Abstract
Aim: To investigate the impact and possible mechanism of action of the rodent malarial parasite on reproduction.
Methods: Male albino mice were infected with 15, 30 and 45%Plasmodium berghei berghei through inoculation with 107 parasitized red blood cells. Each experiment had its own control that was not infected with P. berghei berghei. Mice infected with 15%P. berghei berghei were killed on days 0, 5, 10 and 15; those infected with 30%P. berghei berghei were killed on days 0, 3, 6 and 10; and those infected with 45%P. berghei berghei were killed on days 1–7 after infection. Caudal epididymal sperm motility, counts and morphology, body and wet organ weights and hematological indices were determined.
Results: The results showed a progressive duration dependent decrease in sperm motility, sperm count and viability (P < 0.01) in parasitized mice. There were significant decreases in serum testosterone and increases in cortisol levels (P < 0.05) in the infected mice compared with the controls. There was also a progressive decrease (P < 0.05) in red blood cell count and packed cell volume. However, there was a progressive increase (P < 0.01) in white blood cell count and weight of the spleen and liver. There was no significant change in weight of the testis and epididymides.
Conclusion: The results suggest that the malaria parasite could depress male fertility indices. (Reprod Med Biol 2006; 5: 201–209)
Keywords: malaria, male, mice, Plasmodium, reproduction
INTRODUCTION
MALARIA IS A devastating parasitic disease, which affects many people in the world and it accounts for many deaths annually. Despite the extensive program for drug control and eradication instituted by the World Health Organization, malaria remains one of the greatest causes of illness and death in the world, particularly in Africa.
Malaria chemotherapy has been associated with adverse effects on reproductive functions. For instance, the antifertility activities of chloroquine, quinine, quinacrine and pyrimethamine 1 , 2 , 3 , 4 and the extracts of Azadirachta indica, Quassia amara, Carica papaya, Morinda lucida and Alstonia boonei have been well documented. 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 Chloroquine has been shown to reduce the fertilizing capacity of epididymal sperm in rats, it has also been reported to reduce the mean seminiferous tubular volume and spermatid count per testis. We have recently reported the antifertility effect of artemisinin in male rats. 13 Extracts of Azadirachta indica, Quassia amara, Carica papaya and Morinda lucida are used in folkloric medicine to treat malaria and they have been shown to possess potent antimalarial properties. 14 , 15
Despite the expanding studies on the impact of antimalarial drugs on male reproductive functions, there appears to be no information in the literature on the impact of the malaria parasite on reproductive functions in males. In view of the negative impact of the manifestations of malaria, such as fever, 16 , 17 investigation into the impact of the malaria parasite on male reproduction is imperative. Therefore, the present study reports the impact of Plasmodium berghei berghei on the fertility of male mice. Serum levels of testosterone and cortisol were measured with a view to explaining the probable mechanism of action of the parasite on male reproduction. Plasmodium berghei berghei was identified in the middle of 19th century as the first Plasmodium of rodents such as mice, rats and hamsters. It has since been used to identify the effective therapeutic action of antimalarial compounds. 15 , 18
MATERIALS AND METHODS
Animals
MALE SWISS ALBINO mice weighing between 18 and 22 g were used for the present study. The animals were obtained from the animal house of the Lagos University Teaching Hospital, Idi‐Araba, Lagos, Nigeria. They were housed in groups of five mice per cage on a bed of sterilized wood shavings at about 25°C room temperature. The animals had free access to mouse cubes (Ladokun feeds, Ibadan, Nigeria) and water ad libitum.
Parasites
Anka 1 N‐strain of the malaria parasite, P. berghei berghei, maintained in mice by serial passaging from mouse to mouse was used throughout the experiment. The donor mouse was obtained from National Institute of Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria. The method of blood‐induced Plasmodium infection in mice first described by Peters 19 and used by others 15 was employed in the present study.
Other materials
Analar grade 0.9% saline, disposable plastic syringes and needles (B.B. Pharmaceuticals, Manati, Puerto Rico, USA), Olympus research light microscope, microscope slides and cover slips (Mono International, Surulere, Lagos, Nigeria), improved Neubauer hemocytometer, weighing balance (Microwa Swiss, Greifensee, Switzerland), Giemsa's/Nigrosin stain (Fisher Scientific Company, AL, USA) and Eosin (BDH Chemicals, Poole, UK) were used in the present study.
