Gas chromatography-mass spectrometry-based quantitative method using tert-butyldimethylsilyl derivatization for plasma levels of free amino acids and related metabolites in Japanese Black cattle.

The quantification of amino acid and related metabolite levels is important for evaluating amino acid metabolism and function in animals. However, a useful quantitative method is not enough. In this study, we developed and validated tert-butyldimethylsilyl derivatization method using gas chromatography-mass spectrometry to quantify plasma levels of free amino acids and related metabolites in Japanese Black cattle. Of the 51 metabolites examined, 24, including 20 amino acids, one amine, and three keto acids, could be quantified. Compared with the trimethylsilyl derivatization method using gas chromatography-mass spectrometry, which has been used for untargeted metabolomic analysis, the present method had higher analytical reliability. This method is advantageous for assessing branched-chain amino acid (BCAA) metabolism because it enables the quantification of not only BCAA levels (valine, leucine, and isoleucine) but also their bioactive metabolite keto acid levels (2-ketoisovaleric acid, 2-ketoisocaproic acid, and 2-keto-3-methylvaleric acid) in the plasma. In addition, this method can quantify the plasma levels of not only tryptophan but also its bioactive metabolites kynurenine and serotonin. These results suggest that this quantitative method has the potential to further our understanding of amino acid metabolic processes and their functions in Japanese Black cattle.

Ocimum basilicum essential oil in pacu Piaractus mesopotamicus: anesthetic efficacy, distribution, and depletion in different tissues.

This study aimed to evaluate the anesthetic activity of Ocimum basilicum essential oil and the distribution and depletion of its major compounds in different tissues of the pacu, Piaractus mesopotamicus. Juveniles (319.08 ± 9.14 g) were individually anesthetized with six concentrations of essential oil from O. basilicum (150, 180, 210, 240, 270, and 300 mg L-1), while in a second experiment, fish (492.39 ± 51.51 g) were subjected to a 10 min immersion bath with essential oil from O. basilicum (300 mg L-1). After anesthetic recovery, blood and tissue samples of the brain, gills, liver, spleen, and white muscle were collected at 0, 0.5, 1.0, 3.0, 6.0, 12.0, and 24 h. A 300 mg L-1 concentration induced anesthesia in the shortest time (193.11 ± 9.31), while at 270 and 300 mg L-1 concentrations, the anesthetic recovery period was the longest (244.33 ± 12.44) Methyl chavicol and linalool were quantified in all tissue samples. The plasma concentrations of methyl chavicol differed (p < 0.05) at all evaluated times. Linalool decreased (p < 0.05) from 0 to 1 h and decreased again only after 12 h. Reduction percentages in 24 h were 92.9% for methyl chavicol, and 97.2% for linalool. Elimination of the compounds methyl chavicol and linalool is slower in the gills, where lower elimination constants (0.03 and 0.15 per h) and longer half-lives (25.84 and 4.53 h), respectively, are noted. In general, essential oil from O. basilicum compounds was readily eliminated, showing promising potential for use as an anesthetic in aquaculture.

Unraveling the immune and metabolic changes associated with metritis in dairy cows.

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1ß. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

Gas chromatography-mass spectrometry-based untargeted metabolomics analysis reveals circulating biomarkers related to wooden breast myopathy in broilers: a preliminary study.

Approaches for the diagnosis of wooden breast (WB) myopathy in live birds are urgently required before applying intervention strategies to reduce occurrence and severity for the poultry industry. The objective of this study was to characterize the serum metabolic profiles in male broilers affected by WB and to identify biomarkers related to this myopathy. Broilers were categorized into normal (CON) and WB groups based on gross scoring and histological evaluation. Gas chromatography-mass spectrometry-based metabolomics, multivariate analysis, and orthogonal partial least squares discriminant analysis revealed a clear separation between CON and WB. A total of 73 significantly different (P < 0.05) metabolites with 17 upregulated and 56 downregulated were identified, which were mainly involved in pathways of alanine, aspartate, and glutamate metabolism, carbohydrate metabolism, and taurine and hypotaurine metabolism. By using the nested cross-validation function of random forest analysis, 9 significantly altered (P < 0.05) metabolites (cerotinic acid, arabitol, phosphoenolpyruvate, terephthalic acid, cis-gondoic acid, N-acetyl-d-glucosamine, 4-hydroxymandelic acid, caffeine, and xanthurenic acid) were identified as biomarkers with an excellent discriminant performance for WB myopathy. Collectively, this study provides new insights for a deeper understanding of the pathogenesis and provides metabolites as biomarkers for diagnostic utilization of WB myopathy.

