Influence of Graded Dose of Moringa oleifera Seed Extract Administered Orally on Testicular Pathology, Gonadal and Extra Gonadal Sperm Reserves of Wistar Rats Experimentally Infected with Trypanosoma brucei brucei

Introduction

Animal trypanosomosis is one of the major factors limiting the development of the African livestock industry. (Lemu et al., 2019). Trypanosomosis has continued to cause havoc on animal production in developing countries, both economically and socially. (Abdullahi et al., 2018; Odeniran et al., 2018). African trypanosomosis is a parasitic disease caused by a protozoan parasite of the Trypanosoma genus. (Opaluwa et al., 2015). Tsetse flies transmit this organism, which is a major cause of livestock morbi-dity and mortality (Abubakar et al., 2016). This sco-urge is still a concern for African scientists, who are working on a roadmap to develop prevention and treatment options (Karshima et al., 2016). Trypanosomosis, also known as sleeping sickness or nagana in humans and animals, is caused by African trypanosomes. The trypanosome organisms that affect people and their pets have been classified into two categories (Odeyemi et al., 2015) The haematinic group (Trypanosoma congolense, T. vivax) is contained in plasma, while the tissue invading group (Trypanosoma brucei, T. evansi, T. gambiense, T. rhodesiense, and T. equiperdum) is found in extravascular and intravascular spaces (Krinsky, 2019; Lemu et al., 2019).

As recorded in Yankasa rams by Iliyasu et al. (2014) parasitic disease is characterized by damage to central metabolic organs such as the liver and reproductive organ degeneration. Oxidative stress caused by trypanosome and macrophage behaviors is one of the main factors involved in disease pathogenesis. (Lokugamage et al., 2020). Oxidative stress has been observed in rabbits infected with different trypanosome species in an experimental setting (Ibrahim et al., 2019). Exogenous antioxidants such as ascorbic acid and vitamin E may be provided to infected rats and rabbits to reduce oxidative stress (Lokugamage et al., 2020). In trypanosome-infected animals, this vitamin therapy greatly decreased the degree and incidence of degeneration of tissues and organs, as well as parasitemia and anemia in some cases (Erol et al., 2019). M. oleifera is a highly nutritious plant that contains large amounts of vitamin B6, vitamin C, pro-vitamin A in the form of beta-carotene, magnesium, and protein, among other nutrients. (Chaparro and Suchdev, 2000; Chhikara et al. 2020).

Moringa oleifera (M. oleifera) seed is being tested in a pilot study to see whether its nutrients and phytochemicals agents will help fight tissue damage caused by disease (Iliyasu et al., 2020a). Antibacterial activity and improved glucose tolerance in a rat model of diabetes, inhibition of Epstein-Barr virus activity in vitro, and reduction of skin papilloma in mice are just a few of the pharmacological properties of M. oleifera seeds (Vargas-Sánchez et al., 2019). M. oleifera Lam is one of fourteen species in the Moringaceae family. M. oleifera has anti-cancer, anti-inflammatory, hypoglycemic, and thyroid-status-regulating properties. The study aimed to see if graded doses of M. oleifera aqueous seed extract influenced testicular damage, gonadal and extragonadal sperm reserves, and testicular damage in Wistar rats infected with T. brucei brucie.

Materials and Methods

Twenty-five adult Wistar rats with an average body weight of 120 ± 0.7g were purchased from the Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Maiduguri. The rats were acclimatized for 2 weeks in the Theriogenology Postgraduate Laboratory, University of Maiduguri Faculty of Veterinary Medicine. Thereafter, the twenty-five Wistar rats were randomly divided into five groups: A, B, C, D, and E, and each group has a total of five Wistar rats. The rats were fed with commercial pelleted poultry grower mash (Vital feed®) throughout the experiment, water was available at all times. The rats were housed in grated metal rat cages throughout the experimental period in the Theriogenology Postgraduate Laboratory, Faculty of Vet-erinary Medicine, University of Maiduguri, Nigeria.

