Infection of West African dwarf rams with Trypanosoma brucei brucei and Trypanosoma congolense significantly alter serum electrolytes, redox balance, sperm parameters, and gonadal morphology

Trypanotolerance of the West African dwarf (WAD) breeds may not rule out significant pathophysiological changes that may affect productivity. In this study, the effects of infection of WAD rams with Trypanosoma brucei brucei (Tbb) and Trypanosoma congolense (Tc) on their serum levels of electrolytes [calcium, phosphorus, sodium, potassium]; oxidative stress markers [superoxide dismutase (SOD), malondialdehyde (MDA)]; and sperm parameters [sperm count, motility, vitality, and morphology] were investigated. Fifteen WAD rams, assigned to 3 groups (A, B & C) of 5 rams each, were used for the study. Group A rams were infected with Tbb, while Group B rams were infected with Tc, both intraperitoneally, at the dose of 106 trypanosomes/animal. Group C rams served as the uninfected control. The infections were monitored for 70 days. Serum calcium levels were significantly (p < 0.05) lower in Tbb and Tc infected rams compared to the control throughout the study. Serum sodium was significantly (p < 0.05) higher in the Tb infected rams compared to the Tc infected and control rams on days 14 and 28 PI. Serum SOD activity decreased while MDA levels increased in both infected groups of rams. Tbb infected rams were azoospermic, while Tc infected rams had lower sperm motility, vitality and concentration, and higher number of abnormal sperm cells compared to the control. Necrotic and inflammatory lesions occurred in the testis and epididymis of both infected rams. These results suggest that despite trypanotolerance, trypanosome infections in the WAD rams significantly impact on health and reproduction.


Introduction
African animal trypanosomosis (AAT) is a vector-borne parasitic disease of serious economic importance to livestock production in sub-Saharan Africa, where the disease is endemic (Mulenga et al. 2020). It is caused by extracellular protozoa (trypanosomes), which are cyclically transmitted by several species of tsetse fly (Glossina spp) (Serranito et al. 2021). The tsetse distribution area, or tsetse fly "belt," covers 39 African countries, whose economic development is thus significantly impacted (Alsan 2015). The effects of the disease in livestock are mitigated by chemotherapeutic intervention, which is the most effective and widely applied method for the control of AAT in sub-Saharan Africa (Giordani et al. 2016). Other trypanosomosis control methods involving the use of trypanotolerant livestock and direct tsetse control are sparingly utilized in Africa owing to their expensive and laborious nature (Obi et al. 2019).
Routinely, little importance is attached to trypanosomosis in small ruminants compared to cattle (Griffin 1978). Most available data on the disease in small ruminants are sourced from abattoir reports (Kalejaiye et al. 1995;Nawathe et al. 1995;Dadah et al. 1997). On the other hand, it has been suggested that great economic losses occur in small ruminants due to trypanosome infections (Griffin 1978;Griffin and Allonby 1979;Kalu and Edeghere 1985). Also, these animals have been incriminated as sources/reservoirs of infection to other animals and man (Mahmaud and Elmalik 1977;Scott et al. 1983). This neglect may be due to the misplaced emphasis on trypanotolerance. It was estimated that approximately 32% of sheep and 47% of goats in West and Central Africa are trypanotolerant and this is so because the West African dwarf (WAD) breeds are predominant in these regions (Agyemang 2005;Geerts et al. 2009). Trypanotolerance is defined as a multigenic trait that confers on some breeds of cattle, sheep and goats the ability to survive, reproduce and remain productive under trypanosomosis risk without the need for the use of chemicals to control the vector or drugs to control the parasite, while susceptible breeds die without treatments (Murray et al. 1984;d'Ieteren et al. 1998;Geerts et al. 2009). It had been reported that phenotypically, trypanotolerant livestock can control parasitaemia and anaemia and are less affected by weight loss during infection (Hanotte et al. 2003;Berthier et al. 2016). Despite trypanotolerance, however, pathological changes that may significantly impact on productivity may occur in trypanosome infected WAD breeds.
Calcium, phosphorus, sodium and potassium are electrolytes with diverse biological functions and which are essential for optimal growth, health and reproduction in mammals (Yasothai 2014;Escalera-Valente et al. 2021). These electrolytes participate in the formation of bones and teeth, muscle contraction, permeability of cell membranes, blood clotting, enzymatic reactions, secretion of hormones, metabolism of lipids, carbohydrates and proteins, genetic transmission of nucleic acids, maintenance of normal osmotic pressure, and neural transmission (Devlin 1992;McDowell 1992;Shaker and Deftos 2000). As regards reproductive function, while extracellular sodium, Na ( +), suppresses hyper-activation of sperm motility via Na ( +)-Ca (2 +) exchanger (Takei and Fujinoki 2016), calcium is necessary for spermatogenesis, sperm motility, capacitation, acrosome reaction, and fertilization (Li et al. 2016;Beigi Harchegani et al. 2019).
The fact that imbalances in these mineral elements coupled with oxidative stress, which also affects sperm morphology and physiology, may impact negatively on optimal health and productivity in mammals makes it pertinent to evaluate them in trypanosome-infected WAD rams. Such study may unveil "silent" but significant impacts of the disease in the WAD rams in regions where surveillance, vector control and chemotherapeutic interventions are overlooked while trypanotolerance is emphasized. Moreover, in susceptible animals, trypanosomes have been reported to cause testicular degeneration and inflammation, subnormal sperm count, and structural damage to spermatozoa (Wada et al. 2016;Amin et al. 2020). If similar reproductive lesions occur in the trypanotolerant WAD rams, it would imply that trypanotolerance of the WAD sheep may be overrated. The objective of this study was, therefore, to evaluate the effects of T. brucei brucei (Tbb) and T. congolense (Tc) infections on some serum electrolytes, oxidative stress markers, sperm parameters, serum testosterone levels, and gonadal morphology in WAD rams.