Study protocol
The present study was divided into three experiments. Three donor mice with parasitemia levels of 15, 30 and 45% were used for experiments 1, 2 and 3, respectively. Blood was obtained from each donor mouse by cardiac puncture using a sterilized syringe containing 10 IU of heparin. This blood was suitably diluted with sterile normal saline so that the final inoculum (0.2 mL) for each mouse contained the required number of 107 parasitized red blood cells as described by Peters, 20 and Knight and Williamson. 21
Experiment 1
Twenty male albino mice were divided into four equal groups. Group I was the control group and received 0.2 mL normal saline intraperitoneally (i.p.). The innoculum as 0.2 mL from 15% parasitemia mouse donor was introduced i.p. into each of the mice in groups II, III and IV. The i.p. route of administration has been found to be suitable since Thurston's work. 22 Mice in groups II, III and IV were killed on days 5, 10 and 15 after P. berghei berghei infection, respectively. The control mice were also killed on day 15.
Experiment 2
Twenty male albino mice divided into four equal groups were used. Group I was the control group and received 0.2 mL of normal saline i.p. The innoculum as 0.2 mL from 30% parasitemia mouse donor was introduced i.p. into each of the mice in groups II, III and IV. Mice in groups II, III and IV were killed on days 3, 6 and 10 after P. berghei berghei infection, respectively. The control mice were also killed on day 10 after P. berghei berghei infection.
Experiment 3
Forty male albino mice were divided into eight equal groups of five mice each. Group I was the control group and the mice were not infected with P. berghei berghei. The innoculum as 0.2 mL from 45% parasitemia mouse donor was introduced i.p. into each of the mice in the remaining groups (II–VII). The test mice were infected on day 1 and killed on days 1, 2, 3, 4, 5, 6 and 7, respectively, after P. berghei berghei infection. The control mice were also killed on day 7.
Autopsy
The percentage parasitemia levels and bodyweight of each mouse was determined daily in the three experiments. At the end of each experimental period, mice were killed by cervical dislocation. Blood was collected by carefully transecting the caudal vein. Microscope slides were smeared with a thin film of the blood and the percentage parasitemia level was determined daily. The packed cell volume, red blood cell and white blood cell counts were determined. The liver, heart, spleen, kidneys, testes and epididymides were removed and weighed. Epididymal sperm was collected for sperm characteristics evaluation as earlier described. 9 , 10 , 11 , 12 , 13 Serum samples collected were stored at −20°C until assayed. Serum levels of testosterone and cortisol were measured using the tube based enzyme immunoassay (EIA) technique. The EIA kits used were obtained from Immunometrics (London, UK), and contained testosterone and cortisol EIA substrate reagents and EIA quality control samples. A quality control sample was run for each hormone at the beginning and at the end of the assay to ascertain acceptability with respect to bias and within assay variation. The EIA kit used had a sensitivity level of 0.3 nmol/L (0.1 g/mL). The intra and interassay variations were 10.02% and 10.12%, respectively, for testosterone and 9.01% and 10.0% for cortisol.
Statistical analysis
Data were expressed as mean ± standard error of the mean (M ± SEM) and analyzed by using the Student's t‐test and anova where appropriate. P < 0.05 was accepted as statistically significant.
RESULTS
Parasitemia levels
THE RESULTS SHOWED that once the infection was established, the parasitemia level in the animals increased sharply towards the peak (Fig. 1). The average parasitemia levels in 15%, 30% and 45%P. berghei berghei infected mice on days 15, 10 and 7, respectively, was about 50%.
Figure 1.
Percentage parasitemia levels in adult mice infected with Plasmodium berghei berghei. (□) 45%Plasmodium, (▪) 30%Plasmodium, () 15%Plasmodium.
Body and organ weight
1, 2, 3 show that there was no significant change in the bodyweight of the test mice when compared with the control. All mice gained almost the same weight. Similarly, there was no significant change in the weight of the heart and testes. There was a progressive and significant increase (P < 0.01) in that of the spleen, liver and kidney in the P. berghei berghei infected mice compared with the control.
Table 1.