Experimental Design

The rats were separated into groups A to E at random and inoculated with 1×10⁹ virulent T. brucei brucei intra-peritoneal as describe by (Odeyemi et al., 2015) rats were allowed to stay one week post-infection to exhibit clinical signs before the commencement of the treatment with M. oleifera seed extract. The rats were treated daily around 10:00 AM for five weeks with 75 mg, 100 mg, 125 mg, and 150 mg of M. oleifera seed extract for groups A, B, C, and D respectively. While group E was untreated control received 0.5 ml of water. Blood samples were collected every Monday between 10:00-11:00 AM for hematological indices as described by (Iliyasu et al., 20014; Opaluwa et al., 2015). All of the rats were humanely sacrificed at the end of the 6-week experiment, and their gonadal and extragonadal sperm reserves were assessed as described by (Iliyasu et al., 2014) testicular organs were harvested for the histopathology of the organs.

Plant Collection and Identification

The whole plant with fruits was collected during the dry season (November- March) at Anguwan Yusi, Sabon-gari Local Government Area of Kaduna State. The plant was authenticated and assigned with Voucher number 0571 by a taxonomist at the Herbarium Unit, Department of Biological Sciences, Faculty of Science, Ahmadu Bello University (ABU), Zaria, Nigeria. The Ahmadu Bello University Committee on Animal Use and Care gave clearance to all experimental protocols with clearance number ABUCAUC/2017/029.

Aqueous Extraction of Moringa oleifera Seed

The seeds M. oleifera were obtained from the fruits that dried under shade for 14 days to ease the shedding of the seeds. The dried seeds were made into powdered form (40 g) was weighed using a weighing balance and transferred into a one-liter beaker. Three hundred milliliters (300 ml) of distilled water were added to the powder and allowed to stand for 48 hours. Thereafter it was heated on a water bath at (60 °C) for 3 hours. Warm water was added continuously to the residue and subsequently filtered. The procedure was repeated three times at 10-15 minutes intervals and then the filtrate was evaporated to dryness on a water bath at (60 °C). The liquid extract was concentrated to dryness in vacuo at 40 °C using a rotary evaporator. The dried extract was kept at 4°C until it was required for use.

Acute Toxicity Study

The acute toxicity effects of an aqueous extract of M. oleifera seeds were investigated according to the method described by Lorke (1983). The experiment was done in two phases. In the phase I trial, nine rats were divided into three groups of three rats each at random. The first, second, and third groups were assigned and administered with M. oleifera aqueous seeds extracts at dose rates of 10, 100, and 1000 mg/kg, respectively, orally. In the second phase (II) of the trials, three rats were placed at random into three groups of one rat each. The groups were individually treated with three different doses of M. oleifera aqueous seeds extract based on the outcome of the initial trial. The median lethal dose (LD50) of M. oleifera aqueous seeds extract as an indication of its acute toxic effect was determined by taking the geometric mean of the highest dose that did not produce death and the lowest dose that produced death.

Source of Parasites

1. brucei brucei were sourced from the Department of Veterinary Parasitology and Entomology, University of Maiduguri, Nigeria. The parasites were injected into apparently healthy Wis-tar rats intraperitoneally at a dose rate of 10⁹ cfu/mL and maintained in other rats by repeated passage according to the method described by Adeyemi et al. (2009). The parasitemia was monitored by preparing a thin film of blood smear obtained from the infected rat tail and viewed under light microscope ×100 magnification in agreement with the method described by Adeyemi et al. (2009).

Monitoring of Infected and Control Rats

The experimental rats were monitored regularly for the development of trypanosomosis clinical symptoms, such as morbidity and mortality. Every four days, parasitemia was first observed, and the degree of parasitemia was estimated by using rapid matching technique identified by (Herbert and Lumsden, 1976).

Detection of Parasitemia

Blood samples were obtained from the cardiac puncture of the rats to evaluate parasitemia following inoculation of the respective infected groups with T. brucei brucei. Wet mount and hematocrit buffy coat methods were used to test the blood samples, while the "rapid matching technique" was used to determine the degree of parasitemia. As described by Herbert and Lumsden, (1976) every four days of the experimental period.

Buffy Coat Examination

Blood was collected in two heparinized microhematocrit capillary tubes filled up to 3/4^th of their volume from each rat. One end of the tube was sealed

with crystal sealant. The tube was sealed at the outer end and put in the microhematocrit centrifuge. The blood was centrifuged for 5 minutes at 10,000 g. To calculate the PCV, the tubes were removed from the hematocrit centrifuge and put on the microhematocrit reader. Anemic animals were described as those with a PCV value of less than 20 % as described by Murray et al. (1983). After reading the PCV, the capillary tubes were cut 1 mm below the buffy coat, including the top layer of the RBC, and the contents were expressed onto a slide and examined for trypanosomes using a dark ground-phase contrast microscope as described by Murray et al. (1983).

