Pollen beetle (Astylus atromaculatus)-associated gastroenteric disease in cattle: report of 6 natural outbreaks.

Astylus atromaculatus is a pollen beetle native to South America, commonly found in crop flowers. Experimental intoxication of sheep and guinea pigs by this beetle resulting in fibrinonecrotizing enteritis has been reported. We describe here 6 natural outbreaks of intoxication in cattle associated with consumption of alfalfa (5 of 6) and mixed native (1 of 6) pastures heavily contaminated with A. atromaculatus. The outbreaks occurred during the summer (January-February) of 2023 in Argentina (n = 4) and Uruguay (n = 2), in beef cattle under extensive or semi-extensive rearing systems, with overall cumulative incidence and mortality of 22.3% and 17.8%, respectively. The main clinical signs included acute onset of anorexia, lethargy, hyperthermia, hindlimb weakness, reluctance to move, and diarrhea, for up to 15 d. In 2 outbreaks, sudden death was observed. Eight Hereford, Angus, and/or crossbreed heifers, cows, steers, and/or calves were autopsied. Gross and microscopic findings included multifocal necrosis with fibrinous pseudomembranes in the forestomachs and/or small and large intestines. Fragments or whole specimens of A. atromaculatus were identified in the ruminal content of all animals. Testing for multiple gastroenteric pathogens was negative as was testing of A. atromaculatus for cantharidin and batrachotoxin. GC-MS and LC-MS/MS performed on the beetles did not identify any known toxic compounds. Based on the exposure to A. atromaculatus-contaminated pasture, gross and microscopic lesions, and negative results of all testing for multiple gastroenteric pathogens, a diagnosis of intoxication by A. atromaculatus is proposed. Disease caused by A. atromaculatus consumption has not been reported previously in cattle, to our knowledge.

Yellow oleander (Thevetia peruviana) toxicosis in 4 goats.

Four alpine goats developed diarrhea soon after the owner placed plant clippings believed to be yellow oleander (Thevetia peruviana) into their pen on a suburban property near Palm Desert, CA, USA. A 1-y-old female goat died suddenly ~1 h after eating the plant clippings and was submitted to the San Bernardino Branch of the California Animal Health and Food Safety Laboratory System for postmortem examination. The main autopsy and histopathologic findings were myocardial hemorrhage and necrosis, consistent with cardiac glycoside intoxication. Rumen contents were analyzed by LC-MS/MS; peruvoside, a cardiac glycoside, was detected, but oleandrin, the cardiac glycoside of common oleander (Nerium oleander), was not. An LC-high-resolution MS (LC-HRMS) analysis revealed the presence of peruvoside and neriifolin in the rumen contents and in a tested plant fragment, indicating that the plant was a member of the Thevetia genus. A clipping from the plant fed to the goats and submitted by the owner was identified as yellow oleander, Thevetia peruviana (also known as Cascabela thevetia).

Effects of extended transition milk feeding on blood metabolites of female Holstein dairy calves at 3 weeks of age: a liquid chromatography with tandem mass spectrometry-based metabolomics approach.