Trypanosomes species/strain used
The trypanosome parasites used were the Federer strains of Tbb and Tc (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 Tbb, while Group B rams were infected with Tc, both intraperitoneally, at the dose of 10 6 trypanosomes/ animal. Group C rams served as the uninfected control.

Estimation of parasitaemia
Parasitaemia was checked daily, beginning from day 2 post-infection of the Tbb-and Tc-infected rams. 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 min 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 min. 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 were 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 h 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 min until adequately dispersed for analysis. Sperm motility (%) was evaluated by examining the sample at × 100 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 eosinnigrosin vital stain (Seed et al. 1996). Briefly, equal volume of caudal epididymal sperm suspension and eosinnigrosin stain were mixed for 30 s 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 microscope (Motic B3; Motic, Carlsbad, CA, USA) at × 1000 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 × 1000 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 h before transferring them to 70% alcohol. The epididymides were fixed in 10% neutral-buffered formalin for 48 h. Both tissues were routinely processed and sectioned at 5 µm 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.

Parasitaemia
All the infected rams became parasitaemic between days 5 and 6 PI and remained parasitaemic till the end of the study. Tbb-infected rams were significantly (p < 0.05) more parasitaemic than Tc-infected rams on days 14, 35 and 56 PI (Fig. 1).

Serum minerals and electrolytes
The mean serum calcium concentrations of the infected rams were significantly (p < 0.05) lower than that of the uninfected control throughout the experiment, starting from day 14 PI for the Tbb-infected rams and day 28 PI for Tc-infected rams (Fig. 2).
Serum phosphorus levels of the Tbb-infected rams were significantly (p < 0.05) lower than those of the control on days 14 and 28 PI. On days 42 and 56 PI, the mean serum phosphorus levels of the Tc-infected rams became significantly (p < 0.05) higher than those of the Tbb-infected rams (Fig. 3). Calcium/phosphorus ratio (Ca:P) was not affected in the Tbb-infected rams (normal Ca:P = 1-2:1) but tilted towards hyperphosphataemia in Tc-infected rams, from day 28 PI (Ca:P = 1:1.2) to day 70 PI (Ca:P = 1:1.5).
Serum sodium levels of Tbb-infected rams were significantly (p < 0.05) higher than those of Tc-infected rams and the control on days 14 and 28 PI. Beyond day 28 PI, there were no significant variations in serum sodium concentrations across the groups (Fig. 4). Potassium concentration in serum did not also vary significantly (p > 0.05) across the groups throughout the period of the experiment.