Body and organ weights of 15%Plasmodium berghei berghei infected mice
| Weight (g) | Group I (control) | Group II Day 5 | Group III Day 10 | Group IV Day 15 |
|---|---|---|---|---|
| Bodyweight | 19.50 ± 0.80 | 20.00 ± 0.75 | 20.50 ± 0.90 | 21.00 ± 0.90 |
| Testis | 0.71 ± 0.03 | 0.72 ± 0.02 | 0.74 ± 0.03 | 0.75 ± 0.02 |
| Spleen | 0.40 ± 0.03 | 0.63 ± 0.02 | 0.70 ± 0.04 | 0.75 ± 0.04 |
| Liver | 0.65 ± 0.05 | 0.71 ± 0.04 | 0.80 ± 0.05 | 0.94 ± 0.06 |
| Kidney | 0.10 ± 0.03 | 0.13 ± 0.03 | 0.15 ± 0.04 | 0.19 ± 0.04 |
| Heart | 0.09 ± 0.01 | 0.09 ± 0.02 | 0.10 ± 0.02 | 0.11 ± 0.03 |
Table 2.
Body and organ weights of 30%Plasmodium berghei berghei infected mice
| Weight (g) | Group I (control) | Group II Day 3 | Group III Day 6 | Group IV Day 10 |
|---|---|---|---|---|
| Bodyweight | 20.00 ± 0.90 | 20.50 ± 0.80 | 21.00 ± 0.90 | 21.50 ± 0.90 |
| Testis | 0.70 ± 0.03 | 0.73 ± 0.02 | 0.74 ± 0.03 | 0.76 ± 0.02 |
| Spleen | 0.40 ± 0.03 | 0.53 ± 0.02 | 0.62 ± 0.03 | 0.74 ± 0.04 |
| Liver | 0.69 ± 0.05 | 0.76 ± 0.05 | 0.90 ± 0.05 | 1.00 ± 0.04 |
| Kidney | 0.10 ± 0.02 | 0.14 ± 0.04 | 0.17 ± 0.03 | 0.19 ± 0.04 |
| Heart | 0.10 ± 0.02 | 0.11 ± 0.03 | 0.11 ± 0.02 | 0.12 ± 0.04 |
Table 3.
Body and organ weights of 45%Plasmodium berghei berghei infected mice
| Weight (g) | Control | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
|---|---|---|---|---|---|---|---|---|
| Bodyweight | 19.00 ± 0.80 | 19.30 ± 0.80 | 19.35 ± 0.80 | 20.00 ± 0.90 | 20.20 ± 0.90 | 20.25 ± 0.95 | 20.50 ± 0.90 | 21.00 ± 0.95 |
| Testis | 0.70 ± 0.02 | 0.70 ± 0.03 | 0.71 ± 0.02 | 0.72 ± 0.03 | 0.71 ± 0.02 | 0.71 ± 0.03 | 0.73 ± 0.04 | 0.75 ± 0.03 |
| Spleen | 0.38 ± 0.05 | 0.40 ± 0.05 | 0.48 ± 0.04 | 0.52 ± 0.05 | 0.61 ± 0.04 | 0.68 ± 0.04 | 0.71 ± 0.05 | 0.79 ± 0.02 |
| Liver | 0.68 ± 0.04 | 0.70 ± 0.04 | 0.71 ± 0.05 | 0.75 ± 0.04 | 0.78 ± 0.05 | 0.80 ± 0.05 | 0.88 ± 0.06 | 1.02 ± 0.02 |
| Kidney | 0.10 ± 0.02 | 0.11 ± 0.01 | 0.12 ± 0.02 | 0.13 ± 0.02 | 0.14 ± 0.02 | 0.15 ± 0.03 | 0.16 ± 0.05 | 0.18 ± 0.04 |
| Heart | 0.09 ± 0.03 | 0.09 ± 0.04 | 0.10 ± 0.03 | 0.09 ± 0.04 | 0.09 ± 0.02 | 0.10 ± 0.01 | 0.10 ± 0.02 | 0.10 ± 0.03 |
Hematology
Hematological studies showed that the packed cell volume and red blood cell count decreased progressively (P < 0.01) from the beginning to the end of the experiment in 15, 30 and 45%P. berghei berghei infected mice. However, there was a progressive increase (P < 0.05) in white blood cell count when the control was compared with the P. berghei berghei infected mice in the three experiments (4, 5, 6).
