tuberculosis complex (MTBC) members directly from tissue samples has been pursued. The present study describes the development, optimization and validation of a Real-Time PCR based on the mpb70 gene to detect MTBC members in clinical tissue samples from cattle. Specific primers and a hybridization probe were used to amplify MTBC-specific sequences in order to avoid cross-reaction with non-MTBC species. An Internal Amplification Control (IAC) was included in order to assess the presence of PCR inhibitors in the samples. The PCR was optimized to achieve maximum efficiency, and the limit of detection, limit of quantification and dynamic range of the reaction were determined. The specificity of the reaction was tested against 34 mycobacterial and non-mycobacterial species. The diagnostic sensitivity, specificity and positive and negative predictive values (PPV and NPV) of the method were assessed on 200 bovine tissue samples in relation to bacteriological culture. The dynamic range of the reaction spanned from 5 ng/reaction (106 genome equivalents) to 50 fg/reaction (10 genome equivalents). The efficiency of the reaction was 102.6% and the achieved R2 was 0.999. The limit of detection with 95% confidence was 10 genome equivalents/reaction. No cross-reactions with non-MTBC species were observed. The diagnostic sensitivity and specificity values of the mpb70 specific Real-Time PCR respect to culture were 94.59% (95% CI: 86.73–98.51%) and 96.03% (95% CI: 90.98–98.70%), respectively, with a PPV of 93.33% (95% CI: 85.55–97.07%) and a NPV of 96.80% (95% CI: 92.10–98.74%). The concordance of the Real-Time PCR based on mpb70 is comparable to that of culture (K = 0.904) showing a great potential for the detection of members of the MTBC in animal tissues.

Cultivated meat, an alternative to conventional meat, holds great promise in alleviating environmental and ethical concerns. Skeletal muscle stem cell isolation is a critical phase in cultivated meat production, and efficiency is a major determinant in the final differentiated muscle cell yield. The conventional enzymatic dissociation method for cell isolation presents drawbacks, including added costs and the destruction of vital extracellular matrix components. We developed an alternative cell isolation technique, explant cell isolation, to isolate muscle stem cells from muscle tissue. The present protocol yields myogenic cell populations, mainly composed of skeletal muscle stem cells without the use of enzymes, and through a simplified process. Overall, the explant method allows for propagation of cells in their natural environment, preserving intricate cell-cell and cell-matrix interactions, resulting in both economic efficiency and consistent generation of high-quality cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In forensic entomology, the minimum and maximum post‐mortem interval (PMI) is estimated on the basis of the developmental stages of necrophagous Diptera larvae on a corpse. The nutritional composition of the decomposing tissue, which can influence larval development rates, may impact the precision of this estimate. We compared the post‐embryonic development of Chrysomya albiceps reared on bovine muscle and swine lung. Forty newly hatched fly larvae, per treatment, were placed on 60 g of the following tissues: bovine muscle (control) and swine lung (T1). Each treatment had three replicates. The experiments were maintained under controlled conditions (28°C day/26°C night, 70 ± 10% RH and 12 h photophase). Flies reared on bovine muscle took significantly longer to complete their larval phase (8.1 days) than did larvae reared on swine lung (6.8 days). The total duration of development in bovine muscle was significantly slower (12.3 days) than in swine lung. Larvae that were reared on swine lungs were significantly heavier than larvae reared on bovine muscle. Total larval viability was high on pig lung (over 49%). The sex ratio was closer to the 1:1 ratio in bovine muscle (rs = 0.55), with a slightly greater proportion of female larvae. The percentage of abnormality remained within the predicted range. The results show that the different tissues influenced larval development. This underlines the importance of considering the type of cadaveric tissue when larval development is used to calculate the minimum PMI in forensic investigations. [ABSTRACT FROM AUTHOR]

