Trypanosomes species/strain used
The trypanosome parasites used were the Federer strains of T. brucei brucei and T. congolense (Savannah subgroup), which were sourced from the Nigerian Institute for Trypanosomiasis Research (NITR), Kaduna State, Nigeria and maintained in rats via passages.
Animals
Fifteen West African dwarf rams were used for the study. They were procured from local breeders in Nsukka Nigeria, and allowed to acclimatize for 3 weeks, during which period they were dewormed. All the rams were screened for trypanosomes and/or other haemoparasites, by buffy coat examination. They were placed on fresh forage and drinking water ad libitum. They were further randomly assigned to three groups (A, B & C) of five rams each. Group A rams were infected with T. brucei brucei, while Group B rams were infected with T. congolense, both intraperitoneally, at the dose of 106 trypanosomes/animal. Group C rams served as the uninfected control.
Estimation of parasitaemia
Parasitaemia was checked daily, beginning from day 2 post-infection of groups A and B. Once the infection was established, the levels of parasitaemia in the infected groups were determined weekly by wet mount and scored by rapid matching method (Paris et al. 1982; Herbert and Lumsden 1976).
Serum sample collection and analyses
Blood samples were collected before infection (day 0) and on days 14, 28, 42, 56 & 70 post infection (PI). The blood samples collected (4 ml) were dispensed into clean labelled plain test tubes and allowed 45 minutes to clot. Sera used for analyses were obtained by centrifuging the clotted blood in the test tubes at 3000 revolutions per minute (rpm) for 10 minutes. The serum samples were split into two aliquots and refrigerated. One aliquot was used for the assay of calcium, phosphorus, sodium, potassium, superoxide dismutase (SOD) and malondialdehyde (MDA), using commercially available test kits and a Diatek Blood Chemistry Analyzer (Diatek Instruments Co. Ltd., Wuxi, China) following manufacturers’ instructions. The other aliquot was used to assay for serum testosterone concentration using sheep-specific testosterone test kit (MBS701270) (Mybiosource Inc., California, USA).
Specifically, serum sodium was determined by the magnesium-uranyl acetate method (Scott et al. 2008), using a sodium test kit (Teco Diagnostics, Anaheim California, USA). The serum potassium was determined by the direct turbidimetric spectrophotometric method (Hillman and Beyer 1967; Scott et al. 2008), using a potassium test kit (Teco Diagnostics, Anaheim, California, USA). Serum calcium levels were determined by the ortho- cresolphthalein direct method (Connerty and Briggs 1966; Endres and Rude 2008), while the level of phosphorus in serum was evaluated based on the Fiske-SubbaRow method (Fiske and SubbaRow 1925; Goodwin 1970; Endres and Rude 2008), using Quimica Clinica Applicada (QCA) Calcium and inorganic Phosphorus test kits (QCA, S. A. Spain), respectively. Serum superoxide dismutase (SOD) activity was evaluated by hydroxylamine method (Weydert and Cullen 2010), while serum malondialdehyde levels was measured by the modified thiobarbituric acid method (Plaser and Cushman 1966; Draper and Hadley 1990), using the Elabscience SOD and Malondialdehyde Assay Kits (Elabscience Biotechnology Co. Ltd., South Africa), respectively. All the analyses were completed within 24 hours of sample collection.
Epididymal sperm evaluation: motility, morphology, vitality and concentration
Three rams from each group were castrated on day 70 PI under lignocaine anaesthesia. Both testes and epididymis were removed from each ram. Sperm motility was determined using the sperm diffusion method (Seed et al. 1996). Briefly, the epididymis was dissected from the testis. The cauda epididymis was sectioned from the vas deferens and the end of the tubule segment was immersed in a drop of pre-warmed phosphate buffered saline (PBS; pH 7.4, 37 °C) on a clean glass slide to facilitate sperm dispersion into the buffer. The sperm cells were allowed to diffuse into the medium for 2 min. The tissue was removed, and the sperm cells were incubated for 5 minutes until adequately dispersed for analysis. Sperm motility (%) was evaluated by examining the sample at x100 magnification using a phase-contrast microscope (Motic B3; Motic, Carlsbad, CA, USA) equipped with a stage slide warmer set at 37 °C (TCS-100; Amscope, Ivrine, CA, USA). A total of 200 sperm cells were counted and the number of motile sperm cells recorded as percentage epididymal sperm motility.
Sperm vitality was determined after staining with eosin-nigrosin vital stain (Seed et al. 1996). Briefly, equal volume of caudal epididymal sperm suspension and eosin-nigrosin stain were mixed for 30 seconds and a thin smear made on a microscope slide and air-dried. Live sperm (unstained head) and dead sperm (red-stained head) were identified using light microscopy (Motic B3; Motic, Carlsbad, CA, USA) at x1000 magnification under oil immersion. A total of 200 sperm cells were counted and the number of live sperm cells recorded as percentage vitality of the sample. All micrographs were captured using Moticam 2.0 image system (Motic, Carlsbad, CA, USA).
For sperm morphology, a wet mount of caudal epididymal sperm suspension was evaluated for sperm morphology and structural abnormalities at x1000 magnification using phase-contrast microscopy (Motic B3; Motic, Carlsbad, CA, USA). Sperm morphology was also determined using eosin-nigrosin staining method. A total of 200 sperm cells were counted and the number of abnormal sperm cells was recorded. This was expressed in percentage as epididymal total sperm abnormalities (TSA) (Seed et al. 1996).
To determine the sperm concentration, the cauda epididymal tissue was thoroughly minced and homogenized in 2 ml of PBS (pH 7.4). A 1:200 dilution of the homogenate was made in sperm dilution fluid containing formalin and gentian violet stain followed by counting of sperm cells using a haemocytometer (Weber, England). Total sperm count was expressed as number of spermatozoa/g epididymal tissue (Seed et al. 1996).
Histopathology
The testes were fixed in Bouin’s fixative for 8 hours before transferring them to 70 % alcohol. The epididymides were fixed in 10 % neutral-buffered formalin for 48 hours. Both tissues were routinely processed and sectioned at 5µm thickness and stained with haematoxylin and eosin (H&E).
Statistical analysis
The data were subjected to one way analysis of variance (ANOVA) using SPSS version 21. Parasitaemia scores were analysed by Student’s t-test. The variant means were separated post hoc using the least significant difference method. Probability, p < 0.05 was considered statistically significant.