Wet Mount Examination

Wet blood films were prepared by placing a drop of blood on a clean, grease-free glass slide and placed a clean coverslip. The film was examined systematically for the presence of trypanosomes using × 40 magnification of the light microscope (Olympus, Japan).

Rapid Matching Technique for the Examination of T. brucei brucei Parasitaemia

Following Wet blood films preparation and examination, several parasites in each field under the microscope were matched with the standard reference pictures according to the method described by Herbert and Lumsden (1976). Each count per field was matched with logarithmic figures obtained from the reference tables. The logarithmic figures were converted to antilog and subsequently converted to absolute numbers, which reflected the number of trypanosomes per ml.

Histological preparations.

Rats in the control and treatment groups had their testes dissected. Before slide preparation, testicular organs were fixed in Bouin's fluid for 48 hours. They were again dehydrated in ethanol at different concentrations, cleared in xylene solution, and embed-ded in paraffin wax Luna (1960). The sections were then cut into 5 μm thick sections, placed on glass slides, and stained with hematoxylin and eosin (H&E) before being examined under light microscopy at ×40 magnifications. Gionee M2, Images plus 2.0 digital camera was used to capture photomicrographs (Motic China Group Ltd. 2014).

Statistical Analyses

Data collected for hematological analysis, gonadal and extragonadal sperm reserves were subjected to one-way ANOVA using GraphPad version 5.0 to determine significant difference P<0.05 among the groups.

Results

On days 7 and 10 after inoculation, the infected rats in groups A and B showed a loss of appetite and marked weakness. The rats in groups C and D were dull and sluggish compared to the rats in Group E. (control group). The infected groups continued with some discharges from the eyes up to day 17 post-inoculation when one of the infected rats in group A died. There was a progressive decreased in body weight and some of the reproductive organs of the infected groups compared to the control group as presented in (Table 1). There was a progressive increase in gonadal and extragonadal sperm reserves of the infected treated groups compared to the control group as presented in (Table 2). Similarly, the mean PCV, WBC, WBC differential and RBC counts, were decreased significantly in group A later

progressively increased in groups C and D compared to the control group as presented in (Table 3).

Table 2. Effects of aqueous seed extract of M. oleifera on gonadal and extragonadal sperm reserves of Wistar rats experimentally infected with T. brucei brucei

Values with different superscript within column are significantly different at P<0.05         
Key: MOSE= M. oleifera seed extract

Table 3. Effects of aqueous seed extract of M. oleifera of haematological parameters of Wistar rats experimentally infected with T. brucei brucei

Values with different superscript within column are significantly different at P<0.05         
Key: PCV=packed cell volume; WBC= white blood cell; RBC= red blood cell; MOSE= M. oleifera seed extract

Histology of the testes

Rats in the control group showed normal seminiferous tubules and interstices in their testes. (Figure 1). The morphology of the testes of rats treated with M. oleifera aqueous seed extract showed a significant reduction in the activity of spermatogenic cells in the seminiferous tubules, as well as hyperemia and fluid exudation into the interstices.

In all of the groups infected with T. brucei brucei, testicular pathology was found, and the severity of the pathology was dose-dependent. The germinal epithelium of the seminiferous tubules of rats given a high dose of M. oleifera aqueous seed extract was found to be involved in spermatogenesis, and the interstices were improved. (Figure 2).

Figure 1. Microphotography of Testis of control group (E) rat shows seminiferous tubules (A) with normal spermatogenic cells and interstitial spaces (B) containing Leydig cells.

Figure 2. Microphotography of testis of the group (A) rats treated with aqueous seed extract of M. oleifera at a dose rate of 75 mg/kg (T) showing spermatogonia with poor seminiferous tubules activities.

Figure 3. Microphotography of testis of the group (D) rats treated with aqueous seed extract of M. oleifera at a dose rate of 150 mg/kg (A) showing spermatogonia with effective seminiferous tubules activities.

Similarly, spermatogonia and seminiferous tubules activities were efficient as shown in (Figure 3).