Table 4.
Hematological parameters of 15%Plasmodium berghei berghei infected mice
| Blood parameter | Group I (control) | Group II Day 5 | Group III Day 10 | Group IV Day 15 |
|---|---|---|---|---|
| PCV (%) | 44.00 ± 2.50 | 39.10 ± 2.00 | 32.00 ± 2.40 | 24.50 ± 3.00 |
| RBC ×106/mL | 7.10 ± 0.80 | 6.50 ± 0.75 | 4.20 ± 0.70 | 3.00 ± 0.60 |
| WBC ×103/mL | 4.50 ± 0.18 | 5.10 ± 0.30 | 5.60 ± 0.25 | 6.00 ± 0.30 |
PCV, packed cell volume; RBC, red blood cell count; WBC, white blood cell count.
Table 5.
Hematological parameters of 30%Plasmodium berghei berghei infected mice
| Blood parameter | Group I (control) | Group II Day 3 | Group III Day 6 | Group IV Day 10 |
|---|---|---|---|---|
| PCV (%) | 43.60 ± 3.15 | 38.52 ± 3.40 | 27.50 ± 3.00 | 25.00 ± 3.00 |
| RBC ×106/mL | 7.50 ± 0.72 | 6.70 ± 0.64 | 4.65 ± 0.54 | 3.20 ± 0.50 |
| WBC ×103/mL | 4.66 ± 0.30 | 5.12 ± 0.25 | 5.40 ± 0.30 | 5.61 ± 0.31 |
PCV, packed cell volume; RBC, red blood cell count; WBC, white blood cell count.
Table 6.
Hematological parameters of 45%Plasmodium berghei berghei infected mice
| Blood parameter | Control | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
|---|---|---|---|---|---|---|---|---|
| PCV (%) | 44.40 ± 3.00 | 40.40 ± 3.41 | 38.21 ± 3.15 | 35.20 ± 2.87 | 30.11 ± 3.00 | 28.00 ± 2.90 | 26.51 ± 3.00 | 25.60 ± 2.50 |
| RBC ×106/mL | 8.15 ± 0.64 | 8.05 ± 0.53 | 7.50 ± 0.54 | 6.60 ± 0.59 | 5.48 ± 0.60 | 4.00 ± 0.67 | 3.15 ± 0.60 | 3.05 ± 0.57 |
| WBC ×103/mL | 4.71 ± 0.19 | 4.90 ± 0.20 | 5.00 ± 0.21 | 5.15 ± 0.22 | 5.20 ± 0.19 | 5.35 ± 0.21 | 5.40 ± 0.22 | 5.50 ± 0.21 |
PCV, packed cell volume; RBC, red blood cell count; WBC, white blood cell count.
Sperm characteristics
7, 8, 9 show the sperm characteristics of 15, 30 and 45%P. berghei berghei infected mice, respectively. There was a significant decrease (P < 0.01) in sperm motility in the three parasitemia levels from the second day of infection to the end of the experiment. The sperm count also decreased significantly immediately after the infection. The viability percentage of spermatozoa decreased significantly (P < 0.01) on the 6th and 7th day of infection in the 45%P. berghei berghei infected mice. In contrast, however, the epididymal volume and morphology of the spermatozoa were unchanged (7, 8, 9).
Table 7.
Sperm characteristics of 15%Plasmodium berghei berghei infected mice
| Sperm Characteristics | Group I (control) | Group II Day 5 | Group III Day 10 | Group IV Day 15 |
|---|---|---|---|---|
| Sperm Motility (%) | 85.00 ± 1.82 | 76.00 ± 2.90 | 65.40 ± 2.07 | 51.0 ± 2.00 |
| Epididymal Volume (%) | 1.50 ± 0.19 | 1.51 ± 0.10 | 1.52 ± 0.10 | 1.50 ± 0.10 |
| Morphology (%) | 92.50 ± 1.31 | 93.41 ± 1.63 | 93.00 ± 1.50 | 90.70 ± 1.04 |
| Sperm Counts (106/mL) | 74.50 ± 0.89 | 69.00 ± 1.10 | 60.01 ± 2.30 | 45.0 ± 4.21 |
| Viability (%) | 89.8 ± 1.30 | 84.40 ± 1.38 | 80.00 ± 0.46 | 70.50 ± 1.60 |
Table 8.
