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Reproductive toxicologic evaluations of Bulbine natalensis Baker stem extract in albino rats
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Theriogenology 72 (2009) 322–332 www.theriojournal.com
Reproductive toxicologic evaluations of Bulbine natalensis Baker stem extract in albino rats M.T. Yakubu, A.J. Afolayan * Phytomedicine Research Centre, Department of Botany, University of Fort Hare, Alice 5700, South Africa Received 24 September 2008; received in revised form 28 January 2009; accepted 31 January 2009
Abstract The effects of oral administration of aqueous extract of Bulbine natalensis Baker stem at daily doses of 25, 50, and 100 mg/kg body weight on the reproductive function of Wistar rats were evaluated. The indices of mating and fertility success as well as quantal frequency increased after 7 days of treatment in all the dose groups except the 100 mg/kg body weight group. The number of litters was not statistically different (P > 0.05) from the control. Whereas the absolute weights of the epididymis, seminal vesicle, and prostate were not affected, that of the testes was significantly increased. The epididymal sperm count, motility, morphology, and viscosity were not different from the control after 7 days of treatment. The male rat serum testosterone, progesterone, luteinizing hormone, and follicle-stimulating hormone significantly increased in the 25 and 50 mg/kg body weight groups, whereas the estradiol concentration decreased significantly at all the doses. The extract dose of 100 mg/kg body weight decreased the serum testosterone and progesterone levels in male rats. The prolactin concentration was not affected by all the doses. All the indices of reproduction, maternal, embryo/fetotoxic, teratogenic, and reproductive hormones in the female rats were not statistically different from that of their control except the resorption index, which increased at the dose of 100 mg/kg body weight of the extract. Histologic examination of the cross section of rat testes that received the extract at all the doses investigated revealed well-preserved seminiferous tubules with normal amount of stroma, normal population of spermatogenic and supporting cells, as well as normal spermatocytes within the lumen. The results revealed that the aqueous extract of Bulbine natalensis stem at doses of 25 and 50 mg/ kg body weight enhanced the success rate of mating and fertility due to increased libido as well as the levels of reproductive hormones in male rats. The absence of alterations in the reproductive parameters of female rats at doses of 25 and 50 mg/kg body weight of Bulbine natalensis stem extract suggest that the extract is ‘‘safe’’ for use at these doses by females during the organogenic period of pregnancy, whereas the extract dose of 100 mg/kg body weight portends a negative effect on some reproductive functions of male and female rats. # 2009 Elsevier Inc. All rights reserved. Keywords: Bulbine natalensis; Embryo; Fertility; Pregnancy; Reproductive hormones; Reproductive functions
1. Introduction One of the fundamental areas of human life is fertility and conception. Therefore, attention is being
* Corresponding author. Tel.: +27 82 202 2167; fax: +27 866 282 295. E-mail address: [email protected] (A.J. Afolayan). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.01.026
given to plants with antifertility properties. These may act through effects upon sperm motility and viability, modification of uterine lining functions, implantation of fertilized egg, or a rejection effect within the uterus. The aphrodisiac, fetotoxic, antifertility, and fertility-enhancing properties of some plant extracts have been reported in previous studies. Fadogia agrestis, Piper guineense, Aframomum melegueta, and Lepidium meyenii have an aphrodisiac effect, and the root of L.
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meyenii has been shown to have fertility-enhancing properties [1–4]. The fertility-reducing effects of Ruta graveolens and Gossypol as well as the fetotoxic potential of Globularia arabica and Globularia alypum have also been reported [5–7]. However, one plant that is claimed to be used as an aphrodisiac in the folklore medicine of South Africa without information on its effect on fertility and pregnancy in animals is Bulbine natalensis. Bulbine natalensis Baker (Asphodelaceae) is locally known as ibhucu (Zulu), rooiwortel (Afrikaans), and ingcelwane (Xhosa). It is widely distributed in the eastern and northern parts of South Africa [8]. The leaf sap is widely used in the management of wounds, burns, rashes, itches, ringworm, and cracked lips. The infusion of the roots is taken orally to quell vomiting, diarrhea, convulsion, venereal diseases, diabetes, and rheumatism [9]. The aqueous extract of the stem contains alkaloids (0.200%), tannins (0.481%), saponins (1.970%), cardiac glycosides (0.887%), and anthraquinones (0.152%) [10]. It has been reported that the administration of aqueous extract of B. natalensis stem to male Wistar rats (Rattus norvegicus) significantly increased penile erection indices, frequencies of mount, intromission, and ejaculation, as well as postejaculatory interval [10]. This thus justified the acclaimed folkloric use of the stem as an aphrodisiac and sexual invigorator. However, studies have shown that plants with aphrodisiac potential could have fertility-enhancing properties [1,2]. Therefore, there is the need to provide information on the effects of the stem extract on the reproductive function in animals, which to the best of our knowledge has not been documented in scientific literature. The current investigation was designed to evaluate the effect of the plant stem extract on reproductive function indices such as success of mating and fertility, reproductive hormones, pregnancy and embryo lethality using albino rats as model.