Discussion

The dimension of anemia is a reliable indicator of disease status and efficient output in trypanosome infected animals (Stijlemans et al., 2018; Abdullahi et al., 2018; Shousha et al., 2020). According to Joachim et al. (2019), trypanosome infection causes anemia in animals as a result of massive erythrophagocytosis by the host's enlarged and active mono-nuclear phagocytic system (MPS). In the current study the low PCV showed in the treated groups may be attributed to acute hemolysis due to the parasites' rapid growth. The hematological findings are consistent with those of previous research (Odeniran et al., 2018; Chaparro and Suchdev, 2019; Goel and Maurya, 2020). The RBC concentration increases in a dose dependant fashion following administration of the M. oleifera aqueous seeds extract. The increase in RBC concentration could be to the antio-xidant properties of the extract and this is consistent the findings of a previous research which showed that trypanosome infection increases the vulnerability of red blood cell membranes to oxidative damage, likely as a result of glutathione depletion or reduction on the surface of red blood cell RBCs as reported by Revin et al. (2019) and Narayan et al. (2019). It was also reported by Raftery et al. (2020) that the degree and duration of parasitemia, and perhaps even the disease status, are generally a reflection of severity of anemia which is in consonant with the findings of the present study where the rats exhibited a protracted parasitemia. The presence of T. brucei brucei, in the current study might by responsible for the decrease in the mean values of PCV, RB, and Hb estimations. Al-Otaibi et al. (2019) and Shahrajabian et al. (2019) reported that the presence of anemia is similarly indicated by the decline in these hematological parameters which is also proven by the findings of the current study.

In the present study, the infected rats showed signs of fatigue and loss of appetite after being inoculated. The rats were dull and uninterested in comparison to the control group and there was an increase in the white blood cell (WBC) count in the infected rats which signified sign of infection, it might be attributable to the augmented role of antioxidant and im-mune stimulant activities of M. oleifera aqueous extract which aided the optimal body immune response mechanism to combat invading parasites by increasing the release of more WBC into circulation which is in accord with the findings of Shittu et al. (2018).

In the present study, there was an increase in gonadal and extragonadal sperm reserves which could stimulate sexual drive of the treated rats and this might also be attributable to the nutritional value and antioxidant properties of M. oleifera aqueous seeds extract as reported by (Sekoni, 1994) and Iliyasu et al. (2020) which was also in lined with the report of Abou-Elkhair et al. (2020) in respect to the traditional herbalists findings in Asia and Africa that used the seed of M. oleifera extract to treat sexual inadequacy, stimulate sexual vigor, and treat trypa-nosomiasis in livestock, without regard for the scientific validity of the claims. Clinical toxicity symp-toms such as respiratory distress, salivation, anemia, extreme weight loss, and hair change were not observed in all groups, although one death was reco-rded in group A during the second week of the trial, which could be attributed to a decline in immune status which was similar to the finding of Shahrajabian et al. (2019).

In addition, the T. brucei brucei infection damaged the morphology of the testes, which was characterized by a decrease in spermatogenic activity, oligospermia in the lumen of the seminiferous tubules, and the presence of interstitial exudates is also similar to the findings of Mutwedu et al. (2019). These findings are also in line with a previous study reported by Obidike et al. (2007) and Mohamed et al. (2019). In the present study, it was observed that M. oleifera seed extract at a dose rate of 150 mg/kg could enhance the gonadal sperm reserve characteristics and cause substantial amelioration of T. brucei brucei infection. This effect was also observed in group given 100 mg/kg of M. oleifera aqueous seeds extract as against 75 mg/kg of M. oleifera aqueous seeds extract in Wistar rats.

Conclusion

The current findings showed that ingestion of M. oleifera aqueous seed extract improves reproductive success in adult male rats and mitigates the effects of T. brucei brucei on rat testes. It also supports traditional healers' claims about the use of M. oleifera seeds as a semen quality enhancer or medicine. As a result, this study has shown that restoring the spermatogenic activities of the testes during trypanoso-masis is successful and healthy, as well as serving as an alternative treatment for trypanosomiasis in rats.

Acknowledgments

We sincerely appreciate the Head of Surgery and Theriogenology College of Veterinary Surgeon Nigeria, Zaria Study Centre, Prof. Hassan, A. Zoaka for his wonderful support and encouragement.

Conflict of Interest

The authors declared no conflicts of interest exist.