Cage-Free Pullets Minimally Affected by Stocking Density Stressors.

Management choices during the pullet phase can affect behavior, welfare, and health later in life, but few studies have evaluated the pullet phase, particularly in extensive housing systems. This study was a 2 × 2 factorial randomized complete block design (RCBD) with two strains and two stocking densities. The Lohmann LB-Lite and Lohmann LSL-Lite were housed on the floor at high-stocking density (619-670 cm2/bird) and low-stocking density (1249-1352 cm2/bird), which changed with age from 2 to 16 weeks of age (WOA). Bird-based measures of appearance, blood parameters, organ measurements, and production values were evaluated. Stocking density alone affected (p < 0.05) only relative bursal weight (% of body weight)-3.32% in the low-density versus 3.08% in the high-density group. High-stocking density was correlated with decreased uniformity (high-89.33 ± 0.24%; low-90.41 ± 0.24; p < 0.02) and worse feather coverage in the brown strain. High-stocking density was correlated with greater uniformity (High-90.39 ± 0.24%; Low-88.47 ± 0.24%; p < 0.001) and better feather coverage in the white strain. This study's feed conversion ratio (FCR) was improved by 0.07 in the low-stocking density for both strains. The remaining parameters were affected by strain and age only. Thus, while stocking density effects vary slightly depending on the strain used, cage-free pullets had limited negative effects at both the high and low-stocking densities tested in this study; there were few to no changes in the numerous bird-based welfare parameters tested.

Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses.

BACKGROUND: Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown. HYPOTHESIS: Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner. ANIMALS: Eight healthy horses. METHODS: Randomized placebo-controlled crossover study. Each horse received 4 single-dose IV treatments including AA at 25, 50, and 100 mg/kg and saline (placebo) with each treatment separated by ≥1 week. Blood was collected at baseline, 2 and 6 hours for assessment of plasma dROM and PAC via photometer, intraerythrocytic ROS by flow cytometry, and stimulant-induced neutrophil ROS by a fluorometric assay. Plasma AA concentrations were measured by high-performance liquid chromatography/electrochemical detection. RESULTS: Ascorbic acid at 100 mg/kg resulted in decreased dROM 2 hours after treatment (P = .03, 95% CI 5.51-121.2, point estimate 63.3). There was no effect of AA on basal or stimulant-induced intraerythrocytic ROS (P = .88, 95% CI -0.156 to 0.081, point estimate -0.037; P = .93, 95% CI -0.123 to 0.112, point estimate -0.006, respectively), basal or stimulant-induced neutrophil ROS (P ≥ .12, 95% CI -644.9 to 56.2, point estimate -294.4), or PAC (P ≥ .64, 95% CI -1567 to 463.4, point estimate -552.0) at any dose or timepoint. Plasma AA concentrations increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL IMPORTANCE: High-dose administration of AA might provide antioxidant benefits in horses.

Marked paraneoplastic basophilia in a cat with alimentary T-cell lymphoma.

An 8-year-old, spayed female domestic shorthair cat was presented for acute weight loss, hyporexia, intermittent vomiting, and loose stools. A caudal abdominal mass and thickened intestinal loops were palpated on initial examination. An abdominal ultrasound identified a circumferential intramural jejunal mass with complete loss of wall layering, diffuse thickening of the jejunal muscularis, and jejunal and ileocecal lymphadenomegaly. Initial routine bloodwork revealed mild monocytosis and minimal lymphopenia with reactive lymphocytes. Cytologic evaluation of the jejunal mass and enlarged lymph nodes was consistent with lymphoma (intermediate cell size), and PCR for antigen receptor rearrangement revealed a clonal T-cell receptor rearrangement consistent with T-cell lymphoma. Chemotherapy (CHOP protocol) was initiated, but despite initial improvement of clinical signs, a repeat ultrasound examination 5 weeks after initiation of treatment revealed no improvement in the lymphadenomegaly or reduction in the size of the jejunal mass. At this visit, the cat also developed a marked basophilia (basophils 12.28 × 103 /µL, RI 0.00-0.10) with low numbers of circulating atypical lymphocytes; no concurrent eosinophilia was noted. Heartworm disease, ectoparasites, and allergic diseases were evaluated for and considered unlikely. The chemotherapy protocol was changed to L-asparaginase, followed by lomustine. The basophilia was significantly reduced 2 days after the initial dose of L-asparaginase and remained within the reference interval for 40 days before an eventual decline in the cat's health. To the authors' knowledge, this is the first report of paraneoplastic basophilia without concurrent eosinophilia in a cat with T-cell lymphoma.

Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses.

BACKGROUND: A systemic and dysregulated immune response to infection contributes to morbidity and mortality associated with sepsis. Peripheral blood-derived mesenchymal stromal cells (PB-MSC) mitigate inflammation in animal models of sepsis. Allogeneic PB-MSC administered IV to horses is well-tolerated but therapeutic benefits are unknown. HYPOTHESIS: After IV lipopolysaccharide (LPS) infusion, horses treated with PB-MSC would have less severe clinical signs, clinicopathological abnormalities, inflammatory cytokine gene expression, and oxidative stress compared to controls administered a placebo. ANIMALS: Sixteen horses were included in this study. METHODS: A randomized placebo-controlled experimental trial was performed. Sixteen healthy horses were assigned to 1 of 2 treatment groups (1 × 109 PB-MSC or saline placebo). Treatments were administered 30 minutes after completion of LPS infusion of approximately 30 ng/kg. Clinical signs, clinicopathological variables, inflammatory cytokine gene expression, and oxidative stress markers were assessed at various time points over a 24-hour period. RESULTS: A predictable response to IV LPS infusion was observed in all horses. At the dose administered, there was no significant effect of PB-MSC on clinical signs, clinicopathological variables, or inflammatory cytokine gene expression at any time point. Antioxidant potential was not different between treatment groups, but intracellular ROS increased over time in the placebo group. Other variables that changed over time were likely due to effects of IV LPS infusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of allogeneic PB-MSC did not cause clinically detectable adverse effects in healthy horses. The dose of PB-MSC used here is unlikely to exert a beneficial effect in endotoxemic horses.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Incidental diagnosis of a spindle cell type gastrointestinal stromal tumor in a dog with ethylene glycol intoxication.

A 6-year-old castrated male American Pit Bull Terrier dog was presented for evaluation of acute onset of tonic-clonic seizures, anorexia, and vomiting. On physical examination, neurologic signs, such as generalized proprioceptive ataxia, salivation, circling to the right, and absent patellar reflexes bilaterally, were noted. A complete blood cell count revealed mild hemoconcentration and an inflammatory leukogram, while a chemistry panel showed severe azotemia, marked hypochloremia, and a severe titrational metabolic acidosis, suggesting possible ethylene glycol intoxication. However, an irregularly round, small mass was identified in the large intestine on abdominal ultrasound. Additionally, bilateral hyperechoic renal cortices with medullary rim sign were suggestive of acute nephritis or tubular necrosis. The cytologic evaluation of a fine-needle aspiration biopsy of the abdominal mass revealed a large population of mesenchymal cells, suggesting the presence of neoplasia. Due to the worsening of symptoms, the dog was humanely euthanized. Necropsy confirmed ethylene glycol intoxication, and the incidental finding of a neoplastic intestinal mass was diagnosed as spindle cell sarcoma. Immunohistochemical staining showed strong, diffuse positivity for CD117, smooth muscle actin, and S-100, indicating the final diagnosis of a spindle cell type gastrointestinal stromal tumor (GIST). This report briefly discusses the classifications of nonlymphoid, nonangiogenic intestinal mesenchymal tumors, characteristics of GISTs, and the importance of the immunohistochemical classification of mesenchymal tumors of the gastrointestinal tract.

