Ethical clearance
Ethical clearance was obtained before the commencement of the experiment from the Animal Use and Welfare Committee of the Ahmadu Bello University, Zaria, Nigeria with reference number ABUCAUC/2019/25
Study area
The study was conducted in the National Animal Production Research Institute (NAPRI), Shika – Zaria, Nigeria located, in the Northern Guinea Savanna zone of Nigeria with latitude 11o 12/N, longitude 7o 33/E and an altitude of 610 m above the sea level (Mohammed et al. 2007).
Experimental Animals
A total of 15 adult male donkeys with an average weight of 100.1 ± 1.29 kg, aged between 4 – 5 years and belonging to the Equine and Camel Research Programme, NAPRI, Shika- Zaria Nigeria were used for the study. Ten (10) of them were apparently healthy and devoid of helminth infection confirmed via faecal analysis in the helminthology laboratory, Department of Veterinary Parasitology, Faculty of Veterinary Medicine, ABU, Zaria Nigeria. The other five were infested with roundworm (Strongyles), Ivermectin® at a dose rate of 0.2 mg/kg per os was administered to the infested donkeys to ensure complete helminth-free condition in the donkeys before commencement of the research.
Experimental design and treatments
Fifteen of the donkeys were divided into three groups: A, B and C each containing five donkeys. Each donkey in all the three groups were engaged in packing kg of load covering a distance of 10 km. No treatment was given to Group A, while Groups B and C received 7.5 mg/kg of levamisole per os.
Measurement of body weight and packing
Body weights were measured using an Avery weighing scale (W & T Avery Ltd United states of America) for large animals in the animal pen at Dairy Research Programme, NAPRI, Shika-Zaria. Packing was performed for 10 km (5 km going and 5 km returning), between 6 and 11 am in the morning to allow laboratory analysis of blood samples collected within the same day.
Training of the donkeys on load carrying
The donkeys were trained for a period of four weeks on load carrying before commencement of the experiment. The estimated weight of load (sand), which was 40 % (40 kg) of each donkey’s body weight was placed in “mangala” bags which are specially designed for load carrying by pack animals.
Determination of vital parameters
Respiratory rate (cycles/min), pulse rate (beats/min) and rectal body temperature (oC) were determined by the method described by Mealey (2019). Respiratory rate was determined by gently placing a stethoscope on the thoraco–lumbar region to auscultate respiratory sounds and movements (for one minute), the pulse rate was counted by gently placing one or two fingers to feel the pulse on the mandibular artery for one minute, and a digital rectal thermometer (iProven manufactured in the United States of America) was used to measure the body temperature by gently placing it on the mucosa of the rectum of each donkey for one minute.
From each donkey, five millilitres of blood sample was collected via jugular venepuncture, and 2 mL was placed into potassium ethylenediamine tetra-acetic acid vacutainers. The remaining 3 mL was placed in test-tube and allowed to clot. The 2-mL blood sample was used for blood cellular analysis while the harvested serum was used for biochemical analyses. Faecal analysis was carried out using the McMaster technique with the 5 g faeces collected form each donkey into sterile polythene bags (Cringoli et al. 2010).
The vital parameters and blood collection were carried out before packing and upon return from packing between 6 and 11 am in the morning.
Determination of haematological parameters and erythrocyte osmotic fragility (EOF)
Blood cellular components of packed cell volume (PCV), red blood cell counts (RBCs), absolute and differential leucocyte counts were determined as described by Schlam et al. (1975). Erythrocytic indices were calculated using standard formulae.
Erythrocyte osmotic fragility was determined as described by Faulkner and King (1970). Briefly, 0.02 mL of whole blood was placed in each of 6 tubes, containing increasing concentrations; 0, 0.1, 0.3, 0.5, 0.7, 0.9% of phosphate-buffered sodium chloride (NaCl) solution at pH 7.4. Thereafter, the contents of the tubes were gently mixed and incubated at room temperature (37 0C) for 30 minutes and centrifuged at 1500 g per minute for 10 minutes. Optical density of the supernatant was determined spectrophotometrically at 540 nm using a spectrophotometer (Spectrolab 23 A®, Labomed Incorporated, Culver City, California, USA). Haemolysis in each tube was expressed and calculated as a percentage, assuming haemolysis in distilled water (0% NaCl) as 100%.
Biomarkers of oxidative stress
The concentration of malondialdehyde (MDA) and activities of Superoxide dismutase (SOD) and Catalase (CAT) were determined as described by Okhawa et al. (1979) for MDA, Fridovich (1989). for SOD and Aebi (1974) spectrophotometric method for Catalase.
Data Analyses
The data obtained were expressed as mean ± SEM. Values were subjected to two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test to compare the differences between the means. Values of P < 0.05 were considered as significant.