Transition milk (TRM) is a rich source of bioactive components that promotes intestinal development and growth, and reduces the susceptibility to diarrhoea in calves. The objective of this study was to characterise the effects of replacing pasteurised waste milk (none-saleable milk containing antibiotic and/or drug residues) with pasteurised TRM for 3 wk on blood metabolites of dairy calves at 21 d of age. A total of 84 healthy newborn female Holstein calves was blocked by birth order and assigned randomly to four treatment groups with partial replacement of pasteurised waste milk by TRM (second milking after parturition) at 0 (0 L/day TRM + 6 L/day milk), 0.5 (0.5 L/day TRM + 5.5 L/day milk), 1 (1 L/day TRM + 5 L/day milk), or 2 L (2 L/day TRM + 4 L/day milk) for a 21-day period. Serum metabolome was determined by liquid chromatography with tandem mass spectrometry-based metabolomics analysis on a subset of 26 randomly selected individuals from calves fed pasteurised waste milk (CON, 6 L/d milk; n = 13) or TRM (2 L/d TRM + 4 L/d milk; n = 13) at 21 d of age. The identified metabolites (194 out of 265) were categorised according to chemical class and the number of metabolites per class in the serum, amongst which glycerophospholipids 16% (n = 43), fatty acyls 7% (n = 19), organic acids 7% (n = 18), organic heterocyclic compounds 5% (n = 13), benzenoids 5% (n = 12), sphingolipids 5% (n = 12), organic oxygen compounds 4% (n = 11), and nucleic acids 3% (n = 9), were the predominant types. Significant differences in metabolites were determined by the volcano plot. Applying the volcano plot, only two metabolites (ceramide and phosphatidylserine) were significantly different between CON and TRM. Overall, our results suggested that prolonged TRM feeding for 3 wk had little effect on the serum metabolome of the dairy calves. We speculate that the potential effects of feeding TRM for 3 wk compared with waste milk were spatially limited to affect the composition of the local gut microbial community and the growth or function of the intestinal epithelium, not allowing detection of the likely effects in the serum through a metabolomic approach.

Novel holistic pharmacokinetic model applied to plasma and urine concentrations of 2,5-dihydroxybenzene sulphonate following administrations of calcium dobesilate and etamsylate to exercised horses.

Calcium dobesilate (CD) is a synthetic venoactive drug used in veterinary medicine to treat equine navicular disease. Etamsylate is a haemostatic agent used in horses for the treatment of exercise-induced pulmonary haemorrhage. Both etamsylate and CD dissociate in the circulatory system with 2,5-HBSA as the active drug. The aim of the research was to be able to provide detection time (DT) advice from pharmacokinetic (PK) studies in Thoroughbred horses to better inform trainers, and their veterinary surgeons, prescribing these substances for treatment of Thoroughbred racehorses. Two (pilot study) and six (final study) horses were given 28 and 9 repeated dose of CD (3 mg/kg BID) respectively. Two horses were each given a single intravenous (IV) dose of etamsylate (10 mg/kg). Plasma and urine 2,5-HBSA concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CD pilot study revealed that steady state could be reached with a few days and that 2,5-HBSA in plasma and urine shows instability during storage at -20°C but appears stable at -80°C. A novel holistic non-linear mixed-effects three-compartmental PK model was developed that described both plasma and urine concentrations of 2,5-HBSA, from either CD or etamsylate administration. Typical values for 2,5-HBSA clearance and bioavailability were 2.0 mL/min/kg and 28% respectively. Using the parameters obtained from this PK model, in conjunction with methodology developed by Toutain, afforded a possible screening limit (SL) that can regulate for a DT of 3 days in urine; however, a corresponding SL in plasma would be below current levels of detection. However, it is the responsibility of the individual racing authorities to apply their own risk management with regard to SLs and DTs.

Characterization of sow milk N-linked glycoproteome over the course of lactation.

Milk proteins serve as nutrition and affect neonate development and immunity through their bioactivity. Post-translational modifications of proteins affect their bioactivity. Glycosylation is the attachment of sugar moieties to proteins, with attachment of glycans to asparagine indicated as N-linked glycosylation. Our objective was to characterize N-linked glycosylated proteins in homogenate swine milk samples collected from sows (n = 5/6) during farrowing to represent colostrum and on days 3 and 14 post-farrowing to represent transitional and mature milk, respectively. Glycopeptides were isolated with lectin-based extraction and treated with Peptide N-glycosidase F (PNGase F) to identify N-linked glycosylation sites. Purified glycopeptides were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant software was used to align spectra to Sus scrofa Uniport database to identify proteins and measure their relative abundances. Analysis of variance and Welch's t-test analysis identified glycoproteins differentially abundant between colostrum, transitional, and mature milk (false discovery rate <0.05). Shotgun proteome analysis identified 545 N-linked and glutamine, Q, -linked, glycosylation (P > 0.75 for deamidation) sites on 220 glycoproteins in sow milk. Glycoproteins were found across all three phases of swine milk production and varied by number of glycosylation sites (1-14) and in abundance and distribution between colostrum, transitional, and mature milk. Polymeric immunoglobulin receptor was the most glycosylated protein with 14 sites identified. Also highly glycosylated were casein and mucin proteins. These data are described and the relevance of glycosylated milk proteins in neonate development, such as protection against pathogens, is discussed.

