Characterisation of the oral glucose and sugar tolerance tests and the enteroinsular axis response in healthy adult donkeys.

BACKGROUND: Insulin dysregulation (ID) is diagnosed in horses and ponies using oral glucose (OGTT) and oral sugar (OSTT) tolerance tests. The enteroinsular axis plays a major role in postprandial glucose disposal and insulin response in horses, ponies and foals. The insulin and incretin response to oral carbohydrate challenges has not been characterised in donkeys. OBJECTIVES: (a) To characterise OGTT and OSTT, and (b) to assess the plasma incretin response to OGTT and OSTT in healthy donkeys. STUDY DESIGN: In vivo experiments. METHODS: Six healthy adult female Andalusian donkeys were challenged with OGTT (1 g/kg glucose, 20% solution by nasogastric tube) and OSTT (0.45 mL/kg corn syrup orally by syringe) with a 1-week washout. Blood samples were collected for glucose (spectrophotometry), insulin (radioimmunoassay), glucose-dependent insulinotropic polypeptide (GIP, ELISA) and active glucagon-like peptide-1 (aGLP-1, ELISA) determination over 6 hours. Curves were analysed and proxies calculated. RESULTS: Glucose and insulin concentrations peaked at 180 minutes in OGTT, but at 300 and 150 minutes in OSTT, respectively. Plasma GIP concentrations increased in the OGTT and OSTT (peaked at 180 and 360 minutes, respectively), but aGLP-1 increased only in OGTT (240 minutes). MAIN LIMITATIONS: Single breed, narrow age and sample, diet, season and not having donkeys with evidence of ID to provide clinical validation. CONCLUSIONS: Donkeys have a functional enteroinsular axis that is activated by enteral carbohydrates. Donkeys have evident endocrine differences with horses, supporting the validation of the OSTT and OGTT to assess insulin sensitivity in this species to avoid extrapolation from horses.

Reference intervals of acute phase proteins in healthy Andalusian donkeys and response to experimentally induced endotoxemia.

BACKGROUND: Assessment of acute phase proteins (APPs) may allow prompt detection of diseases in donkeys, that otherwise may be missed because of the stoic behavior of donkeys. Reference intervals (RIs) of APPs measured using immunoassays and a comparison of the response of these biomarkers to a controlled inflammatory insult are lacking in donkeys. OBJECTIVES: (a) To describe the RIs for APPs in healthy Andalusian donkeys, (b) to study the effects of sex and age on APPs, and (c) to assess the early response of APPs to experimentally induced endotoxemia. ANIMALS: Seventy-three healthy Andalusian donkeys (67 for RIs and 6 for endotoxemia). METHODS: Serum amyloid A (SAA), haptoglobin (Hp), C-reactive protein (CRP), ceruloplasmin (Cp), α1-acid glycoprotein (AGP), procalcitonin (PCT), ferritin (Ft), and fibrinogen (Fb) RIs were determined. Endotoxemia was induced and samples for APP determination were obtained at regular intervals for 4 hours. RESULTS: The RIs in Andalusian donkeys were: SAA (0.1-0.6 mg/L), Hp (75-2261 mg/L), CRP (1.3-7.0 mg/L), Cp (0-745 mg/L), AGP (0-884 mg/L), PCT (0-504 pg/mL), Ft (26.9-31.8 µg/L), and Fb (115-466 mg/dL). Concentrations of SAA were higher (P < .05) in jacks. Donkeys <5 years old had higher Cp, AGP, and PCT compared to older donkeys. Concentrations of SAA and Hp were significantly increased in endotoxemic donkeys from 2 hours postinduction. CONCLUSIONS AND CLINICAL IMPORTANCE: We illustrated the importance of using species-specific RIs for APPs in donkeys and the effect of age and sex on APP concentrations. Concentrations of SAA and Hp appear to be the most useful biomarkers in donkeys in the early stages of endotoxemia.

Blood Storage Conditions Affect Hematological Analysis in Samples From Healthy Donkeys and Donkeys With Experimentally-Induced Endotoxemia.