Insecticidal activity of essential oil of Cannabis sativa against the immature and adult stages of Ctenocephalides felis felis.

Essential oil (EO) of Cannabis sativa (C. sativa) was evaluated against the egg, larval, pupal, and adult stages of the flea Ctenocephalides felis felis. The chemical composition of EO was determined by gas chromatography with flame ionization and mass spectrometry. EO mainly comprised γ-elemene (16.2%) and caryophyllene oxide (14.2%) as major compounds. To evaluate the mortality of flea stages in vitro, filter paper tests were performed at different concentrations. EO of C. sativa showed insecticidal activity (100% mortality at the highest concentrations) for flea control at egg, larval, pupal, and adult stages, with lethal concentrations (LC50) of 32.45; 91.61; 466.41 and 927.92 µg/cm2, respectively. EO of C. sativa indicated the potential for the development of ectoparasiticide for veterinary use, especially for fleas in egg and larval stages.

TECHNICAL NOTE: Analysis of volatile fatty acids in rumen fluid by gas chromatography mass spectrometry using a dimethyl carbonate extraction.

Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ionization detector (FID). Aqueous samples are highly problematic because of the large vapor volume that can lead to poor peak shape and contamination of inlets, potentially causing sample carryover. Methods using aqueous samples are not well suited for use in a mass spectrometer (MS) detector system. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (500 g/L), and 2-ethylbutyrate (internal standard; 8.5 mM) were added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture was thoroughly vortexed and centrifuged. The organic layer (top) was removed and placed in a GC vial. The DMC extract was injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105 °C for 5 min, increased at 10 °C/min to 150 °C, then 65 °C/min to 240 °C, and held constant for 10 min. The peak area of acetate relative to the internal standard is linear from approximately 2 mM to at least 130 mM and encompasses the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.10) or the amount of VFA spiked (P > 0.19) for acetate, propionate, isobutyrate, or butyrate. There was an interaction of amount of VFA spiked and the diet of the animal (P = 0.021) for valerate and a tendency for an interaction (P = 0.051) for isovalerate, due to the recovery of the VFA being lower in the medium spike amount in rumen fluid from cattle on wheat pasture. Overall, recovery was greatest for propionate (101.9 ± 1.67%) and lowest for valerate (95.7 ± 1.95%). Including the 10-min hold at 240 °C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantifies rumen fluid VFA.

Scientists need to measure the end-products of microbial digestion called volatile fatty acids in the gastrointestinal tract of ruminants for many reasons. The methods currently available for the analysis of volatile fatty acids have evolved over time along with new technology. Many of the methods available are either not compatible with a mass spectrometer detector or require hazardous chemicals to prepare the samples. The method described here uses a less-hazardous chemical to prepare the samples for analysis with a mass spectrometer detector. The method tested in this manuscript was adequate in preparing samples for volatile fatty acid analysis.

Control of felinine-derived malodor in cat litter.

OBJECTIVES: Malodors stemming from soiled cat litter are a major frustration for cat owners, despite the widespread use of absorbent litters with claims of odor control. Technologies for effective litter odor control have not been rigorously evaluated. Here, we report on the effectiveness of a novel litter formulation of 1-monochlorodimethylhydantoin (MCDMH)-modified clinoptilolite zeolite (MCDMH-Z) to control the odors of 3-mercapto-3-methylbutanol (3M3MB) and ammonia, the principal products generated by the enzymatic breakdown of felinine and urea, respectively. METHODS: The efficacy of MCDMH-Z for the odor control of 3M3MB was determined by solid-phase microextraction and gas chromatography mass spectrometry analysis, colorimetric analysis and a sensory panel. Enzyme inhibition was monitored by a colorimetric coupled assay for ammonia. The antimicrobial properties were measured by a reduction in colony-forming units (CFUs). RESULTS: 3M3MB proved highly susceptible to modification by MCDMH-Z granules. Headspace above litter exposed to MCDMH-Z showed no detectable 3M3MB; levels >59 ng were detected in commercially available products. Urease activity decreased by >97% after incubation with MCDMH-Z to 0.14 mg/ml. Cat litter F showed comparable inhibition (0.13 mg/ml); others showed less inhibition, producing up to 4.8 mg/ml of ammonia. MCDMH-Z reduced the CFUs of Proteus vulgaris by six log reduction values in 30 mins; in the same amount of time, no reduction was seen with commercial products tested. The odor control capability of the MCDMH-Z granules was further supported by a sensory panel scoring 3M3MB-spiked litters. CONCLUSIONS AND RELEVANCE: Samples of commercially available litter products showed an effect on malodor, or inhibition of urease, or contained antimicrobial activity; no samples were capable of accomplishing these concurrently. In contrast, MCDMH-Z granules were effective in all three test categories. Control of felinine-derived odors, in particular, has the potential to improve cat owner satisfaction, and may beneficially affect cat behaviors provoked by pheromonally active sulfurous metabolites deposited in the litter.