Milk is essential for healthy growth and development of neonates, with proteins in milk serving as key nutrients and regulators of these processes. Protein activity is affected by modifications made to their structure including the addition of sugar groups called glycans. Here we present the characterization of sow milk proteins modification with glycan groups on asparagine and glutamine amino acids in colostrum, transitional, and mature milk of pigs. We found 220 high confidences (found in at least two sows on one day) glycoproteins, and that the abundance of glycosylated proteins varied by stage of milk production and number of glycosylated sites.

Carcass traits and meat characteristics of grazing Nellore cattle submitted to different supplementation strategies in the tropics.

This study explored the effects of different supplementation strategies during the dry and rainy seasons in the tropics on the carcass traits and meat quality of Nellore cattle produced under grazing conditions. Additionally, a cost assessment of the supplementation strategies was conducted to define the most suitable ones from an economic standpoint. Twenty-eight non-castrated male animals (18 months) with an initial body weight of 327.9 ± 4.2 kg were used. The animals were equitably distributed in a randomized complete design thorough four supplementation strategies as follows: (i) mineral supplementation (MS) in both dry and rainy seasons (MS/MS), (ii) MS during the dry season and concentrate supplementation (CS) during the rainy season (MS/CS), (iii) CS during the dry season and MS during the rainy season (CS/MS), and (iv) CS in both dry and rainy seasons (CS/CS). Thereafter, carcass traits, primary carcass cut yields, meat quality traits, and chemical composition of the meat of cattle produced across different supplementation strategies were determined. Data revealed that animals under CS/CS showed the greatest (P < 0.01) hot carcass weights among the other supplementation strategies evaluated. Conversely, supplementation strategy did not affect (P > 0.10) the carcass traits (the ribeye area, final pH, and forequarter), meat quality traits (shear force, myofibrillar fragment index, sarcomere length, and color), and meat chemical composition (crude protein, fat, and moisture) of the animals. A cost assessment of the supplementation strategies revealed that CS/CS had the highest production costs. Nevertheless, CS/CS had the greatest income and profit, while MS/MS had the lowest ones. In conclusion, data suggest that cattle grazing on tropical forage under CS during at least one season (i.e., dry or rainy) produce similar meat quality traits and chemical composition of meat to those observed for animals under CS in both seasons. Additionally, the last supplementation strategy revealed the greatest profit indicators among the other explored.

Anticancer activity of an Artemisia annua L. hydroalcoholic extract on canine osteosarcoma cell lines.

Since ancient times, Artemisia annua (A. annua) has been used as a medicinal plant in Traditional Chinese Medicine. In addition, recent studies have investigated the cytotoxic effects of A. annua extracts towards cancer cells. The leading aim of the present research is to evaluate the cytotoxic effects of an hydroalcoholic extract of A. annua on two canine osteosarcoma (OSA) cell lines, OSCA-8 and OSCA-40, focusing on the possible involvement of ferroptosis. The quantitative determination of artemisinin concentration in the extract, culture medium and OSA cells was carried out through the use of an instrumental analytical method based on liquid chromatography coupled with spectrophotometric detection and tandem mass spectrometry (LC-DAD-MS/MS). OSCA-8 and OSCA-40 were exposed to different dilutions of the extract for the EC50 calculation then the uptake of artemisinin by the cells, the effects on the cell cycle, the intracellular iron level, the cellular morphology and the lipid oxidation state were evaluated. A concentration of artemisinin of 63.8 ± 3.4 µg/mL was detected in the extract. A dose-dependent cytotoxic effect was evidenced. In OSCA-40 alterations of the cell cycle and a significantly higher intracellular iron content were observed. In both cell lines the treatment with the extract was associated with lipid peroxidation and with the appearance of a "ballooning" phenotype suggesting the activation of ferroptosis. In conclusion the A. annua idroalcoholic extract utilized in this study showed anticancer activity on canine OSA cell lines that could be useful in treating drug resistant canine OSAs.