Development and validation of a flow cytometric assay for detecting reactive oxygen species in the erythrocytes of healthy dogs.

OBJECTIVE: To validate the use of a flow cytometric assay that uses 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure reactive oxygen species in the erythrocytes of healthy dogs. ANIMALS: 50 healthy adult dogs. PROCEDURES: Erythrocytes were incubated with DCFH-DA or a vehicle control (dimethyl sulfoxide), then incubated with (stimulated) or without (unstimulated) hydrogen peroxide. The flow cytometric assay was evaluated for specificity with increasing concentrations of DCFH-DA and hydrogen peroxide, and a polynomial regression line was applied to determine optimal concentrations. For precision, samples were analyzed 5 consecutive times for determination of intra- and interassay variability. Stability of samples stored at 4°C for up to 48 hours after blood collection was determined with flow cytometric analysis. Coefficient of variation (CV) was considered acceptable at 20%. Baseline measurements were used to determine an expected range of median fluorescence intensity for unstimulated erythrocytes incubated with DCFH-DA. RESULTS: Erythrocytes were successfully isolated, and stimulated samples demonstrated higher median fluorescence intensity, compared with unstimulated samples. The intra-assay CV was 11.9% and 8.9% and interassay CV was 11.9% and 9.1% for unstimulated and stimulated samples, respectively. Unstimulated samples were stable for up to 24 hours, whereas stimulated samples were stable for up to 48 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry for the measurement of reactive oxygen species in the erythrocytes of healthy dogs by use of DCFH-DA had acceptable specificity, precision, and stability. Flow cytometry is a promising technique for evaluating intraerythrocytic oxidative stress for healthy dogs.

Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses.

BACKGROUND: Oxidative stress refers to the accumulation of reactive oxygen species (ROS). Most assays for ROS detection are costly, laborious, and usually use indirect markers. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a possible alternative. This substance becomes a fluorochrome when oxidized by ROS, with the resultant fluorescence proportional to ROS concentration. Erythrocytes are highly exposed to ROS, resulting in cell damage and consequently impaired oxygen delivery. The effects of this exposure in physiologic and pathologic conditions necessitate an improvement in ROS detection methods. OBJECTIVE: We aimed to validate intraerythrocytic ROS detection by flow cytometry using DCHF-DA in healthy horses. METHODS: Erythrocytes from 31 healthy horses were isolated, incubated with DCFH-DA, and either left unstimulated or stimulated with hydrogen peroxide (H2 O2 ). For specificity, each cellular component of blood was separated and plotted according to its size and complexity. Samples were run in triplicate for intra-assay precision and five consecutive times for inter-assay repeatability. Stability was determined by analyzing the same sample up to 48 hours after blood collection. The acceptable coefficient of variation (CV) was ≤20%. RESULTS: The intra-assay CV was 1.7% and 13.3%, and the inter-assay CV was 4.8% and 17.8% for unstimulated and stimulated samples, respectively. Unstimulated and stimulated samples were stable for up to 48 and 24 hours, respectively. Stimulated samples had greater fluorescence than unstimulated samples (P < .0001). CONCLUSIONS: This flow cytometric assay demonstrated adequate specificity, precision, and stability and is, therefore, a promising technique with multiple applications for studying oxidative stress in horses.

Reactive oxygen species, glutathione, and vitamin E concentrations in dogs with hemolytic or nonhemolytic anemia.