Abstract 1 1 Maximum residue limit (MRL), tulathromycin marker residue (TLM), European Union (EU), liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography (LC), electrospray ionization (ESI), selected reaction monitoring (SRM), polypropylene (PP), limit of detection (LOD), limit of quantification (LOQ), relative standard deviation (RSD). A reliable and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed to quantify total tulathromycin residues in bovine tissues. Specifically, the above method relied on the quantification of CP-60,300, a marker produced by tulathromycin hydrolysis, for which maximum residue limits (MRLs) were established by the European Union and several other countries. Sample preparation and LC-MS/MS conditions were thoroughly optimized to allow for accurate quantification. The optimized procedure involved sample homogenization with 2 mol/L hydrochloric acid and ethyl acetate, heating of the resulting aqueous layer to convert tulathromycin and its metabolites into the marker residue, cleanup by a polymer-based cation-exchange cartridge, and subsequent analysis by LC-MS/MS. The developed method was validated for tulathromycin A and the marker residue in bovine muscle, fat, and liver at two levels, namely at the MRL set in Japan and at 0.01 mg/kg. Excellent analytical performance was observed, with the average recoveries of tulathromycin A and the marker residue ranging from 98 to 107%, and relative standard deviations ranging from 1 to 3%. Matrix effects were negligible, and analyte loss during sample preparation was minimal for all matrices tested, which allowed for accurate determination by external standard calibration using a solvent standard. No interfering peaks were observed close to the retention time of the marker residue for all matrices, which was indicative of high specificity. Overall, the developed method was proven suitable for regulatory purpose analysis of total tulathromycin residues. Highlights • LC-MS/MS method for the determination of total tulathromycin residues is developed. • Hydrolyzed marker residue CP-60,300 is used for tulathromycin residue analysis. • Sample preparation and LC-MS/MS conditions are carefully optimized. • The developed method shows excellent analytical performance. • Accurate quantification is achieved by external calibration using solvent standards. [ABSTRACT FROM AUTHOR]

The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogen–host interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C) from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3) were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.

The proliferation and differentiation of skeletal muscle satellite cells is regulated by multiple regulatory factors including non-coding RNAs. It has been reported that miR-133b regulates myogenesis. In this study, we detected a novel lncRNA, lnc133b, which is completely complemented by mature miR-133b, indicating that lnc133b may regulate the expression of miR-133b by “sponge” miR-133b. A luciferase report assay confirmed that lnc133b interacts with miR-133b in regions complemented by miR-133b. We successfully constructed lnc133b gain/loss-of-function cell models by infecting LV-1nc133b and transfecting si-lnc133b into satellite cells. Results of quantitative real-time polymerase chain reaction (qRT-PCR) and 5-ethynyl-2′-deoxyuridine (EdU) assays showed that overexpression or inhibition of lnc133b could promote the proliferation or inhibition of satellite cell differentiation. The qRT-PCR results also showed that lnc133b negatively regulates miR-133b expression and a Western blot assay showed that lnc133b positively regulates IGF1R expression, indicating that the lnc133b/miR-133b/IGF1R axis is a potential pathway for promoting satellite cell proliferation and repressing their differentiation through the ceRNA mechanism. Building on the findings of previous reports, we constructed the lnc133b/miR-133b/FGFR1 & PP2AC pathway to improve the lnc133b regulation network regulating the proliferation and differentiation of satellite cells. The current study provides a new perspective for understanding the mechanism regulating satellite cell proliferation and differentiation through the interaction of miR-133b and lnc133b. [ABSTRACT FROM AUTHOR]