Preanalytical factors such as storage time and temperature are proved to induce marked artifactual changes in hematological parameters in horses, small animals and humans. These errors can mirror findings typical of endotoxemia, leading to dangerous misdiagnosis. Since donkeys are common in warm climates and remote regions, blood samples from this species can be subjected to long lasting travels from the farm to the nearest laboratory, frequently under suboptimal conditions. Moreover, as other equids, donkeys are prone to suffer endotoxemia. Nonetheless, stability has not been evaluated in samples for hematology in this species. The aim of this study was to characterize the effect of temperature and storage time in hematological parameters from healthy donkeys and donkeys with induced endotoxemia. Blood samples were collected from six healthy female Andalusian donkeys and stored for 6, 12, 24, and 48 h at several temperatures (4, 24, and 35°C). Endotoxemia was induced in the same animals by an intravenous LPS infusion and samples obtained 30 min post-infusion were handled similarly. Hematological analysis was performed using a laser-based analyzer and blood smear examination. Storage at 24°C caused significant neutropenia after 48 h as well as morphological changes typical of endotoxemia in blood from healthy donkeys as soon as 24 h post-storage. Samples kept at 35°C displayed more profound and earlier artifactual variations. Conservation at 4°C did not cause any significant change in blood parameters. Prolonged (48 h) storage of samples from animals with induced endotoxemia at 24 and 35°C accentuated pre-existing leukopenia and neutropenia. These findings highlight that donkey samples should be stored at 4°C, instead of 24°C as recommended for horses. Moreover, blood smear interpretation should be cautious in samples stored for longer than 24 h and could be misleading when blood is kept at 35°C.

Meloxicam ameliorates the systemic inflammatory response syndrome associated with experimentally induced endotoxemia in adult donkeys.

BACKGROUND: Little information is available about endotoxemia in donkeys. Characterizing the systemic inflammatory response (SIRS) to lipopolysaccharide (LPS) in donkeys would provide valuable clinical and therapeutic information. The effects of meloxicam on endotoxemia have not been studied in this species. OBJECTIVES: To study the pathophysiology and gene expression associated with experimentally induced endotoxemia, and evaluate the effects of meloxicam on experimentally induced endotoxemia in donkeys and in equine monocyte cultures. ANIMALS: Six healthy adult female donkeys. METHODS: Endotoxemia was induced by an IV infusion of LPS for 30 minutes. Animals either received 20 mL of saline or 0.6 mg/kg of meloxicam IV after LPS infusion. The experiments lasted 6 hours. Blood samples were collected serially for hematology, serum biochemistry, interleukin measurement, and leukocyte gene expression analysis. Vital signs were recorded throughout the study. Monocyte cultures were used to test the effects of meloxicam on LPS-activated monocytes. RESULTS: Lipopolysaccharide induced fever, leukopenia, and neutropenia of similar magnitude in both groups, but meloxicam attenuated increases in plasma lactate, tumor necrosis factor-alpha (TNFα), and interleukin 1ß concentrations compared to controls. No differences were detected between groups for cytokine mRNA expression. Furthermore, meloxicam decreased TNFα release in LPS-activated monocyte cultures. CONCLUSIONS AND CLINICAL IMPORTANCE: Meloxicam could be a feasible option for the treatment of endotoxemia and SIRS in donkeys. Additional studies are necessary to investigate possible meloxicam-related posttranscriptional regulation and to compare this drug with other nonsteroidal anti-inflammatory drugs (NSAIDs) in animals with endotoxemia.

Pharmacokinetics of the anti-androgenic drug flutamide in healthy stallions.

Alternatives to surgical castration are necessary for controlling the sexual behaviour of stallions with breeding potential in training and competition. Flutamide is a potent selective non-steroidal androgen receptor competitive antagonist that has been used in human beings as an anti-androgenic drug. In this study, the pharmacokinetics and bioavailability of flutamide and its main active metabolite, 2-hydroflutamide, were determined in seven healthy mature stallions. Single doses of flutamide (1mg/kg intravenously, 1mg/kg orally in fasted horses, 5mg/kg orally in fasted horses and 5mg/kg orally in fed horses) were administered randomly at intervals of 2 weeks. All horses had full physical examinations and blood samples were collected for pharmacokinetics, complete blood counts and biochemistry before and after drug administration. Administration of flutamide did not result in any abnormalities on physical examination or in blood parameters. After intravenous administration of flutamide, the volume of distribution was 0.83L/kg and clearance was 1.20L/h/kg. Flutamide and its metabolite had high protein binding values (93-97%). After oral administration, flutamide was rapidly transformed to 2-hydroxyflutamide, with areas under the concentration-time curve ratios of metabolite:drug ∼7. Oral bioavailability was 6.63% after 1mg/kg flutamide in fasted horses, 6.50% after 5mg/kg flutamide in fasted horses and 6.95% after 5mg/kg in fed horses. Half lives of flutamide were close to 1h after intravenous administration and 2h after oral administration. Half lives of 2-hydroxyflutamide were 4.79-6.84h for all routes and doses. After oral administration, oral flutamide reached plasma concentrations that could be effective as an anti-androgenic agent in horses, but further studies are needed to determine whether flutamide has clinical value as an alternative to castration for controlling sexual behaviour in stallions.