BACKGROUND: Red blood cells (RBC) are uniquely susceptible to oxidative injury. Oxidative stress is both a cause for, and effect, of anemia in people but this has been minimally documented in dogs. OBJECTIVE: To describe direct and indirect markers of oxidative stress in anemic dogs. HYPOTHESIS: Anemic dogs will have oxidative stress when compared to healthy dogs. ANIMALS: Forty-seven dogs with anemia (10 with hemolytic anemia) and 70 healthy control dogs. METHODS: Prospective, cross-sectional study. Anemic dogs were identified from the patient population, and medical records were reviewed to classify the anemia as hemolytic or nonhemolytic. Flow cytometry was used to detect reactive oxygen species (ROS) in erythrocyte isolates. Reduced glutathione (GSH) concentrations were measured in both plasma and hemolysate samples, and vitamin E was measured in serum. RESULTS: Anemic dogs (both hemolytic and nonhemolytic) had significantly lower median RBC hemolysate GSH concentrations (3.1 µM [0.4-30.8]) when compared to healthy dogs (7.0 µM [0.5-29.7]; P = .03). Dogs with hemolytic anemia had significantly higher median plasma GSH (7.6 µM [0.4-17.8]) when compared to dogs with nonhemolytic anemia (1.6 µM [0.01-7.1]; P = .04) and healthy dogs (2.8 µM [0.1-29.9]; P < .0001). Reactive oxygen species were detectable in all samples, but there was no difference in ROS or vitamin E between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Oxidative stress is present in anemic dogs. Derangements in biomarkers of oxidative stress are different in dogs with hemolytic anemia and nonhemolytic anemia.

Pharmacokinetics and Pharmacodynamics of an Oral Formulation of Apixaban in Horses After Oral and Intravenous Administration.

Horses with inflammatory and infectious disorders are often treated with injectable heparin anticoagulants to prevent thrombotic complications. In humans, a new class of direct oral acting anticoagulants (DOAC) appear as effective as heparin, while eliminating the need for daily injections. Our study in horses evaluated apixaban, a newly approved DOAC for human thromboprophylaxis targeting activated factor X (Xa). Our goals were to: (1) Determine pharmacokinetics and pharmacodynamics of apixaban after oral (PO) and intravenous (IV) administration in horses; (2) Detect any inhibitory effects of apixaban on ex vivo Equid herpesvirus type 1 (EHV-1)-induced platelet activation, and (3) Compare an anti-Xa bioactivity assay with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) for measuring apixaban concentrations. In a blinded placebo-controlled cross-over study, five horses received a single dose (0.2 mg/kg) of apixaban or placebo PO or IV. Blood was collected before and at 3 (IV) or 15 (PO) min, 30 and 45 min, and 1, 2, 3, 4, 6, 8, and 24 h after dosing for measuring apixaban UPLC-MS concentrations and anti-Xa activity. Pharmacodynamic response was measured in a dilute prothrombin time (dPT) assay. Flow cytometric EHV-1-induced platelet P-selectin expression and clinical pathologic safety testing were performed at baseline, 2 and 24 h and baseline and 24 h, respectively. We found no detectable apixaban in plasma PO administration. After IV administration, plasma apixaban levels followed a two-compartment model, with concentrations peaking at 3 min and decreasing to undetectable levels by 8 h. The elimination half-life was 1.3 ± 0.2 h, with high protein binding (92-99%). The dPT showed no relationship to apixaban UPLC-MS concentration and apixaban did not inhibit EHV-1-induced platelet activation after IV dosing. Apixaban anti-Xa activity showed excellent correlation to UPLC-MS (r 2 = 0.9997). Our results demonstrate that apixaban has no apparent clinical utility as an anticoagulant for horses due to poor oral availability.

Subcutaneous Administration of Low-Molecular-Weight Heparin to Horses Inhibits Ex Vivo Equine Herpesvirus Type 1-Induced Platelet Activation.