Comparison of the improving embryo development effects of Sasa quelpaertensis Nakai extract, p-coumaric acid, and myricetin on porcine oocytes according to their antioxidant capacities.

This study investigated the antioxidant activities of Sasa quelpaertensis Nakai extract (SQE), p-coumaric acid (PCA) and myricetin (MY), and their effects on the in vitro maturation and developmental ability of porcine oocytes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed that 1 mg of SQE contained 3.92 µg of PCA and 0.19 µg of MY. The concentrations required to inhibit 50% of DPPH radicals were 2732.8 ppm, 38.8 mg/mL, and 0.110 mg/mL for SQE, PCA, and MY, respectively. The reducing power increased as the concentration increased, and the reducing power of MY was higher than that of PCA. The polar body extrusion rate was highest upon treatment with 1250 ppm SQE and 10 µM MY. The reactive oxygen species and glutathione levels were significantly decreased and increased, respectively. In a normal or peroxidative environment, the embryo development rate upon parthenogenetic activation was increased, and the total cell number, apoptosis rate, and development-related gene expression were altered to enhance embryonic development. The embryo development rate and total cell number upon somatic cell nuclear transfer did not differ between the groups. These results show that the antioxidant effects of SQE and MY enhance the in vitro maturation and subsequent embryonic development.

Quantitative analysis of differentially expressed milk fat globule membrane proteins between donkey and bovine colostrum based on high-performance liquid chromatography with tandem mass spectrometry proteomics.

This study was designed to provide novel insights into milk fat globule membrane (MFGM) proteins in donkey colostrum (DC) and bovine colostrum (BC) using quantitative proteomics. In total, 179 (DC) and 195 (BC) MFGM proteins were characterized, including 71 shared, 108 DC-specific, and 124 BC-specific proteins. Fifty-one shared proteins were selected as differentially expressed MFGM proteins, including 21 upregulated and 30 downregulated proteins in DC. Gene ontology analysis showed that these proteins were mainly enriched in cellular components, including the extracellular exosome, extracellular space, and plasma membrane. Additionally, they were further involved in metabolic pathways, including cholesterol metabolism, the peroxisome proliferator-activated receptor signaling pathway, and purine metabolism. Furthermore, several key protein factors with high connectivity were identified via protein-protein interaction analysis. These results provide more comprehensive knowledge of differences in the biological properties of MFGM proteins in DC and BC as well as pave the way for future studies of the nutritional and functional requirements of these important ingredients toward the development of dairy products based on multiple milk sources.

Shotgun proteomics of homogenate milk reveals dynamic changes in protein abundances between colostrum, transitional, and mature milk of swine.