Oxidative stress markers
The mean serum superoxide dismutase (SOD) activity of the Tbb-infected rams was significantly (p < 0.05) lower T. congolense than those of both the Tc-infected rams and the uninfected control on days 14, 28 and 42 PI. However, by days 56 and 70 PI, it was significantly (p < 0.05) lower than only that of the control. On the other hand, the mean serum SOD activity of the Tc-infected rams was significantly (p < 0.05) lower than that of the control from day 28 PI to the termination of the experiment (Fig. 5).
The mean serum malondialdehyde (MDA) levels of the Tbb-infected rams were significantly (p < 0.05) higher than that of the control on days 42 and 70 PI. Similarly, the mean serum MDA levels of the Tc-infected rams were significantly (p < 0.05) higher than that of the control on days 14 and 42 PI and also significantly (p < 0.05) higher than that of Tbb-infected rams on days 14 and 28 PI (Fig. 6).

Serum testosterone concentration
The mean serum testosterone levels did not significantly vary across the groups from day 14 to day 56 PI, although that of Tc-infected rams dropped steadily from day 28 to day 42 PI compared to that of Tbb-infected rams and the control. However, by day 70 PI, the mean serum testosterone concentrations of the infected rams (Tbb and Tc) were significantly (p < 0.05) lower than the control (Fig. 7).

Sperm parameters: concentration, motility, vitality, and morphology
No spermatozoon was found in the epididymal fluid of the Tbb-infected rams. Sperm cells were present in the epididymides of Tc-infected rams and the control group of rams. The mean serum epididymal sperm count of Tc-infected rams was significantly (p < 0.05) lower than that of the control. The mean percentage sperm motility and vitality of the Tc-infected rams were significantly (p < 0.05) lower than those of the control, while the mean percentage abnormality of spermatozoa of Tc-infected rams were significantly (p < 0.05) higher than that of the control group (Table 1).
Stained smear of the epididymal f luid of Tbbinfected rams did not reveal any sperm cells, only necrotic debris was observed. Motile trypanosomes were found in the epididymal semen of Tc-infected rams (Fig. 8a). The sperm morphological abnormalities observed in Tc-infected rams were headless sperm cells, dead sperm cells, coil-tailed spermatozoa, short-tailed spermatozoa, and cytoplasmic droplets (Fig. 8b). Tc-infected rams had fewer and mainly abnormal sperms cells (Fig. 8b), while control group of rams had a mixture of normal and abnormal sperm cells (Fig. 8c).

Histopathology
The testicular tissues of the Tbb-infected rams showed severe degeneration and necrosis of the germ cells of the seminiferous tubules and mild infiltration of mononuclear cells in the interstices (Fig. 9a). Tc-infected rams also showed severe testicular degeneration and subcapsular oedema with severely thickened interstices due to infiltration of mononuclear inflammatory cells (Fig. 9b). The uninfected rams showed normal germinal cells of the seminiferous tubules and normal interstices (Fig. 9c).
The epididymis of Tbb-infected rams showed severe necrosis and desquamation of the tubular epithelial cells and severe infiltration of mononuclear inflammatory cells into the ductular walls and interstices (Fig. 10a). Tc-infected rams had similar lesions in the epididymis; but the tubular cells were rather vacuolated and the connective tissues were severely thickened due to more severe infiltration of mononuclear inflammatory cells (Fig. 10b). The epididymis of the control group of rams appeared normal with clumps of spermatozoa in the tubules (Fig. 10c).