Stearoyl-CoA desaturase 1 (SCD1) greatly contributes to the unsaturated fatty acids present in milk and meat of cattle. The SCD1 enzyme introduces a double bond into certain saturated fatty acyl-CoAs producing monounsaturated fatty acids (MUFA). The SCD1 enzyme also has been shown to be active in the bovine mammary gland converting t11 18 : 1 (vaccenic acid) to c9 t11 conjugated linoleic acid (CLA). The objective of this study was to determine any association between the gene expression of SCD1 and occurrence of its products (c9 14 : 1, c9 16 : 1, c9 18 : 1, and c9 t11 18 : 2) in various bovine tissues. Tissue samples were obtained from lactating Holstein cows (n=28) at slaughter, frozen in liquid nitrogen and stored at −80 °C. Total RNA was extracted and converted to complementary DNA for quantitative real time polymerase chain reaction (PCR) analysis of the SCD1 gene. Extracted lipid was converted to fatty acid methyl esters and analysed by GC. Tissues varied in expression of SCD1 gene with mammary, cardiac, intestinal adipose, and skeletal muscle expressing greater copy number as compared with lung, large intestine, small intestine and liver (371, 369, 328, 286, 257, 145, 73, and 21 copies/ng RNA, respectively). Tissues with high mRNA expression of SCD1 contained greater SCD1 protein whereas detection of SCD1 protein in tissues with low SCD1 mRNA expression was very faint or absent. Across tissues, the desaturase indices for c9 18 : 1 (r=0·24) and sum of SCD products (r=0·20) were positively correlated with SCD1 gene expression (P<0·01 for both). Within each tissue, the relationship between SCD1 gene expression and the desaturase indices varied. No correlation was detected between SCD1 expression and desaturase indices in the liver, large and small intestines, lung, cardiac or skeletal muscles. Positive correlations, however, were detected between SCD1 expression and the desaturase indices in intestinal adipose tissue (P<0·02 for all) except 14 : 1, whereas only c9 18 : 1, c9 t11 18 : 2 and sum of all desaturase indices were positively correlated with SCD1 expression in mammary tissue (P⩽0·03). Overall, the relationship between SCD1 gene expression and occurrence of its products seems to be tissue specific. [ABSTRACT FROM PUBLISHER]

In this study, desorption electrospray ionisation (DESI) linear ion trap tandem mass spectrometry (MSn) was applied for the confirmation and three-dimensional profiling of anabolic steroid esters in an injection site of bovine muscle. The spatial resolution of the DESI-MSnwas demonstrated by scanning hormone esters and marker ink lines drawn at various distances on a microscopic slide at set distances, using an x-scanner with manual y and z adjustment. Tissue slices of bovine muscle injected with a hormone cocktail were analysed. All anabolic steroid esters could be directly detected in the sample and confirmed on the basis of identification points awarded for selected MS/MS transitions according to the performance criteria given in Commission Decision 2002/657/EC. Moreover, the injection site could be mapped by two-dimensional and three-dimensional imaging MS, showing a horizontal and vertical distribution through the muscle tissue. This DESI approach offers potential for analysis of injection sites of steroid esters from illegally treated animals; moreover, direct analysis by ambient imaging DESI-MS still allows conventional extraction and analysis of the whole tissue for further confirmatory or contra-analysis afterwards. [ABSTRACT FROM PUBLISHER]

We present a sensitive and selective method for detecting imidocarb in bovine tissues and milk samples using a solid-phase extraction (SPE) with diatomaceous earth column and liquid chromatography with electrospray tandem mass spectrometry (LC-ESI-MS/MS) on positive mode. Chromatographic separation was performed on a TSK-GEL ODS 100 V column using 0.1% formic acid in water/methanol. Limit of detection (LOD) was 0.5 ng/mL for standard solution. Limit of quantitation (LOQ) was ranged from 1.0 to 10 µg/kg for bovine tissue and milk samples. The linearity of the calibration curve was excellent in matrix matched standards, and yielded the coefficients (r2 = 0.997–0.999, range from LOQ to 250, 750, or 5000 µg/kg) of imidocarb. Recoveries were in the range of 73.3–98.6% with associated precision values (within-day: 1.0–6.7%, n = 6, and between-day: 2.0–7.7% for 3 days) for repeatability and reproducibility. This developed method is well adapted for residue analysis of imidocarb in bovine tissues and milk based on maximum tolerated residue levels. [ABSTRACT FROM AUTHOR]