Milk is an easily digestible source of nutrients and bioactive factors, its composition reflects the neonate's needs, and changes from colostrum to transitional and mature milk. Our objective was to measure milk fat, lactose, total carbohydrate, and protein content in parallel with global proteome of homogenate milk samples to characterize changes across the three phases of swine lactation. Milk samples were collected from multiparous sows (n = 9) on postnatal day 0 (D0; colostrum), 3 (D3; early transitional), 7 (D7; late transitional), and 14 (D14; mature). On D3, percent fat (16 ± 2.1) and lactose (3.8 ± 0.3) were higher (P < 0.05) than on D0 (10 ± 3.9 and 1.5 ± 0.3, respectively). Levels of fat and lactose were not different between D3 and D14. Percent total protein decreased (P < 0.05) between D0 (11 ± 2.1) and D3 (5 ± 0.7), but there was no significant change in percent protein between D3 and D14. Total carbohydrates increased (P < 0.05) between D3 (944 ± 353 µg/mL) and D14 (1,150 ± 462 µg/mL). Quantitative proteomic analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) of homogenate D0, D3, and D14 milk samples (n = 6) identified 772 protein groups which corresponded to 501 individual protein-coding genes. A total of 207 high confidence proteins were detected in n = 3 sows/day. Of the high confidence proteins, 81 proteins were common among all 3 days of lactation. Among the proteins that decreased between the days (false discovery rate; FDR < 0.05) were multiple apolipoproteins and XDH which decreased between D0 to D3. Proteins that increased across the days (FDR < 0.05) were complement factors and 14-3-3 proteins (YWHAQ, YWHAE). Our data provide a good characterization of milk proteome changes that likely reflect mammary function as well as the neonate's phase-specific developmental needs. This data may be useful in developing approaches to enhance the health and welfare of swine.

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. HYPOTHESIS/OBJECTIVES: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses. ANIMALS: Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses. METHODS: The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog. RESULTS: Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

Heat treatment of bovine colostrum: II. Effects on calf serum immunoglobulin, insulin, and IGF-I concentrations, and the serum proteome.

In-depth analysis of colostrum components has identified hundreds of proteins, but data are sparse regarding their systemic uptake in the newborn calf. Moreover, heat treatment may influence these colostral components and their absorption. Our objectives were to describe the serum proteome of newborn calves before and after colostrum feeding and the possible effects of colostral heat treatment. Newborn Holstein heifer calves (n = 22) were randomized within pair and fed heat-treated (n = 11; 60°C, 60 min) or raw (n = 11) colostrum at 8.5% of birth body weight by esophageal feeder within 1 h of birth. After the single colostrum feeding, calves were not fed until after the 8-h time point, when milk was offered free-choice. Blood samples were taken immediately before feeding (0 h), as well as 4, 8, and 24 h after feeding. Whole blood packed cell volume (%), serum Brix percentage, and plasma glucose concentrations were determined for all time points. Plasma insulin and insulin-like growth factor-I concentrations were determined by radioimmunoassay for selected time points. Serum IgA and IgG were measured by radial immunodiffusion at 24 h. The serum proteome was analyzed using nano-scale reverse-phase chromatography and tandem mass spectrometry (nano LC-MS/MS) in 0- and 8-h samples. For proteomics analysis, ratios of results for 8-h to 0-h samples were analyzed with false discovery rate adjustment. For all other outcomes, repeated-measures ANOVA was performed with the fixed effects of group, time, and their interaction, and random effect of pair. Serum Brix percentage and glucose concentrations increased over time and were independent of colostrum treatment. Serum IgG and IgA concentrations at 24 h did not differ between groups. Nano LC-MS/MS identified a total of 663 unique proteins in serum, of which 261 increased in abundance, whereas 67 decreased in abundance after feeding in both groups. Among serum proteins that increased in abundance and that were previously identified in colostrum, many belonged to those involved in immune response, coagulation, the classical complement pathway, or the antimicrobial peptide class of cathelicidins. Serum proteins that decreased in abundance and that were identified in colostrum belonged to the alternative complement pathway and the membrane attack complex. Thirty-eight proteins differed in calves that were fed heat-treated colostrum compared with those fed raw colostrum. Decreased abundances in calves fed heat-treated colostrum included several enzymes involved in glycolysis or glycogenolysis, whereas the incretin gastric inhibitory polypeptide and serum insulin were increased in this group. Our findings point to important innate immune defense pathways associated with colostrum ingestion in newborn calves. Furthermore, calves fed heat-treated colostrum showed differences in serum proteins and enzymes associated with carbohydrate metabolism.

Tandem mass tag-based quantitative proteomics analysis and gelling properties in egg albumen of laying hens feeding tea polyphenols.