2. Materials and methods 2.1. Plant material and authentication Samples of B. natalensis, collected from a single population in the Eastern Cape, were authenticated by Prof. D.S. Grierson of the Department of Botany, University of Fort Hare, South Africa. A voucher specimen of the plant (Yakmed. 2008/1) was deposited at the Giffen Herbarium of the university.
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2.2. Experimental animals Male albino rats (Rattus norvegicus) of Wistar strain weighing 270.00 5.21 g and females weighing 252.08 4.37 g were obtained from the Animal House of the Agricultural and Rural Development Research Institute (ARDRI), University of Fort Hare. The animals were housed individually in separate clean metabolic cages of dimension 48.9 cm 33.5 cm 22.5 cm, placed in well-ventilated house conditions. They were also allowed free access to Balanced Trusty Chunks (Pioneer Foods (Pty) Ltd, Huguenot, South Africa) and tap water. The cleaning of the cages was done on a daily basis. 2.3. Hormonal assay kits and other reagents The assay kits for testosterone, prolactin, progesterone, luteinizing hormone, and follicle-stimulating hormone were obtained from Roche Diagnostic GmbH (Mannheim, Germany). All other reagents used were of analytical grade and were supplied by Merck Chemicals (Pty) Ltd. (Bellville, South Africa). 2.4. Preparation of extract The plant stem was carefully washed in tap water, oven-dried at 40 8C for 48 h, and pulverized with an electric blender. Twenty grams of the powder was extracted in 1000 mL distilled water for 48 h at room temperature with constant shaking on an Orbital Shaker (Stuart Scientific Co. Ltd., Essex, United Kingdom, model no: SO1). The extract was suction filtered with Whatman No. 1 (Maidstone, UK) filter paper, and the resulting filtrate was freeze-dried (Savant Refrigerated Vapor Trap, RV T4104, California, USA) to give a yield of 5.81 g. This was reconstituted separately in distilled water to give the required doses for each experiment. 2.5. Animal grouping and extract administration Eighty albino rats (40 each of males and females) were completely randomized into 4 groups of 10 each and orally administered with the extracts as follows: On a daily basis, Group A (control) was repeatedly administered 1 mL distilled water, and Groups B, C, and D were treated like the control except they received doses of 25, 50, and 100 mg/kg body weight of the extract, respectively. In the male rat fertility study, the administration was done orally on a daily basis for 7 days using metal oropharyngeal cannula, whereas the administration was done during the organogenic period
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(between Days 7 and 14 of pregnancy) in the female rat fertility and embryonic study. The dose typically used as obtained from our ethnobotanical survey of the plant is 50 mg/kg body weight. The study was carried out after approval from the Ethical Committee on Animal Care and Use of the University of Fort Hare, South Africa. 2.6. Male rat fertility study The effect of the extract on the fertility of male rats was assessed for 7 days. Ten animals in each treatment group, housed individually in metabolic cages, were paired with a receptive female after their treatment doses in a ratio of 1:1. The precoital sexual behaviour was then observed for 60 min. Successful mating was confirmed by the presence of sperm in the vaginal smear the following morning. The gestation period and number of litters were noted. The following reproductive parameters were then computed: percent mating success ([number mated/number paired] 100); quantal frequency ([number pregnant/ number mated] 100); percent fertility success ([number pregnant/number paired] 100). 2.7. Epididymal semen analysis The procedure described by Amelar et al. [11] was used for the epididymal semen analysis. Briefly, cauda epididymides were removed from the freezer after storing it for 12 h at 20 8C to maintain the longevity of the sperm cells. This was allowed to thaw slowly in a refrigerator at 4 8C for one-half hour. The distal cauda epididymides were thereafter minced with scissors to release the epididymal content into a 35-mm Petri dish containing 2 mL phosphate buffer (0.1 M, pH 7.4). The samples were maintained at 37 8C for 30 min before determining the sperm parameters with a Neubauer Improved Haemocytometer (Marienfeld Laboratory Glassware, Lauda-Ko¨nigshofen, Germany) as described for sperm count [12], motility [13], morphology [14], and turbidity [15]. 2.8. Female rat fertility and embryonic study The procedure described by Costa-Silva et al. [16] was used. The extract at doses of 25, 50, and 100 mg/kg body weight was administered during the organogenic period (Days 7 to 14 of pregnancy) [17]. The rats (n = 10) in each treatment group, housed individually in metabolic cages, were observed for survival, changes in behaviour, and signs of vaginal bleeding. The maternal weights were also recorded on Days 1 and 20.