Discussion
The finding in the present study that mean serum calcium for the infected groups were significantly lower than the values for the control throughout the study, starting from day 14 PI in Tbb-infected group and day 28 PI in Tcinfected group, agrees with the reports of Allam et al. (2011) in pigs in which persistent decline in serum calcium was observed and also with the reports of Da Silva et al. (2011) on rabbits, in which decline in serum calcium was observed on day 35 PI. It is possible that the trypanosomes utilized extracellular calcium for their intracellular metabolism. Calcium is said to be important for flagella function and life cycle of trypanosomes (Docampo and Huang 2015) and for microtubule assembly, release of variant surface glycoprotein and activation of adenylate cyclase (Voorheis and Martin 1981;Bowles and Voorheis 1982;Dolan et al. 1986). The thought that trypanosome infections may have caused disruptions in the functions of the parathyroid glands involved in minute-to-minute fine regulation of blood calcium as was shared in another report of a study in pigs experimentally infected with T.  brucei, in which persistent decline in serum calcium level was observed (Allam et al. 2011) seems likely.
In the later stages of the infections (i.e. from day 42 PI) the mean values for serum phosphorus of the Tc-infected rams were significantly higher than the values for Tbbinfected rams, which in turn was significantly lower than those of the control on days 14 and 28 PI. A previous study (Da Silva et al. 2011) reported a reduction in serum phosphorus level on day 35 PI, which compared favourably with the phosphorus level for Tbb-infected rams in this study, but does not agree with the values for Tc-infected group, in which normal Ca:P ratio (2:1) tilted towards hyperphosphatemia (1:1.5). This implies that calcium/ phosphorus homeostasis regulated by the parathyroid gland may be differently altered in Tc and Tbb infections in the WAD rams.
Mean serum sodium levels of the Tbb-infected rams were significantly higher than the values for the Tc-infected rams on days 14 and 28 PI. There were no significant differences in the mean serum potassium levels of the infected rams and control throughout the study. These results differ from a previous study in rabbits (Da Silva et al. 2011) in which decreased serum levels of both sodium and potassium were reported. The lack of agreement with the present study may be due to animal species differences or possibly a more effective regulation of Na/k homeostasis in the infected WAD rams.
Oxidative stress in the infected rams was characterized by significantly lower SOD activity in both infected groups throughout the study period, and significantly higher MDA levels at the early phase of the infection in Tc-infected rams and at the later phase in Tbb-infected rams. The findings of this study, especially as regards Tc-infected rams, agree with other studies in camels and cattle, which are trypanosusceptible, in which increased MDA levels and decreased SOD activities were reported following T. evansi infection (Saleh et al. 2009;Mishra et al. 2017).
Necrotic lesions in the seminiferous and epididymal tubules were more severe in the Tbb-infected rams. Inflammatory lesions, however, predominated in the testicular and epididymal stromal tissues in the Tc-infected rams. These results agree with previous reports of trypanosome-induced testicular and epididymal degeneration and inflammation in Yankassa rams (Okubanjo et al. 2014). These lesions in the testis and epididymis in addition to the lowered serum testosterone levels may have also contributed to azoospermia reported for Tbb-infected rams and the sperm abnormalities observed in the Tc-infected group, which agree with previous reports of aspermatogenesis in Tbb infection in sheep (Ikede and Losos 1972), gazelle (Mbaya 2007) and sperm abnormalities in cattle infected with Tc (Sekoni et al. 2004). It should be noted also that testosterone is required for normal spermatogenesis (Smith and Walker 2014) and the inflammatory lesions in the testicular stroma, which were more severe in the Tc-infected rams than in the Tbb-infected rams, may have correspondingly affected serum testosterone levels. Furthermore, the finding of motile trypanosomes in the epididymal semen of the Tc-infected rams suggests that this parasite may also be tissue invasive and not strictly haematic (Abebe et al. 1993). Its presence in the semen may have equally caused severe stress to sperm cells, hence, contributing to the reported sperm morphological and physiological alterations.

Conclusion
The findings of this study have revealed that despite trypanotolerance, severe pathological changes, capable of affecting fertility, occurred in WAD rams infected separately with Tbb and Tc. The pathomechanism involved direct stress to sperm cells due to invasion of the cauda epididymis by Tc; degeneration, necrosis and inflammation of the testis and epididymis; and infection-induced redox imbalance and serum electrolyte perturbations, especially calcium. Therefore, trypanosome infections in the trypanotolerant WAD rams significantly affect health and reproduction even in the absence of mortality or clear signs of morbidity.