The tea polyphenol (TP) can improve the egg albumen quality in laying hens; however, our understanding of the molecular mechanisms and proteomic changes in the egg albumen remains limited. A total of 720 layers (35-wk-old) were allocated into 5 treatments with TP and were added at 0 (control), 200 (TP200), 400 (TP400), 600 (TP600), and 800 (TP800) mg/kg. It showed that 400 mg/kg TP increases albumen height and Haugh unit (quadratic effect, P < 0.01), while 400 mg/kg TP decreases gel strength, hardness, gumminess, and chewiness value in a quadratic manner (P = 0.01). Eggs from TP400-fed layers had highest reducing power and oxygen radical absorbance capacity, and lowest albumen malondialdehyde content (quadratic effect, P < 0.05). Through Tandem Mass Tag-based quantitative proteomics analysis, 258 proteins were identified and 31 differentially accumulated proteins in egg white affected by 400 mg/kg TP compared to control group, with 19 proteins upregulated and 12 proteins downregulated. A total of 11 binding proteins (A0A1D5PZE3, F1NTQ2, Q7SX63, F1NRV5, P24802, A0A1L1RM02, E1BTX1, A0A1L1RMF4, A0A1D5P1N3, A0A1L1RML6, A0A1L1RQF3), 9 immune response proteins (P10184, R4GI90, P01875, Q6IV20, Q64EU6, P02701, P08110, P0CB50, A0A1D5PQ63), and 3 cell redox homeostasis proteins (P0CB50, P20136, Q8JG64) were changed in albumen of laying hens fed TP400. The differentially expressed proteins mainly involved in pyruvate metabolism, cysteine and methionine metabolism, glutathione metabolism, glycolysis, and protein processing in endoplasmic reticulum pathway. The result gathered in this study suggested that the improving mechanism of TP on albumen quality may act through regulating binding mediation, immune function, and antioxidant activity-related proteins.

Influence of Metabolic Status and Diet on Early Pregnant Equine Histotroph Proteome: Preliminary Findings.

Insulin resistance (IR) is characterized by an increase in biomarkers of systemic inflammation and susceptibility to laminitis in horses. Impacts on reproduction include a lengthened interovulatory period in horses. Dietary omega-3 (docosahexaenoic acid [DHA]) promotes anti-inflammatory processes, has been implicated in health benefits, and can reduce cytokine secretion. This preliminary study investigated the impact of IR as well as the influence of dietary supplementation (DHA) on the uterine fluid proteome in early pregnant horses. Mares were artificially inseminated; uterine fluid and embryos were collected on d 12.5 after ovulation. Uterine fluid was pooled for metabolic and diet categories (n = 8; n = 2 per metabolic and dietary status) and concentrated, and the proteome was analyzed using tandem mass spectrometry (iTRAQ). Five proteins met differential abundance criteria (±1.5-fold change, P < .05) in all comparisons (Control C, IS vs. C, IR; C, IS vs. DHA, IS; C, IR vs. DHA, IR). Serum amyloid A, afamin, and serotransferrin were upregulated in C, IR mares but downregulated in DHA, IR mares when compared to C, IS and C, IR, respectively. Quantitative PCR supported mass spectrometry results. The presence of serum amyloid A and serotransferrin in histotroph of IR mares potentially indicates an inflammatory response not seen in IS counterparts. These preliminary findings provide novel evidence on the potential impact of insulin resistance and DHA supplementation on the secreted equine uterine proteome during early pregnancy.

Characterization of the colostrum and transition milk proteomes from primiparous and multiparous Holstein dairy cows.