Fatty acid profiles of milk from Holstein cows, Jersey cows, buffalos, yaks, humans, goats, camels, and donkeys based on gas chromatography-mass spectrometry.

Due to the diversity and limitation of determination methods, published data on the fatty acid (FA) compositions of different milk samples have contributed to inaccurate comparisons. In this study, we developed a high-throughput gas chromatography-mass spectrometry method to determinate milk FA, and the proposed method had satisfactory linearity, sensitivity, accuracy, and precision. We also analyzed the FA compositions of 237 milk samples from Holstein cows, Jersey cows, buffalos, yaks, humans, goats, donkeys, and camels. Holstein, Jersey, goat, and buffalo milks contained high content of even-chain saturated FA, whereas goat milk had higher content of medium- and short-chain FA (MSCFA). Yak and camel milk are potential functional foods due to their high levels of odd- and branched-chain FA and low ratios of n-6 to n-3 polyunsaturated FA (PUFA). Human milk contained lower levels of saturated FA, MSCFA, and conjugated linoleic acid, and higher levels of monounsaturated FA and PUFA. As a special nonruminant milk, donkey milk contained low levels of monounsaturated FA and high levels of PUFA and MSCFA. Based on the FA profiles of 8 types of milk, nonruminant milk was distinct from ruminant milk, whereas camel and yak milk were different from other ruminant milks and considered as potential functional foods for balanced human diet.

Efficacy and residual effect of Illicium verum (star anise) and Pelargonium graveolens (rose geranium) essential oil on cat fleas Ctenocephalides felis felis.

The essential oils (EOs) of Illicium verum and Pelargonium graveolens were evaluated for lethality, inhibition of development and residual efficacy against the flea Ctenocephalides felis felis. Their chemical composition was characterized by means of gas chromatography with a flame ionization and mass spectrometry detection. Mortality at different immature stages and among adult fleas was measured through in vitro filter paper tests at different concentrations of EOs. The chemical characterization of I. verum volatile oil showed that E-anethole (79.96%) was the major constituent, while the major compounds in P. graveolens were citronellol (29.67%) and geraniol (14.85%). Insecticidal activity against both immature and adult flea stages were observed. The EO of I. verum had insecticidal activity for approximately 18 days, while the EO activity of P. graveolens lasted for 13 days. The pulicidal activity of I. verum remained above 70% for up to 9 days, while the activity of P. graveolens was 41.7% for up to 2 days. Essential oils, especially that of I. verum, showed insecticidal activity for flea control at different life cycle stages and have potential for the development of ectoparasiticides (biopesticides) for veterinary use.

Fish oil rich in eicosapentaenoic acid and docosahexaenoic acid in sow diets modifies oxylipins and immune indicators in colostrum and milk.

Colostrum and milk are the first nutrient sources for newborn piglets. In addition, n-3 fatty acids (FAs) and their oxygenated derivatives (oxylipins) have the capacity to modulate immune components. The aim of the current study was to include a fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in sow diets to promote an increase of anti-inflammatory molecules in colostrum and milk to benefit piglets. Thirty-six sows were randomly assigned from insemination to the end of lactation to either a control diet with animal fat (15 g/kg in gestation and 30 g/kg in lactation) or an n-3 diet in which animal fat was totally (gestation) or half (lactation) replaced by an equivalent amount of solid fish oil. Performance of sows and piglets was monitored during the study. Colostrum and milk samples were obtained after the birth of the first piglet and at weaning, respectively. From all samples (n = 18 per treatment), FAs were quantified by gas chromatography and immunoglobulins and cytokines by ELISA. Three samples per treatment were randomly selected to analyse oxylipin composition by liquid chromatography-tandem mass spectrometry. In colostrum and in milk, the n-3 FA (P = 0.020 and P < 0.001), particularly EPA (P < 0.001 and P < 0.001) and DHA (P < 0.001 and P < 0.001), and also their oxygenated derivatives were increased in samples from sows fed n-3 diet. Fish oil had no effect on immunoglobulin concentrations, but reduced tumour necrosis factor α (TNFα) (P = 0.011) and a tendency to reduce interleukin 10 (IL10) (P = 0.059) were observed in milk. In conclusion, fish oil in sow diets increased n-3 FA, particularly EPA and DHA, and their oxygenated derivatives in colostrum and milk, reducing TNFα and IL10 in milk.

