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profiles were carried out within 24 h of sampling. Blood samples were taken as part of full clinical assessment to ensure that the donkeys were fit for rehoming. Clinical examinations and blood samples were carried out by veterinarians with experience of donkeys. Examination involved thorough physical inspection from head to hoof including ophthalmoscopic examination, chest auscultation and dental assessment. If any significant clinical abnormalities were detected the donkey was excluded from the study. Full clinical records relating to each donkey were reviewed and donkeys excluded from the study if they had suffered from significant health problems within the 6 months prior to sampling. The only pharmaceutically-active agents to have been used during this 6 month period were licensed deworming products. Common clinical conditions including seedy toe, minor dental abnormalities and superficial skin wounds were not considered significant and donkeys with these conditions at the time of sampling or having had them in the 6 months prior to sampling were not excluded from the study. The donkeys were assigned a body condition score (BCS) using a standard scale of 1–5 according to Svendsen (2008) and excluded from the study if they were underweight (BCS≤2) or obese (BCS≥4.5) by this score. In total 138 animals were included in the study; the majority of animals had full haematological and biochemical profiles but where particular parameters were not assessed the total numbers reported reflect this.


Laboratory analysis All samples were transported promptly to the laboratory at the Donkey Sanctuary, centrifuged and separated within 2 h of collection for biochemical analysis and subsequently refrigerated. Samples were analysed in-house within 24 h of sampling. Blood samples in EDTA evacuated tubes were analysed


using an automated cell counter (Sysmex XT 2000i)2 calibrated previously for donkey samples. Parameters assessed by the automated counter included counts of erythrocytes (RBC), total leucocytes (WBC) and platelets (PLT); concentrations of haemoglobin (Hb), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC); mean corpuscular volume (MCV), red cell distribution width (RDW) and haematocrit (calculated PCV). Differentiation of white cells into segmented neutrophils (Neu), lymphocytes (Lym), eosinophils (Eos), monocytes (Mon) and basophils (Bas) was carried out by microscopic examination of Giesma-stained thin blood smears; 200 cells were counted to determine the differential cell count. Automated biochemical analyses (Roche Integra 400


analyser)3 were undertaken on serum samples; analytes included in the panel were triglycerides (Trig), creatine phosphokinase (CPK), aspartate aminotransferase (AST) gamma glutamyl transferase (GGT), glutamate dehydrogenase (GLDH), alkaline phosphatase (ALP), total bilirubin (TBil), total serum protein (TP), albumin (Alb), globulin (Glob), creatinine (Creat), urea, amylase (Amy), lipase (Lip), calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and cholesterol (Chol). Bile acids were evaluated in a limited number of cases (84/138).


Quality assurance In addition to in-house quality control and calibration, all 20 biochemistry analytes tested were validated monthly across 3


external quality assurance (QA) schemes provided by WEQAS. Haematology analyte QA was undertaken using ‘Sysmex e-Check (XE)’ control samples on a daily basis prior to testing.


Data analysis The data from 18 haematological and 20 biochemical analytes were transferred into Excel4 and the mean, standard deviation (s.d.), sample variance, kurtosis, skew, range, count and confidence intervals (90%) calculated. This exploratory analysis of the dataset showed that the observations were, in the majority, non-normally distributed; as a result, nonparametric analyses were undertaken (SPSS 17.0)5. Outliers were identified as values below Q1–1.5 (IQR) or


above Q3 + 1.5 (IQR) and removed if biologically implausible or if credible were included in further analysis. A reference interval was then established from the central 95% interval by removal of the lower and upper 2.5% of the interval for each parameter, thus the 2.5 and 97.5 percentiles were obtained according to The International Federation of Clinical Chemistry (IFCC) protocol for nonparametric data. Ninety percent confidence intervals were calculated for the upper and lower RIs (MedCalc).


Reference interval transference validation Reference interval transference validation of new RIs for donkeys was undertaken to determine transferability with candidate RIs established for mature non-Thoroughbred horses (Anonymous 2011a), previously established RIs for donkeys (French and Patrick 1995), RIs for donkeys in Mexico (de Aluja et al. 2006) and RIs for donkeys in Ethiopia (Lemma and Moges 2009). Reference interval transference validation was undertaken according to American Society for Veterinary Clinical Pathology (ASCVP) guidelines (Anonymous 2011a) on the transference and validation of RIs between laboratories. In short, 40 samples representative of the newly established RIs for donkeys were evaluated against the candidate RI for transference. Using ASCVP guidelines, if ≥4/40 (10%) of the values fall outside of the candidate RI then transference is rejected for the analyte.


Results


In total, blood samples from 138 donkeys were examined. The donkeys ranged in age from 4 to 24 years; the cohort consisted of 29 females (aged 5–24 years, mean 13.1 years) and 111 geldings (aged 4–22 years, mean 12 years). All donkeys included in the study were found to be clinically healthy. The median BCS in the cohort was 3/5 (range 2.5–4). Median reference values, RIs (2.5 and 97.5 percentiles)


and 90% CIs for the quantified haematological and biochemical parameters are shown in Tables 1 and 2.


Reference interval transference To assess the transferability of these new RIs with those developed for other populations reference interval transference validation was undertaken. Reference interval transference validation data for new haematological and biochemical RIs are shown in Tables 1 and 2. This validation showed that 0/15 haematological and 4/20 biochemical RIs were transferrable between these new RIs for donkeys and


© 2015 The Donkey Sanctuary. Equine Veterinary Education published by John Wiley & Sons Ltd. on behalf of EVJ Ltd.


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