Sperm characteristics of 30%Plasmodium berghei berghei infected mice
| Sperm Characteristics | Group I (control) | Group II Day 3 | Group III Day 6 | Group IV Day 10 |
|---|---|---|---|---|
| Sperm Motility (%) | 84.50 ± 1.70 | 74.00 ± 2.80 | 66.10 ± 2.80 | 50.00 ± 2.00 |
| Epididymal Volume (%) | 1.52 ± 0.19 | 1.52 ± 0.10 | 1.51 ± 0.11 | 1.50 ± 0.10 |
| Morphology (%) | 93.50 ± 1.28 | 93.48 ± 1.80 | 93.01 ± 1.50 | 90.70 ± 1.00 |
| Sperm Counts (106/mL) | 72.50 ± 0.89 | 68.40 ± 1.10 | 60.40 ± 2.50 | 44.00 ± 4.20 |
| Viability (%) | 88.8 ± 1.30 | 85.30 ± 1.20 | 80.00 ± 1.40 | 70.60 ± 1.50 |
Table 9.
Sperm characteristics of 45%Plasmodium berghei berghei infected mice
| Sperm Characteristics | Control | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 |
|---|---|---|---|---|---|---|---|---|
| Sperm Motility (%) | 85.6 ± 1.72 | 83.2 ± 1.28 | 78.0 ± 2.96 | 72.2 ± 2.61 | 68.4 ± 2.87 | 68.0 ± 3.11 | 64.5 ± 3.23 | 61.0 ± 2.01 |
| Epididymal Volume (%) | 1.56 ± 0.17 | 1.56 ± 0.16 | 1.52 ± 0.11 | 1.52 ± 0.05 | 1.52 ± 0.12 | 1.55 ± 0.10 | 1.55 ± 0.10 | 1.50 ± 0.10 |
| Morphology (%) | 93.56 ± 1.38 | 94.01 ± 1.50 | 93.48 ± 1.83 | 94.10 ± 1.30 | 93.81 ± 1.50 | 93.64 ± 1.22 | 93.53 ± 1.21 | 93.72 ± 1.01 |
| Sperm Counts (106/mL) | 72.8 ± 0.87 | 71.8 ± 0.72 | 69.4 ± 1.12 | 68.6 ± 1.38 | 63.6 ± 2.50 | 58.5 ± 0.58 | 51.0 ± 1.96 | 46.0 ± 4.24 |
| Viability (%) | 87.8 ± 1.39 | 87.4 ± 1.36 | 87.4 ± 1.29 | 87.2 ± 1.50 | 87.0 ± 0.43 | 85.3 ± 0.48 | 80.5 ± 1.33 | 76.5 ± 1.50 |
Serum testosterone and cortisol levels
As shown in 2, 3, 4, there was a progressive decrease and a similar progressive increase (P < 0.05) in serum levels of testosterone and cortisol, respectively, in mice infected with P. berghei berghei at 15, 30 and 45% levels when compared with the respective control groups.
Figure 2.
Serum testosterone and cortisol levels in mice infected with 15%Plasmodium berghei berghei.
Figure 3.
Serum testosterone and cortisol levels in mice infected with 30%Plasmodium berghei berghei.
Figure 4.
Serum testosterone and cortisol levels in mice infected with 45%Plasmodium berghei berghei.
DISCUSSION
THE RESULTS SHOWED that i.p. inoculation of mice with P. berghei berghei produced a significant level of parasitemia, which increased progressively up to the end of the experiment. The parasites could have penetrated the intraperitoneal wall and entered into the blood stream before parasitemia was established. This is consistent with the finding in a previous study. 23 The increased size of the spleen and the liver could be the result of increased stimulation of phagocytic activity in these organs 24 and extramedullary erythropoeisis. 25 The observed hepatomegaly had also been reported as a common feature of malaria infection in humans, although variable in experimental animals because during the progress of infection, the phagocytic endothelial cells lining the liver sinusoids transform into Kupffer cells, dividing rapidly to increase the number of macrophages. 24 The unchanged weight of the heart appears to be in concordance with the finding of Kreier, 26 who reported no striking gross cardiological changes during P. berghei berghei infection. The leukocytes count, which was found to increase in the test mice, is in agreement with the leucocytosis 27 which could be the result of the immunological response of the host mice to invasion by the P. berghei berghei. Furthermore, the observed morphological distortions in the red blood cells might have led to the destruction of the red blood cells and could explain the decrease in red cell count. Kreier et al. 28 reported that Plasmodium falciparum infected erythrocytes tend to be smaller than normal with some degrees of crenation of erythrocytes in humans.