Atrazine intoxication in cattle, confirmed by gas chromatography-mass spectrometry.

Ten of 40 cows died within 48 h of gaining access to a barn in which various chemicals were stored. Some of the surviving cows exhibited drooling, muscle tremors, and agitation. Postmortem examinations of 2 cows were performed in the field, and revealed nonspecific, moderate-to-severe pulmonary congestion. Liver and rumen contents, each from a different cow, were analyzed using a qualitative, multi-residue GC-MS method validated for the detection of pesticides and other chemical analytes. Using this method, extracts from the liver and rumen content samples were compared to atrazine (neat standard) and matrix-matched, control samples fortified with atrazine. GC-MS analysis detected atrazine at 215 m/z (NIST match >97%) with a retention time of ~13 min in liver and rumen content samples from our case. Detection of atrazine in the samples from the cows in this herd, combined with the clinical history, indicate that atrazine toxicity was the likely cause of clinical signs and death observed in this herd.

Metabolic footprint analysis of volatile metabolites by gas chromatography-ion mobility spectrometry to discriminate between different fermentation temperatures during Streptococcus thermophilus milk fermentation.

Streptococcus thermophilus is widely used in the dairy industry to produce fermented milk. Gas chromatography-ion mobility spectrometry-based metabolomics was used to discriminate different fermentation temperatures (37°C and 42°C) at 3 time points (F0: pH = 6.50 ± 0.02; F1: pH = 5.20 ± 0.02; F2: pH = 4.60 ± 0.02) during S. thermophilus milk fermentation, and differences of fermentation physical properties and growth curves were also evaluated. Fermentation was completed (pH 4.60) after 6 h at 42°C and after 8 h at 37°C; there were no significant differences in viable cell counts and titratable acidity; water-holding capacity and viscosity were higher at 37°C than at 42°C. Different fermentation temperatures affected volatile metabolic profiles. After the fermentation was completed, the volatile metabolites that could be used to distinguish the fermentation temperature were hexanal, butyraldehyde, ethyl acetate, ethanol, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropionic acid. Specifically, at 37°C of milk fermentation, branched-chain AA had higher levels, and leucine, isoleucine, and valine were involved in growth and metabolism, which promoted accumulation of some short-chain fatty acids such as 3-methylbutanoic acid and 2-methylpanprooic acid. At 42°C, at 3 different time points during fermentation, ethanol from glycolysis all presented higher levels, including acetone and 3-methylbutanal, producing a more pleasant flavor in the fermented milk. This work provides detailed insight into S. thermophilus fermented milk metabolites that differed between incubation temperatures; these data can be used for understanding and eventually predicting metabolic changes during milk fermentation.

Efficacy and residual effect of Illicium verum (star anise) and Pelargonium graveolens (rose geranium) essential oil on cat fleas Ctenocephalides felis felis / Eficácia e efeito residual dos óleos essenciais de Illicium verum (anis-estrelado) e Pelargonium graveolens (gerânio rosa) em pulgas de gato Ctenocephalides felis felis

Abstract The essential oils (EOs) of Illicium verum and Pelargonium graveolens were evaluated for lethality, inhibition of development and residual efficacy against the flea Ctenocephalides felis felis. Their chemical composition was characterized by means of gas chromatography with a flame ionization and mass spectrometry detection. Mortality at different immature stages and among adult fleas was measured through in vitro filter paper tests at different concentrations of EOs. The chemical characterization of I. verum volatile oil showed that E-anethole (79.96%) was the major constituent, while the major compounds in P. graveolens were citronellol (29.67%) and geraniol (14.85%). Insecticidal activity against both immature and adult flea stages were observed. The EO of I. verum had insecticidal activity for approximately 18 days, while the EO activity of P. graveolens lasted for 13 days. The pulicidal activity of I. verum remained above 70% for up to 9 days, while the activity of P. graveolens was 41.7% for up to 2 days. Essential oils, especially that of I. verum, showed insecticidal activity for flea control at different life cycle stages and have potential for the development of ectoparasiticides (biopesticides) for veterinary use.

