Evaluation of the Effects of Administration of Aqueous Root Extract of Chrysophylum albidum on Fertility in Male Wistar Albino Rats

This study evaluated the effects of administration of aqueous root extracts of Chrysophyllum albidum on sperm characteristics and histology of the epididymis of experimental rats. Forty-five adult male Wistar rats were randomized into 3 groups of 15 rats each. Each group was further randomized into 3 subgroups of 5 rats each, according to the dose and duration of extract administration. Administration of the extract for 8 weeks resulted in significant (P < 0.05) decreases in the number of progressively motile sperm cells of rats in subgroup B3 (75.00 ± 0.00 %) and C3 (75.00 ± 0.00 %) as compared with the control subgroup A3 (80.00 ± 0.00 %). Also, there were significant (p < 0.05) decreases in the number of morphologically normal sperm cells of rats in test subgroups B3 treated with low dose and C3 treated with high dose. However, there were no significant histological changes in the epididymis of the experimental rats in all the treated groups, at different doses and duration. Administration of aqueous root extract of Chrysophyllum albidium at varying doses and time duration negatively affects sperm motility and morphology which may compromise the fertility of the experimental rats.


Introduction
In Africa, thousands of plants have been used and are still been used for medicinal purposes. The use of these medicinal plants for therapeutic purposes transcends all cultures and traditions. It is a vital approach that is easily accessible to the populace and at little or no cost. An important aspect of reproductive health is the regulation of fertility, i.e. contraception and management of infertility [1]. Over the years, there has been an increase in the search for male contraceptives of natural origin with the lowest possible side effect [2]. Although there are many contraceptive agents with varying side effects, several medicinal plants have been shown to exhibit male contraceptive properties by either suppressing spermatogenesis or by spermicidal action [3].
Medicinal plants are herbal remedies for the treatment and management of different ailments [4]. Chrysophyllum albidum, also known as White Star Apple or African Star Apple, is a popular medicinal plant which belongs to the family Sapotaceae. Phytochemical analyses show that Chrysophyllum albidum contains alkaloids, tannin, saponin, phenol and flavonoid [5]. The different parts of the plants; leaves, bark, seeds and fruits, has its own medicinal value. The leaf is used as a skin moisturizer and for the treatment of skin eruption, stomach ache and diarrhoea [6]. The bark decoction is often used in the treatment of diseases such as yellow fever and malaria [7]. Onyeka et al. [8] reported that the root bark has anti-fertility effects while Adewoye et al. [9] reported that the stem bark has antimicrobial effects. The seed cotyledons are used as ointments in the treatment of vaginal and skin infections [10]. However, the extensive, long-term usage of herbal products without proper monitoring has brought about a number of health related problems like infertility, a

Body and organ weight measurements
The body weights of the rats were taken weekly while their organ weights were taken at the end of the experiment (after sacrifice) using a Top loader weighing balance. The differences in body weights were determined.

Determination of LD 50
The LD 50 of Chrysophyllum albidum root bark as determined by Adewoye et al. [12] is 1850mg/kg body weight.

Sacrifice of animals
At the end of each experimental period (i.e. at week 4, week 6 and week 8), a transverse incision was made through the ventral wall of the abdomen of each rat under slight chloroform anaesthesia. The epididymis were excised and fixed in Bouin's fluid in readiness for routine histological procedure. Sperm cells were collected from the vas deferens of the sacrificed rats, Sperm analysis were conducted immediately the sperm cells were excised from the vas deferens.