The animals were laparotomized under ether anesthesia on Day 20 of pregnancy and their uterine horns removed. The number of implants, resorptions, corpora lutea, and live and dead fetuses was recorded. The fetuses, placentas, and ovaries were observed for any abnormality and their weights noted. From these data, the implantation index ([total number of implantation sites/number corpora lutea] 100), resorption index ([total number of resorption sites/total number of implantation sites] 100), preimplantation loss ([number of corpora lutea – number of implantations/ number of corpora lutea] 100), and postimplantation loss ([number of implantations number of live fetuses/number of implantations] 100) were calculated. The serum was also prepared for the determination of reproductive hormone levels. 2.9. Preparation of serum The procedure described by Yakubu et al. [4] was employed. Briefly, under ether anesthesia, rats were made to bleed through their cut jugular veins, which were slightly displaced (to prevent blood contamination by interstitial fluid) into centrifuge tubes. These were left to clot for 10 min at room temperature. The tubes were centrifuged at 1282 g for 5 min using a Hermle Bench Top Centrifuge (model Z300; Hermle, Hamburg, Germany). The sera were stored frozen and used within 12 h of preparation for the hormonal assay. Immediately thereafter, the testes were excised from the animals, freed of surrounding tissues, and prepared for the histopathologic analysis. 2.10. Determination of serum hormone concentration The serum hormone concentration was carried out using a Roche E170 modular analytics immunoassay analyzer (Roche Diagnostic GmbH) as described for testosterone and follicle-stimulating hormone [18], luteinizing hormone [19], progesterone, prolactin, and oestradiol [20]. The Roche modular analyzer was calibrated for the animal blood. 2.11. Histopathologic examination The left testes were fixed in 10% (v/v) formaldehyde, dehydrated through ascending grades of ethanol (70%, 90%, and 95%, v/v), cleaned in xylene, and embedded in paraffin wax (melting point 56 8C) [21]. Tissue sections were prepared according to the method described by Drury and Wallington [22] and stained with hematoxylin
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Table 1 Effect of Bulbine natalensis stem extract on the fertility of male rats. Parameters
Control
Mating success,* n (%) Quantal frequency,* n (%) Fertility success,* n (%) Number of litters,y n (Mean SD) Average weight of litters,y g (Mean SD) Gestation period,* d (Mean SD)
B. natalensis extract dose (mg/kg body weight)
6 of 10 (60%) 4 of 6 (67%) 4 of 10 (40%) 12.20 1.01a 5.20 0.21a 21.20 0.44a
25
50
100
10 of 10 (100%) 8 of 10 (80%) 8 of 10 (80%) 12.40 1.11a 5.25 0.14a 21.20 0.44a
10 of 10 (100%) 10 of 10 (100%) 10 of 10 (100%) 12.30 1.05a 5.30 0.11a 21.40 0.55a
4 of 10 (40%) 2 of 4 (50%) 2 of 10 (20%) 12.25 2.00a 5.22 0.27a 21.40 0.55a
*n = 10. y n = 10 to 13. a Parameters with different superscripts are significantly different.
and eosin (H & E). The photomicrographs were taken at 100 and 400 with a Canon PowerShot G2 digital camera (Canon Inc., Melville, NY, USA). 2.12. Statistical analysis Results were expressed as the mean SD of 10 replicates except as otherwise stated. One-way ANOVA complemented with Student’s t-tests were used to evaluate significant differences between the extracttreated groups and distilled water control group. Percentage data were transformed to arcsine before analysis. Differences within values at P < 0.05 were considered statistically significant [23]. 3. Results The aqueous extract of Bulbine natalensis at doses of 25 and 50 mg/kg body weight increased the success rate of mating, fertility, and quantal frequency in male rats, whereas the dose of 100 mg/kg body weight reduced these parameters (Table 1). The average weight and number of litters as well as gestation period were not significantly affected in the animals. All the litters were delivered within a few hours after their normal gestation period.