Resumo Os óleos essenciais (OE) de Illicium verum e Pelargonium graveolens foram avaliados quanto à letalidade, inibição do desenvolvimento e eficácia residual contra a pulga Ctenocephalides felis felis. Sua composição química foi caracterizada por meio de cromatografia gasosa com detector de ionização de chama e espectrometria de massas. A mortalidade entre os diferentes estágios imaturos e pulgas adultas foi avaliada por meio de testes in vitro em papel filtro, contendo diferentes concentrações de OEs. A caracterização química do óleo volátil de I. verum mostrou que o E-anetol (79,96%) foi o constituinte majoritário, enquanto os principais compostos de P. graveolens foram citronelol (29,67%) e geraniol (14,85%). Foi observada atividade inseticida contra os estágios imaturos e adulto da pulga. O OE de I. verum teve atividade inseticida por aproximadamente 18 dias, enquanto o de P. graveolens durou 13 dias. A atividade pulicida de I. verum permaneceu acima de 70% até o 9º dia, enquanto a atividade de P. graveolens foi de 41,7% até o 2º dia. Os óleos essenciais, principalmente de I. verum, apresentam atividade inseticida para o controle de pulgas em diferentes estágios do ciclo de vida e têm potencial para o desenvolvimento de ectoparasiticidas (biopesticidas) de uso veterinário.

Comparative analysis of characteristic volatile compounds in Chinese traditional smoked chicken (specialty poultry products) from different regions by headspace-gas chromatography-ion mobility spectrometry.

This article presents investigation of the flavor profile on 5 different regional Chinese smoked chicken samples using gas chromatography-ion mobility spectrometry analysis methods. Five batches of samples were obtained over the course of 6 mo. A total of 34 flavor substances were identified in the 5 smoked chicken samples, including 10 aldehydes, 7 alcohols, 4 ketones, 2 hydrocarbons, 3 heterocyclic compounds, 4 esters, 2 ethers, and 2 phenolic compounds. The whole spectral fingerprint visually displayed flavor differences and relations in 5 types of smoked chicken with local characteristics. Moreover, the orthogonal projections to latent structures discriminant analysis model revealed that these samples could be separately classified into 5 groups. Multivariate statistical analysis showed that 20 chemicals with higher Variable Importance for the Projection values were the key contributors to the differences of flavor in these 5 kinds of smoked chicken. N-nonanal, heptanal, n-nonanal, heptanal, furfurol, and hexanal were the main common flavor compounds in the 5 types of Chinese smoked chicken, whereas linalool, alpha-terpineol, 1,8-cineole, and anethole were the main characteristic flavor compounds of Goubangzi chicken (No. 1); gamma-butyrolactone, 2-acetylfuran, 2-methoxyphenol, 2-acetylpyrrole, and limonene were determined as the key flavor compounds of Liaocheng chicken (No. 2); the concentration of octanal and n-nonanal was higher in Tangqiao chicken (No. 3); butyl acetate was the key contributor to the flavor compounds of Jinshan chicken (No. 4). 2-Heptanone and 2-pentylfuran had a high correlation with Zhuozishan chicken (No. 5). The different raw materials and ingredients used, especially the method of preparation and cultural differences, in different regions of the country in China were the main reasons leading to the similarities and differences of volatile compounds in the 5 kinds of Chinese traditional smoked chicken.

Rhipicephalus microplus (acari: Ixodidae): Clinical safety and potential control by topical application of cottonseed oil (Gossypium sp.) on cattle.