Sperm analysis
Sperm cells were collected from the epididymis during the sacrifice by litigating the extremities of the vas deferens to a length of about 36nm and 6μl normal saline was added to the litigated extremities. The litigated extremities of the vas deferens were placed on a Petri dish and teased to allow the sperm cells diffuse. The section was viewed under the microscope for sperm motility. The results were evaluated using two variables as stated below: 1) Progressive (or qualitative) motility, usually expressed in terms of mean Progressive motility score -Progressive Motility (PM), Non Progressive Motility (NPM), Immotile (IM).
Vitality test was carried out to evaluated living and dead spermatozoa. This was done with a supra-vital stain (eosin Y). Sperm Morphology was evaluated by making a smear of sperm cell on a grease free slide and allowing it to air dry. The sample was stained using the Bryan-Leishman technique [20] for 30 mins. The smear was rinsed with distilled water, bloated and finally air dried. It was then viewed under oil immersion objective lens. The scores or results were recorded as a percentage of normal and abnormal sperm cells. Normal sperm shows a normal sperm characteristic of head, axoneme, middle piece and a tail. Abnormal sperm cells were characterized by large heads; headless, tailless, bulgy mid-piece curved tail and joined head.

Histological procedure
The tissues were fixed in Bouin's fluid for less than 24hours. The tissues were then processed via paraffin wax embed method of Drury and Wallington [13] and Scheehan and Brapchak [14].

Haematoxylin and eosin staining method
Staining of the tissues was done using H & E dyes (Haematoxylin and Eosin dyes).

Photomicrography
The sections were examined under a Leica DM750 research microscope with a digital camera Leica ICC50 attached. Digital photomicrographs of the tissues section were taken at various magnifications.

Statistical analysis
The data generated were analyzed using descriptive and inferential statistics. Data were presented as Mean ± Standard Error of Means (S.E.M). Significance difference of means was determined using one-way analysis of variance (ANOVA) at 95% confidence interval. Least Square Difference (LSD) and post-hoc tests were carried out for all groups. All statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) (version 17) manufactured by International Business Machine Corporation (IBM) in Armonk, New York.

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Vol. 10

Results
The effects of administration of aqueous root extracts of Chrysophyllum albidum on the body weights, organ weights, sperm characteristics and histology of the epididymis of the experimental rats are as shown below; Effect on body weight   Effect on total sperm count Figure 6. Effect of aqueous root extract of Chrysophyllum albidum on the total sperm count of the experimental rats at the 4th week (day 28), 6th week (day 42) and 8th week (day 56) of the experiment At the 4th week of extract administration (Fig. 6), there was no significant difference (p > 0.05) between the total sperm count of the rats in the control group A1 (34.00 ± 4.00 x 106 cells/ml) and those of the treated groups (B1: 36.50 ± 1.5 x 106 cells/ml and C1: 35.50 ± 3.50 x 106 cells/ml).

Volume 10
At the 8th week of administration (Fig. 6), the total sperm count of rats in the control group A3 (34.00 ± 2.00 x 106 cells/ml) was not significantly different (p > 0.05) from those in the treated groups (B3: 32.00 ± 1.0 x 106 cells/ml and C3: 30.00 ± 2.00 x 106 cells/ml). At the 4th week (day 28) of extract administration, there was no significant difference (P > 0.05) between the number of progressively motile sperm cells of rats in subgroup B1treated with low dose (80.00 ± 0.00 %) and that of the control rats A1 (80.00 ± 0.00 %). However, there was a significant (P < 0.05) decrease in the number of progressively motile sperm cells in sub group C1 treated with high dose (75.00 ± 5.00 %) than the control A1 (80.00 ± 0.00 %). Also, there were significant (P < 0.05) decreases in the percentages of non-progressively motile sperm of rats in subgroup B1 (10.00 ± 0.00 %) and subgroup C1 (10.00 ± 0.00 %) than those of rats in the control group A1 (15.00 ± 0.00 %). The administration of the extract for 4 weeks caused significant (P < 0.05) increases in the percentages of immotile sperm of the experimental rats in subgroups B1 and C1 (10.00 ± 5.00 % and 15.00 ± 0.00 % respectively) than those of the control rats A1 (5.00 ± 0.00 %) (Fig. 7).

