Vetnuus | May 2025 21 1. Introduction Donkeys are a unique equine species with many behavioural traits that they have retained from their ancestors, the African Wild Asses [1]. Some of these unique traits include their subtle display of disease. Donkeys often demonstrate only vague, elusive signs of illness or pain, such as dull demeanour, increased frequency or time spent lying down, decreased appetite, or isolation from their herd-mates [2–4]. The changes in behaviour that an astute owner, caretaker, or veterinarian may observe might not even obviously correlate to the body system that is affected or the stage of the disease. For instance, painful hoof disorders in mammoth asses may be more likely to result in recumbency than painful gastrointestinal disorders [4]. Veterinarians must perform thorough physical examinations of donkey patients but often need to also rely heavily on coupling those findings with supporting laboratory data to make an accurate diagnosis. Reference interval (RI) information specific to donkey clinicopathologic data is limited. Furthermore, most of the current donkey RI literature utilizes breed, geographic location, or lifestage-specific populations that may not be applicable to larger and more varied populations [5–12]. One of the most extensive donkey RI studies to date involved 138 samples from donkeys living in a sanctuary in the United Kingdom (UK) [13]. In that study, Burden and colleagues compared their findings to horses and to a previous UK donkey RI study, finding only limited transferability between the various populations. This important study highlights the need for RIs to represent the appropriate species and population and for the intermittent reassessment of reference intervals, as changes in management and other influences may affect intervals over time. The New York State Animal Health Diagnostic Center (AHDC) receives approximately 500 hematologic or serum biochemical test requests on donkey blood samples annually. To the authors’ knowledge, the last extensive RI study involving donkeys (n = 217) in the continental U.S. was published in 1990 [14]. Our goal in this study was to utilize the guidance of the American Society of Veterinary Clinical Pathology (ASVCP) to establish hematologic and serum biochemistry reference intervals at the AHDC for adult donkeys. We hypothesized that our reference intervals would differ from those previously established for donkeys in other publications and those previously established for horses by the AHDC. The authors sought to assess hematologic and biochemical RIs specific to US donkeys to assist practitioners who rely heavily on these objective diagnostic tests to accurately categorize disease states in this stoic species. 2. Materials and Methods 2.1. Ethics Approval This study, under the umbrella protocol “Laboratory test reference intervals, method validation and testing for the diagnostic utility of laboratory assays”, was performed under an approved International Animal Care and Use Committee (IACUC) protocol. Signed owner consent was obtained for all sample collection. 2.2. Animals and Sample Collection First, 120 donkeys were sampled between February 2020 and June 2021 from several locations across the U.S., representing geographic locations typical of submissions to the AHDC. Samples were collected as part of routine/annual exams, herd health visits, and castration procedures. Inclusion criteria were apparently healthy donkeys (as assessed by the veterinarian obtaining the samples) greater than or equal to two years of age, with no known health problems diagnosed in the past six months. Donkeys must not have received any pharmaceutical products other than anthelmintics in the past six months and must have been determined to have a body condition score (BSC) of 2, 3, or 4 on a 5-point scale, by the submitting veterinarian at the time of sample collection [15]. No specific requirements were made regarding diet or time of last meal. When available, the actual age of the donkey was obtained from birthdates recorded in health records; otherwise, it was estimated by the veterinarian, based on dentition. Donkeys were also characterized based on their size as miniature (less than 36 inches at the withers), mammoth (greater than or equal to 56 inches at the withers), or standard (measuring between those sizes at the withers) [16]. The sex of the donkey was also recorded (intact male, castrated male, or female). Venipuncture was performed on each donkey from the left or right jugular. Whole blood was collected into either a 7 mL Covidien Monoject™ Vacutainer K3-EDTA tube (Medtronic, Dublin, Ireland) or a 4 mL BD Vacutainer K2-EDTA tube and a BD Vacutainer® serum tube (Becton Dickinson, Franklin Lakes, NJ, USA). Within one hour of collection, two unstained, air-dried, blood smears were made using the EDTA whole blood sample. For most samples, the serum tubes were centrifuged within four hours of collection, and serum was removed from the blood clot and transferred to another BD Vacutainer® serum tube. A few veterinarians were unable to centrifuge samples themselves, so 10 samples were submitted as clotted whole blood in the serum tube, and centrifugation was performed at the AHDC. The blood and serum samples were kept chilled on ice packs or refrigerated until overnight shipment on ice packs to the AHDC within 48 h of sample collection. The blood smear slides were placed in slide mailers and protected from contact with ice packs during shipment. 2.3. Laboratory Analyses All samples were analyzed within 24 hours of receipt. EDTA whole blood was utilized for all haematology testing, and serum was utilized for all biochemistry testing. The total protein analyte of the complete blood count (CBC) was determined by refractometry. Hematocrit, haemoglobin concentration (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count, mean platelet volume (MPV), and white blood cell count (WBC) were determined using a Siemens Advia 2120i automated haematology analyzer (Siemens Medical Solutions, Malvern, PA, USA). The differential WBC count was derived by a manual count of 100 cells in Wright-Giemsa-stained smears. Biochemical parameters and their associated methods are listed in Table 1 and were measured using a Cobas 501 automated serum chemistry analyzer (Roche Diagnostics, Indianapolis, IN, USA). Both laboratory analyzers are evaluated daily for performance using commercially available quality-control material and calibrated as needed. At the time of testing, the laboratory participated in two external quality assurance programs. >>>22 Article
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