The present study was performed to determine the acaricidal activity of the cottonseed oil (CSO) against cattle tick Rhipicephalus microplus. CSO was analyzed using Gas Chromatograph with high-resolution Mass Spectrometer (GC-HRMS) to identify the presence of active compounds. In vitro bioassays were performed using larval packet test (LPT) and adult immersion test (AIT) by taking different concentrations of CSO (i.e. 0.1, 0.5, 1.5, 2.5, 5, 7.5, 10 and 12.5%). In vivo acaricidal activity of CSO was evaluated by its topical application on red Sahiwal calves for 144 h. Clinical safety of CSO was evaluated by performing skin irritancy test and examination of hematological profile of calves'. GC-HRMS analysis of CSO revealed the presence of many fatty acids including oleic acid, lauric acid, palmitic acid, stearic acid and other components. Results exhibited that all the concentrations of CSO were effective in reducing the number of ticks and their growth. However, CSO at concentrations of 10% (CSO7) and 12.5% (CSO8) exhibited 100% mortality of R. microplus larvae and adults in LPT and AIT, respectively. In vivo acaricidal assay revealed that CSO7 and CSO8 shown 85% and 89% inhibition of ticks, respectively on calves after 144 h as compared to the control group. CSO was clinically safe on calves' skin with mild erythema up to 20 min. Hematological profile of calves revealed no sign of toxicity after treatment with CSO. Thus, CSO can be used as an alternative and safe drug therapy against R. microplus.

Assessment of urinary 15-F2 -isoprostanes in dogs with urothelial carcinoma of the urinary bladder and other lower urinary tract diseases.

BACKGROUND: The 15-F2 -isoprostanes are by-products of oxidative stress and are increased in the urine of people with lower urinary tract diseases (LUTD), especially urinary neoplasia. Urothelial carcinoma (UC) is the most common urinary neoplasm in dogs. Earlier detection of UC by noninvasive means could lead to improved outcomes. Urinary 15-F2 -isoprostanes potentially could provide this means, but have not been evaluated in dogs with UC. OBJECTIVE: The objective of this study was to measure urinary 15-F2 -isoprostanes in dogs with UC and dogs with other LUTD. ANIMALS: One hundred seventeen dogs: 46 dogs with UC, 30 dogs with LUTD, and 25 control dogs. METHODS: Any dog that was presented with dysuria was eligible for inclusion. Diagnosis of UC was confirmed histologically. Urinalysis was performed in each case, and 15-F2 -isoprostanes quantified by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) and normalized to urinary creatinine concentration. RESULTS: Dogs with urinary diseases (UC + LUTD) had higher median urinary 15-F2 -isoprostanes when compared to control dogs (5.92 ng/mg [range, 0.46-31.03] vs 3.73 [range, 1.8-7.98]; P = .02). Urinary 15-F2 -isoprostanes were similar in dogs with UC (5.33 ng/mg [range, 0.46-31.03]) compared to dogs with LUTD (6.29 ng/mg [range, 0.54-18.93]; P = .47) and control dogs (P = .06). Dogs with UC had higher qualitative measures of proteinuria (P = .004), hematuria (P = .01), and epithelial cells on urinalysis (P = .002) compared to the other groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary F2 -isoprostanes are not useful for the detection of UC in dogs. Future research could evaluate urinary 15-F2 -isoprostanes as a marker of inflammation in disease progression and prognosis.

Metabolomics in the study of spontaneous animal diseases.

Using analytical chemistry techniques such as nuclear magnetic resonance (NMR) spectroscopy and liquid or gas chromatography-mass spectrometry (LC/GC-MS), metabolomics allows detection of most endogenous and exogenous metabolites in a biological sample. Metabolomics has a wide range of applications, and has been employed in nutrition science, toxicology, environmental studies, and systems biology. Metabolomics is particularly useful in biomedical science, and has been used for diagnostic laboratory testing, identifying targets for drug development, and monitoring drug metabolism, mode of action, and toxicity. Despite its immense potential, metabolomics remains underutilized in the study of spontaneous animal diseases. Our aim was to comprehensively review the existing literature on the use of metabolomics in spontaneous veterinary diseases. Three databases were used to find journal articles that applied metabolomics in veterinary medicine. A screening process was then conducted to eliminate references that did not meet the eligibility criteria; only primary research studies investigating spontaneous animal disease were included; 38 studies met the inclusion criteria. The main techniques used were NMR and MS. All studies detected metabolite alterations in diseased animals compared with non-diseased animals. Metabolomics was mainly used to study diseases of the digestive, reproductive, and musculoskeletal systems. Inflammatory conditions made up the largest proportion of studies when articles were categorized by disease process. Following a comprehensive analysis of the literature on metabolomics in spontaneous veterinary diseases, we concluded that metabolomics, although in its early stages in veterinary research, is a promising tool regarding diagnosis, biomarker discovery, and in uncovering new insights into disease pathophysiology.