Equine herpesvirus type 1 (EHV-1) is a major cause of infectious respiratory disease, abortion and neurologic disease. Thrombosis in placental and spinal vessels and subsequent ischemic injury in EHV-1-infected horses manifests clinically as abortion and myeloencephalopathy. We have previously shown that addition of heparin anticoagulants to equine platelet-rich plasma (PRP) can abolish ex vivo EHV-1-induced platelet activation. The goal of this study was to test whether platelets isolated from horses treated with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) were resistant to ex vivo EHV-1-induced activation. In a masked, block-randomized placebo-controlled cross-over trial, 9 healthy adult horses received 4 subcutaneous injections at q. 12 h intervals of one of the following treatments: UFH (100 U/kg loading dose, 3 maintenance doses of 80 U/kg), 2 doses of LMWH (enoxaparin) 80 U/kg 24 h apart with saline at the intervening 12 h intervals, or 4 doses of saline. Blood samples were collected before treatment and after 36 h, 40 h (4 h after the last injection) and 60 h (24 h after the last injection). Two strains of EHV-1, Ab4 and RacL11, were added to PRP ex vivo and platelet membrane expression of P selectin was measured as a marker of platelet activation. Drug concentrations were monitored in a Factor Xa inhibition (anti-Xa) bioassay. We found that LMWH, but not UFH, inhibited platelet activation induced by low concentrations (1 × 106 plaque forming units/mL) of both EHV-1 strains at 40 h. At this time point, all horses had anti-Xa activities above 0.1 U/ml (range 0.15-0.48 U/ml) with LMWH, but not UFH. By 60 h, a platelet inhibitory effect was no longer detected and anti-Xa activity had decreased (range 0.03 to 0.07 U/ml) in LMWH-treated horses. Neither heparin inhibited platelet activation induced by high concentrations (5 × 106 plaque forming units/mL) of the RacL11 strain. We found substantial between horse variability in EHV-1-induced platelet activation at baseline and after treatment. Minor injection site reactions developed in horses given either heparin. These results suggest that LMWH therapy may prevent thrombotic sequelae of EHV-1, however further evaluation of dosage regimens is required.

Intravenous 15% isoflurane lipid nanoemulsion for general anesthesia in dogs.

OBJECTIVE: To investigate the efficacy of a new intravenous (IV) nanoemulsified isoflurane formulation for maintenance of general anesthesia in dogs. STUDY DESIGN: Prospective, crossover, experimental study. ANIMALS: Seven healthy, mature, mixed-breed dogs, three male and four female, weighing 11.5 ± 1.5 kg. METHODS: Anesthesia was induced with propofol for instrumentation. Measurements were obtained before administration of either inhaled isoflurane (Iso-I) or IV 15% isoflurane-loaded lipid nanoemulsion (Iso-nano). The minimum alveolar concentration (MAC) of isoflurane was determined using the 'up-and-down' technique. A tail clamp was applied every 15 minutes for a total time of 90 minutes and isoflurane administration was adjusted according to the response. Data were recorded at 30, 60 and 90 minutes for end-tidal isoflurane concentration (Fe´Iso), end-tidal carbon dioxide partial pressure (Pe'CO2), inspired isoflurane concentration (FIIso), arterial hemoglobin oxygen saturation (SaO2), peripheral hemoglobin oxygen saturation (SpO2), respiratory rate (fR), heart rate (HR), arterial blood pH, PaCO2, PaO2, base excess (BE), bicarbonate (HCO3-), systemic arterial pressure (sAP), and biochemical variables of blood urea nitrogen, alanine aminotransferase, creatine kinase and creatinine. RESULTS: No significant differences between treatments were detected for HR, fR, SaO2 or any biochemical variables (p > 0.05). In the Iso-nano treatment, sAP was significantly decreased throughout the study. Significant decreases in pH, Pe'CO2, BE and HCO3- were measured in the Iso-nano treatment. Isoflurane MAC was significantly lower in the Iso-nano than the Iso-I treatment. The dose of isoflurane (g hour-1) required to maintain general anesthesia did not differ significantly between treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of 15% isoflurane-loaded lipid nanoemulsion IV was effective in maintaining general anesthesia in dogs but did not reduce the amount of isoflurane necessary to maintain general anesthesia. Significant hypotension and nonrespiratory acidosis occurred with the injectable form.

Corrigendum: Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

[This corrects the article on p. 99 in vol. 3, PMID: 27909693.].

High-resolution melting analysis for detection of a single-nucleotide polymorphism and the genotype of the myostatin gene in warmblood horses.

OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing. RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.