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1.
Transl Anim Sci ; 7(1): txad111, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37841323

RESUMO

Benzoic acid is a common alternative for antibiotic and zinc oxide use in nursery diets. Free benzoic acid (BZA) is often supplied, but this form is absorbed before it can exert any effect on distal segments of the gut. The study aimed to evaluate the effects of protected benzoic acid on growth performance, nutrient digestibility, plasma metabolites, and gut health indices in starter pigs. A total of 192 pigs were weaned at 28 ± 1 d age (initial body weight, 8.72 ± 1.13 kg). Pens were assigned to one of four treatment diets (n = 8 pens per treatment): (1) no additive (NC), (2) free benzoic acid (BZA; 0.6%), (3) protected benzoic acid (BC50; 0.2%, supplied at a ratio of one to three equivalents of BZA), and (4) antibiotic growth promoter (AGP; Carbadox, 50 ppm). Diets were fed for three weeks over two periods (period 1, 7 d; period 2, 14 d). Body weight and feed intake were measured for each period. Feces were collected at the end of each period to determine apparent total tract digestibility (ATTD) of organic matter (OM), gross energy (GE), and crude protein (CP). One pig per pen was euthanized per period to determine plasma metabolites; jejunum and ileum morphology; jejunum, ileum, and colon cytokine abundance; and jejunum, ileum, and colon tight junction protein expression. The AGP group had increased average daily gain (ADG) and average daily feed intake (ADFI) compared to other groups in period 1 and overall (P < 0.05); however, ADG and ADFI of the BC50 group was intermediate between the NC and BZA groups and the AGP group in period 2. The ATTD of OM, GE, and CP were greater in the AGP group compared to the NC and BC50 groups (P < 0.05), whereas the BZA group was intermediate. Jejunum and ileum villus height and crypt depth increased from period 1 to period 2 (P < 0.01) but were similar across groups. Ileum and colon tumor necrosis factor-α (TNF-α) abundances were greater, whereas colon interleukin (IL)-1ß and colon and ileum IL-8 abundances were less, in the AGP group compared to the BZA group (P < 0.05); the NC and BC50 groups exhibited intermediate TNF-α, IL-1ß, and IL-8 abundance in the ileum and colon. Jejunum cytokine abundance did not vary among groups but declined from period 1 to period 2 (P < 0.05). Tight junction protein expression also did not vary among groups. In summary, protected BZA supported a slight increase in growth performance in starter pigs, suggesting its potential as an alternative feed additive in nursery diets.

2.
J Anim Sci ; 1012023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36630697

RESUMO

Sulfur amino acid nutrition and metabolism are linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), γ-glutamylcysteine (γ-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 µm, 2.1 mm × 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15-1,500 nmol/g for Cys, CysGly, Hcys, and γ-GluCys and 150-15,000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r2 > 0.99) and excellent recovery of amino thiols from spiked samples (mean ± SD across tissues; Cys, 100.0 ± 2.2%; CysGly, 95.4 ± 5.1%; Hcys, 96.6 ± 2.0%; γ-GluCys, 102.2 ± 2.7%; and GSH, 100.6 ± 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for 1 wk after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that despite elevated Cys and maintained GSH concentrations in liver, both γ-GluCys and GSH decline in jejunum of weaned pigs.


The synthesis of glutathione, a major intracellular antioxidant, in animal tissue accounts for a considerable fraction of the intake of the sulfur amino acids methionine and cysteine. Animal scientists accordingly need methods suitable for measuring the abundance of metabolites related to sulfur amino acid metabolism in solid tissue. However, methods currently available are either validated for measuring these metabolites in plasma, serum, or urine, do not fully describe all procedures needed to prepare tissue samples for analysis, or are validated for measuring only cysteine and glutathione in tissue. The focus of this work was to describe the sample preparation and analysis methods needed to measure these metabolites in solid tissue. Sample preparation time is less than 2 h and sample analysis time is less than 5 min. The method is robust and reproducible and is applied to identify weaning-induced differences in sulfur amino acid metabolism in liver and small intestine in pigs. The method will also help evaluate the impact of diet, stress, or inflammation on cysteine and glutathione metabolism on a tissue-by-tissue basis to help optimize levels of sulfur amino acids in swine diets.


Assuntos
Cisteína , Compostos de Sulfidrila , Animais , Suínos , Cromatografia Líquida de Alta Pressão/veterinária , Espectrometria de Massas/veterinária , Tiopronina , Glutationa/análise
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