Colostrum plays a vital role in the nutrition, development, and immunity of a newborn calf. This study aimed to characterize the protein profile of colostrum and to identify changes in the colostrum proteome across parity during the transition to mature milk. Colostrum and transition milk samples were collected at milkings 1, 2, 4, and 14 after calving from multiparous (n = 10) and primiparous cows (n = 10). Samples were skimmed, fractionated, and enriched before analysis for low-abundance proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Changes in protein abundances were analyzed using PROC MIXED in SAS (SAS Institute Inc., Cary, NC) with determination of the adaptive false discovery rate adjustment using a MULTTEST procedure to identify effects of parity (P), milking number (MN), and their interaction (MN×P). We identified 86 proteins through LC-MS/MS, including 3 low-abundance proteins that were affected by P, 78 that were affected by MN, and 36 affected by MN×P. Prominent ontological groupings of proteins affected by MN included defense or immunity proteins, such as immunoglobulins. Proteins involved in the plasminogen activating cascade and more broadly, blood coagulation, were affected by MN×P. The results of this study add to increasing knowledge of the colostrum and transition milk proteomes, and this is the first study to find evidence of different abundances of these proteins when examined across P, MN, and MN×P. These findings aid in the identification of potential milk protein biomarkers for mammary health during the early postpartum period.

Residue concentration of cefquinome taking into account different milk fractions and comparing the performance of two screening tests.

This Research Communication describes the residue concentration of a dry cow antibiotic in two different milk fractions and describes effects of milk fraction and milk composition on the test performance of a rapid screening and a microbial inhibitor test. Thirteen dry cows were treated with an intramammary dry cow antibiotic containing 150 mg cefquinome. Quarter foremilk and stripping samples were collected on the first 10 d postpartum. All milk samples were analyzed for milk composition by the local Dairy Herd Improvement Association and were tested for antibiotic residues using the rapid screening test Milchtest BL and the microbial inhibitor test Delvotest BR Brilliant Plates. The residue concentration of cefquinome was determined in foremilk and stripping samples from milkings 1, 2, 3, 5, and 7 after calving using high performance liquid chromatography - tandem mass spectrometry. The logarithm of cefquinome concentration (logCef) was higher in foremilk than in stripping samples and higher in milk samples with lower lactose content. Furthermore, logCef decreased with the number of milkings (P < 0.001). The Milchtest BL was more likely to be not evaluated (i.e. no test and control line or no control line appeared) in stripping samples and milk samples with higher protein content. In the Delvotest BR Brilliant Plates milk samples with higher protein content were more likely to have a false positive result (i.e. the screening test result was positive, but the HPLC-MS/MS result was below the detection limit of the screening test). These results indicate that foremilk is the recommended milk fraction to be tested for residues of cefquinome and that a high protein content can be a cause of test failure and false positive results when milk during the first 10 d postpartum is tested for antibiotic residues using screening tests.

Effect of twice-daily oral administration of a chondroitin sulfate-containing supplement on urine chondroitin sulfate concentrations in dogs.

OBJECTIVE: To quantify the magnitude and duration of changes in urine chondroitin sulfate concentration (uCS) as a result of oral administration of a chondroitin sulfate-containing supplement in dogs. ANIMALS: 8 healthy privately owned dogs. PROCEDURES: A urine sample was collected from each dog via cystocentesis on day 1; free-catch midstream urine samples were collected once daily on days 2 through 5. Pretreatment uCS was established from those samples. Each dog then received a chondroitin sulfate-containing supplement (20 to 30 mg/kg, PO, q 12 h) for 8 days (on days 7 through 14). Urine samples were collected on days 8 through 12 and day 15. For each sample, uCS was quantified by liquid chromatography-tandem mass spectrometry. Variable urine concentration was accounted for by dividing the uCS by urine creatinine concentration (uCrea) to determine the uCS:uCrea ratio. Pretreatment uCS:uCrea ratios were compared with treatment uCS:uCrea ratios to calculate the fold change in uCS after supplement administration. RESULTS: Among the study dogs, oral administration of the chondroitin sulfate-containing supplement resulted in a 1.9-fold increase in the median uCS:uCrea ratio. Data obtained on days 8 through 12 and day 15 indicated that the daily increase in uCS remained consistent and was not additive. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that oral administration of supplemental chondroitin sulfate to dogs modestly increased uCS within 24 hours; however, subsequent supplement administration did not have an additive effect. A potential therapeutic benefit of persistently increased uCS in preventing recurrent urinary tract infections in dogs warrants investigation.