The results of the present study are indicative of the ability of P. berghei berghei and therefore malaria to inhibit fertility in male mice. The reduction in caudal epididymal sperm motility and sperm counts in the mice substantiates the depressant nature of the malaria parasite on spermatozoa activities. The P. berghei berghei might affect the caudal epididymis directly resulting in decreased epididymal sperm counts due probably to a direct effect of the parasite at the epididymal site by producing spermatoxic agent(s) on maturing or mature spermatozoa. 29 Malaria is caused by Plasmodium. Infection in humans begins with the bite of an infected female anopheles mosquito, which releases Plasmodium sporozoites from its salivary glands into the human blood stream. The sporozoites invade the liver cells (hepatocytes) and differentiate during the next 14 days, resulting in tens of thousands of merozoites which burst, from the hepatocyte. 30 Some merozoites in the erythrocyte develop into gametocytes, which are ingested when a female anopheles mosquito bites the infected person. In the mosquito's gut, the gametes (sperms and eggs) unite to form the zygote that divides repeatedly to form more sporozoites. The sporozoites migrate to the salivary glands of the mosquito and when the mosquito bites another person, the life cycle begins again. The clinical manifestations of malaria, severe chills, fever, sweating, fatigue and intense thirst, are associated with the synchronous rupture of the infected erythrocyte. The pyrogens released during the life cycle of the malaria parasite from the host red blood cells usually leads to fever and an increase in body temperature. 30 The adverse effects of increased temperature on spermatogenesis have been well documented. 17 , 31 , 32 Indeed, exposure to radiant heat had been reported to reduce sperm counts and adversely change reproductive functions. 31 , 32 Thus, the progressive decrease in sperm counts and motility observed in the present study could be mediated through the pyrogens.
The hypothalamic function is usually affected by stress. Furthermore, hormonal alterations have been observed in the parasitized male mice, which could be related to the stress of malaria. In response to the stress of malaria 33 it appears that the negative feedback is abolished, probably leading to a reduction in corticotrophin releasing hormone (CRH) from the hypothalamus, adrenocorticotrophic hormone (ACTH) from the anterior pituitary and an increase in the secretion of cortisol. This could have given rise to a chain of events causing an increase in cortisol secretion as observed in the present study. The adrenal derive increases during stress, thereby complementing the hypothalamic adenohypophyseal gonadal negative feedback action. 34 , 35 The differential androgen threshold requirement in the genital tract and the requirement of a higher androgen threshold in the epididymis is also a plausible factor. It should be noted that all maturational events of spermatozoa taking place in the epididymis are vulnerable to chemical interference. 36 , 37 , 38 Thus, the observed reduction in spermatozoa parameters might be the result of the altered microenvironment in the epididymis, which disturbed the metabolism of spermatozoa in parasitized mice because of the reduction in testosterone secretion. Reduction in testosterone concentration could also be related to hypothalamus‐pituitary feedback on the testis, which was probably affected by the stress of malaria. Recently, Muehlenbein et al. 39 reported a positive relationship between testosterone levels and Plasmodium vivax parasitemia in adult human males. Furthermore, the human males infected with P. vivax exhibited significantly lower testosterone levels and significantly higher cortisol levels than healthy individuals. 39 This report supports the findings in male mice infected with P. berghei berghei in the present study. Whether P. berghei berghei has a direct effect on testosterone and cortisol secreting glands is not known. Further studies are expected to shed more light on this. In conclusion, the malaria parasite could depress reproductive activities at least during the period of infection.
ACKNOWLEDGMENTS
WE EXPRESS OUR gratitude to the University of Ibadan, for financial support through a Senate Research Grant SRG/COM/2000/11A to YR.
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