Effect on sperm morphology
Administration of aqueous root extract of Chrysophyllum albidium for 6 weeks (Fig. 8) resulted in significant (P < 0.05) decreases in number of morphologically normal sperm cells of rats in subgroup B2 treated with low dose of the extract (36.50 ± 1.5 x 106cells/ml; 75.00±5.00 %) and At the 8th week of experiment, administration of the extract caused significant (p < 0.05) decreases in the number of morphologically normal sperm cells of rats in test subgroups B3 treated with low dose (32.00±1.0 x 106 cells/ml; 70.00 ± 0.00 %) and C3 treated with high dose (30.00 ± 2.00 x 106 cells/ml; 70.00 ± 0.00 %) as compared to the control subgroup A3 (34.00 ± 2.00 x 106 cells/ml; 90.00 ± 0.00 %). Also, there were significant (P < 0.05) decreases in the number of morphologically abnormal sperm cells of rats in the test subgroups B3 (32.00±1.0 x 106 cells/ml; 30.00±0.00 %) and C3 (30.00±2.00 x 106 cells/ml; 30.00±0.00 %) as compared to rats in the control subgroup A3 (34.00±2.00 x 106 cells/ml; 10.00±0.00 %). There were no significant histological changes in the epididymis of the experimental rats in all the treated groups, at different doses and duration, as compared to the rats in the control group.

Discussion
The male reproductive system is a complex system which consists of the hypothalamus, anterior pituitary gland, the testes and glands like the epididymis. These structures work together to maintain the potency, fertility, and male secondary sexual characteristics [15]. Spermatogenesis, the first part of sperm production which includes stem cell mitosis and meiosis, is the biological process of gradual transformation of germ cells into spermatozoa over an extended period of time within the seminiferous tubules of the testis [16]. While spermiogenesis is the final differentiation process which occurs in the haploid male germ cells [17], sperm cells are produced in the seminiferous tubules at a rate of about 2 × 108 per day in the young adult. However, spermatogenic process in rats requires 53 days, out of which the spermatozoa spends the last 6 to 7 days in the final transit through the epididymis. 16 17 18 26

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In this study, aqueous root extract of Chrysophyllum albidium was administered to the experimental rats for 28 days (4weeks), 42 days (6weeks) and 56 days (8weeks), at different doses. This was done to ascertain the possible dose and duration effects of Chrysophyllum albidium on the sperm characteristics of the rats, as well as changes in body and organ weights. Our findings show that administration of Chrysophyllum albidium did not cause significant changes in body weights and organ (testes and epididymis) weights of the experimental rats irrespective of the dose and duration of extract administration. Also, the total epididymal sperm count was not significantly affected. These findings, which correlate with non-changing histology of the epididymis, may possibly indicate that the aqueous root extract of Chrysophyllum albidium had no adverse effect on the sperm characteristics of the experimental rats.
However, the effects on sperm motility indicates that increasing dosage and duration of administration of the aqueous root extract of Chrysophyllum albidium caused increased number of non-progressive sperm cells and immotile sperm cells, and decreased number of progressive motile sperm cells. This may portend possible disruption of sperm development by the extract. Disruption in the development of sperm cells could result in the production of more immotile and none progressive motile sperm cell which therefore decreases sperm motility. Sperm morphology, a relative function of the testes, was also affected by increasing dose and duration of extract administration. The number of normal sperm cells decreased while the number of abnormal sperm cells increased. Abnormal development of sperm cells in the testes, possibly caused by the extract, may lead to increase abnormal sperm cells which may compromise the fertility of the experimental rats.
Histology revealed that there were no changes in the epididymis of the test rats irrespective of the dose and duration of extract administration. The epididymis has its own blood epididymis barrier [18], contains substances such as proteins and its structure is quite different from that of the testes [19]. According to Friend and Gilula [20], the zonula occludens of the epididymis is the most highly developed, even more than the testes. These factors possibly played a major role in countering the effects of the extract on the epididymis, causing it to maintain its normal histological appearance.

Conclusion
Administration of aqueous root extract of Chrysophyllum albidium resulted in dose and duration dependent adverse effects on sperm motility and morphology which may compromise the fertility of the experimental rats.