LC-MS/MS measurement of ampicillin residue in chicken tissues at 2 days after in-feed administration.

We assessed ampicillin (ABPC) concentrations of liver, kidney and skin at a 2-day withdrawal period in ten male and ten female White Leghorn chickens fed the diet containing ABPC (ABPC medicated feed 40 mg/kg body weight/day) for a week. The ABPC residues were measured with liquid chromatography-tandem mass spectrometry and the mean recoveries and quantitation limits ranged from 93.0% to 102.7% and from 0.1 to 1.4 ng/g, respectively. The residual ABPC concentrations were ≤7.82 ng/g for the skin and ≤0.64 ng/g for the kidney, suggesting below the Japanese provisional maximum residue limits. These results revealed that the analytical method is developed for residue ABPC and that the withdrawal period is appropriate.

Effect of alpha-lipoic acid on relieving ammonia stress and hepatic proteomic analyses of broilers.

Ammonia in poultry houses not only affects worker health but also induces a variety of poultry diseases. Alpha-lipoic acid (LA) is an effective antioxidant that protects cells against oxidative injury during various toxic and pathological processes. This study was designed to evaluate the mitigating effects of LA supplementation on ammonia stress and hepatic proteome changes in broilers. Male broilers (22 d old) were allocated to 3 groups: (1) a control group without ammonia stress (CTRL); (2) exposure to 70 ppm ammonia (AM); and (3) exposure to 70 ppm ammonia and dietary administration of 300 mg/kg LA (AM+LA). Ammonia exposure significantly decreased broiler growth performance and plasma glutathione peroxidase activity (P < 0.05), and increased plasma malondialdehyde content and glutamic-pyruvic transaminase activity (P < 0.05). These negative effects were eliminated by LA supplementation. Comparative proteomic analyses revealed 291 differentially expressed proteins in the AM group compared to the CTRL and AM+LA groups. A total of 30 proteins were differentially expressed between the AM/CTRL and (AM+LA)/AM groups. The addition of LA restored 24 of these proteins to control levels; these proteins were mainly related to transcription regulation, detoxification, protein translation and degradation, and immune and stress responses. The differentially expressed proteins included the high mobility group box (HMGB) and glutathione S-transferase (GST), which is closely related to immune response and oxidative stress, and collagens, which are implicated in liver injury. The addition of LA to broiler diet may reduce ammonia toxicity by maintaining the antioxidant system, xenobiotic metabolism, and metabolic pathways.

Colostrum protein uptake in neonatal lambs examined by descriptive and quantitative liquid chromatography-tandem mass spectrometry.

Colostrum intake is a key factor for newborn ruminant survival because the placenta does not allow the transfer of immune components. Therefore, newborn ruminants depend entirely on passive immunity transfer from the mother to the neonate, through the suckling of colostrum. Understanding the importance of specific colostrum proteins has gained significant attention in recent years. However, proteomics studies of sheep colostrum and their uptake in neonate lambs has not yet been presented. The aim of this study was to describe the proteomes of sheep colostrum and lamb blood plasma, using sodium dodecyl sulfate-PAGE for protein separation and in-gel digestion, followed by liquid chromatography-tandem mass spectrometry of resulting tryptic peptides for protein identification. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach was subsequently used to provide relative quantification of how neonatal plasma protein concentrations change as an effect of colostrum intake. The results of this study describe the presence of 70 proteins in the ovine colostrum proteome. Furthermore, colostrum intake resulted in an increase of 8 proteins with important immune functions in the blood plasma of lambs. Further proteomic studies will be necessary, particularly using the selected reaction monitoring approach, to describe in detail the role of specific colostrum proteins for immune transfer to the neonate.