Feline urinary F2-isoprostanes measured by enzyme-linked immunoassay and gas chromatography-mass spectroscopy are poorly correlated.

15-F2T-isoprostanes are byproducts of lipid peroxidation and were determined to be the best marker of oxidative injury in a rodent model of oxidative stress. A previous study compared methods for measurement of urinary F2-isoprostanes (gas chromatography and negative ion chemical ionization-mass spectrometry, GC-NICI-MS; and ELISA) and found poor agreement in dogs, horses, and cows. Surprisingly, fair agreement between these methods was identified in a small population of cats. We evaluated the agreement between GC-NICI-MS and ELISA of urinary F2-isoprostanes in the urine of 50 mature cats ranging from healthy to systemically ill. All urine samples had detectable levels of F2-isoprostanes by both methods. Significant proportional bias and poor agreement were identified between the 2 methods (ρ = 0.364, p = 0.009) for all cats, and in subgroup analysis based on health status. The concentration of urinary F2-isoprostanes was significantly lower in systemically ill cats compared to healthy cats when measured by ELISA (p = 0.002) but not by GC-NICI-MS (p = 0.068). Our results indicate that GC-NICI-MS and ELISA have poor agreement when measuring urinary F2-isoprostanes in cats.

Quantification and validation of nine nonsteroidal anti-inflammatory drugs in equine urine using gas chromatography-mass spectrometry for doping control.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine in accordance with the International Screening Limits (ISL) regulated by International Federation of Horseracing Authorities. After basic hydrolysis, samples were extracted with a C18 cartridge using automated solid-phase extraction. Several derivatization reagents were investigated, and trimethylphenylammonium hydroxide/methanol (20/80, v/v) was selected. Analyses were carried out using gas chromatography-mass spectrometry with selected ion monitoring mode, but the method can be applied to a large number of analytes. The within-laboratory reproducibility was not more than 12.8% (≤15%), and mean relative recoveries ranged from 91.1% to 104.1% for inter-day and intra-day precision. The decision limits (CCα) and detection capabilities (CCß) were evaluated at concentrations near the ISL for each therapeutic substance. The validation results demonstrated that the method is highly reproducible, easily applicable, and suitable for the analysis of some NSAIDs in equine urine that have not been previously published. Finally, the method was also applied to known positive samples.

Secretome of the carcinogenic helminth Spirocerca lupi reveals specific parasite proteins associated with its different life stages.

Spirocerca lupi is a parasitic and carcinogenic nematode of canids distributed in tropical and subtropical regions around the world. The excretion-secretion proteins (PES) of S. lupi have been suggested to play a role in the pathogenesis of its infection. We aimed to identify the PES of different stages of S. lupi and search for proteins that would be useful for diagnostic, therapeutic and vaccination purposes as well as understand their functions. A nano-UPLC mass spectrometry de novo analysis was performed on proteins collected from cultures of S. lupi L3 larvae, L4 females, adult females and adult males from naturally infected hosts. A total of 211 proteins were identified in all cultures. Accordingly, 117, 130, 99 and 116 proteins were detected in L3 larva, L4 females, adult females and adult males, respectively, with a strong correlation in the biological replicates (Pearson coefficients > 0.73). Fourty-four proteins were detected in all developmental stages, 64 were stage-specific and 49 were exclusively identified in L4 females. Cell compartment enrichment analysis revealed that proteins common to all stages were cytoplasmatic (p < 9.x10-6), whereas L4 unique proteins were in collagen trimers, and macromolecular complexes (p < 0.00001). Functional enrichment analysis of proteins showed significant enrichment in lipid metabolism in L3-unique proteins (p<0.00005), in mannose metabolism and protein de-glycosylation for L4-unique proteins (p < 0.00004), and in phosphorus metabolism in proteins shared by all stages (p <  2.1 x10-9). Interestingly, annexin 6, associated with cancer in humans, was detected in all life stages, but in a larger abundance in L4 females and adults. These findings indicate that S. lupi establishes complex interactions with its hosts by an arsenal of proteins expressed in different patterns in each life stage which influence the pathogenesis and oncogenesis of S. lupi and may be used as potential targets for diagnostic assays, drug targets or vaccine candidates.