rapid and sensitive method has been developed for the simultaneous determination of four avermectins and one milbemycin residues in bovine tissue. The isolation of the analytes from muscle and liver samples was accomplished utilizing a pressurized solvent extractor. The optimized extraction procedure using acetonitrile/water (40:60, v/v) as extraction solvent, was automatically carried out at 100 °C and 10 MPa, applying two static cycles for 3 min. The extracts were cleaned up on a C solid-phase extraction cartridge and analyzed by liquid chromatography with fluorescence detection after derivatization. Mean recoveries of the five analytes from fortified samples were between 84.8 and 101.8%, with relative standard deviations lower than 10.8%. The limit of detection and quantification were in the ranges of 0.1-0.2 and 0.5-0.6 μg kg, respectively. The application of the newly developed method was demonstrated by analyzing bovine meat samples from market. [ABSTRACT FROM AUTHOR]

Aim: This paper describes the rationale, development and clinical performance of a novel small-diameter vascular prosthesis of non-vascular animal tissue origin.Method: Durable conduits suitable for coronary and peripheral arterial reconstruction are prepared for the first time from non-vascular animal tissue. Australian bovine ureters from young steers from protected herds are processed to form strong, bloodflow-compatible and non-immunogenic biologic grafts (‘Flonova’R, Bionova International Pty Ltd, Melbourne). Fifty-six patients lacking suitable autogenous veins received 62 prostheses (32 bypasses were implanted above knee and 30 below knee). Determination of patency and function included Doppler ultrasound, duplex scanning and angiography.Findings: The primary patency rate at 1, 2 and 5 years, respectively was 67, 59 and 52% and the secondary patency for the same time frames was 88, 73 and 61%. There were no occlusions as a result of intimal hyperplasia. Dilatation of the wall was diagnosed in one prosthesis at 3.5 years, and one at 7 years, and a false aneurysm of unknown aetiology occurred in another at 3 months. There were no primary graft infections.Conclusion: This evaluation shows the chemically treated bovine ureter (CTBU) is a viable medium-term alternative to the currently available artificial vascular prostheses for peripheral revascularisation. [Copyright &y& Elsevier]

Although the post-mortem diagnosis of bovine tuberculosis is mainly achieved through microbiological culture, the development of other techniques to detect Mycobacterium tuberculosis complex (MTBC) members directly from tissue samples has been pursued. The present study describes the development, optimization and validation of a Real-Time PCR based on the mpb70 gene to detect MTBC members in clinical tissue samples from cattle. Specific primers and a hybridization probe were used to amplify MTBC-specific sequences in order to avoid cross-reaction with non-MTBC species. An Internal Amplification Control (IAC) was included in order to assess the presence of PCR inhibitors in the samples. The PCR was optimized to achieve maximum efficiency, and the limit of detection, limit of quantification and dynamic range of the reaction were determined. The specificity of the reaction was tested against 34 mycobacterial and non-mycobacterial species. The diagnostic sensitivity, specificity and positive and negative predictive values (PPV and NPV) of the method were assessed on 200 bovine tissue samples in relation to bacteriological culture. The dynamic range of the reaction spanned from 5 ng/reaction (10 6 genome equivalents) to 50 fg/reaction (10 genome equivalents). The efficiency of the reaction was 102.6% and the achieved R 2 was 0.999. The limit of detection with 95% confidence was 10 genome equivalents/reaction. No cross-reactions with non-MTBC species were observed. The diagnostic sensitivity and specificity values of the mpb70 specific Real-Time PCR respect to culture were 94.59% (95% CI: 86.73-98.51%) and 96.03% (95% CI: 90.98-98.70%), respectively, with a PPV of 93.33% (95% CI: 85.55-97.07%) and a NPV of 96.80% (95% CI: 92.10-98.74%). The concordance of the Real-Time PCR based on mpb70 is comparable to that of culture (K = 0.904) showing a great potential for the detection of members of the MTBC in animal tissues.