Although the significant increase in the absolute weight of the testes was dose-dependent, the epididymis, seminal vesicle, and prostate were not significantly affected (Table 2). Similarly, the count, motility, morphology, and viscosity of the sperm were not significantly altered by the dose levels of the plant extract (Table 3). The extract doses of 25 and 50 mg/kg body weight significantly increased the serum concentrations of testosterone, progesterone, luteinizing hormone, and follicle-stimulating hormone in male rats. The extract dose of 100 mg/kg body weight, however, decreased the serum testosterone concentration, whereas the values of luteinizing and follicle-stimulating hormones were not significantly altered (Table 4). The serum estradiol levels were significantly reduced in a dose-dependent manner, whereas that of prolactin was not significantly affected. Generally, the concentrations of the female reproductive hormones (luteinizing hormone, folliclestimulating hormone, progesterone, and estradiol) compared favorably with those of the control at all the doses (Fig. 1). Histologic examination of the cross section of the testes of extract-treated animals when compared with that of the control did not show any abnormality. The seminiferous tubules were well persevered. The amount
Table 2 Effect of Bulbine natalensis stem extract on the absolute weight of sex organs of male rats. Organs
Control
Weight (g) B. natalensis extract dose (mg/kg body weight) 25
Testes Cauda epididymis Seminal vesicles Ventral prostate
a
3.43 0.17 0.53 0.13a 0.25 0.004a 0.18 0.003a
50 b
3.50 0.14 0.53 0.10a 0.25 0.007a 0.18 0.002a
n = 10, mean SD. a–c Parameters with different superscripts are significantly different.
100 b
3.53 0.19 0.53 0.10a 0.25 0.005a 0.19 0.004a
3.58 0.13c 0.53 0.15a 0.25 0.002a 0.19 0.003a
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Table 3 Effect of Bulbine natalensis stem extract on some characteristics of male rat sperm. Sperm parameters
Control
B. natalensis extract dose (mg/kg body weight) 25
6
Sperm count (10 ) Sperm motility (%) Sperm morphology (%) Viscosity
357.88 14.21 94.21 7.69a 93.42 5.81a Normal
a
50
360.30 15.94 94.48 8.20a 93.63 5.20a Normal
a
100
355.92 16.92 94.53 8.11a 92.94 6.11a Normal
a
359.60 13.20a 93.96 9.34a 91.03 5.14a Normal
n = 10, mean SD. a Parameters with different superscripts are significantly different. Table 4 Effect of Bulbine natalensis stem extract on serum reproductive hormones of male rats. Hormones
Control
B. natalensis extract dose (mg/kg body weight) 25
Testosterone (ng/mL serum) LH (ng/mL serum) FSH (ng/mL serum) Progesterone (ng/mL serum) Prolactin (ng/mL serum) Estradiol (pmol/L)
a
1.50 0.05 0.55 0.07a 7.01 0.20a 7.00 0.30a 0.50 0.02a 88.00 4.21a
50 b
3.90 0.02 1.16 0.03b 9.28 0.14b 9.20 0.11b 0.49 0.04a 70.20 3.80b
100 c
5.20 0.03 1.48 0.02c 11.22 0.08c 11.90 0.58c 0.51 0.02a 57.30 4.20c
1.00 0.02d 0.40 0.01a 6.95 0.05a 4.30 0.14d 0.50 0.03a 64.50 5.21d
n = 10, mean SD. a–d Parameters with different superscripts are significantly different.
of stroma, population of spermatogenic and supporting cells, as well as the spermatocytes within the lumen were normal (Figs. 2 to 5). The extract of Bulbine natalensis did not produce any deaths or clinical signs of toxicity (salivation, diarrhea, changes in behaviour, and signs of vaginal bleeding) in the pregnant rats. Weight gained by the mothers and in the organogenic period, daily food and water intake, number and mass of live fetuses, number of dead fetuses, masses of ovary and placenta were unaffected by the plant extract (Table 5). Similarly,
there was no significant change in the number of corpora lutea, sites of implantation and resorption, or pre- and postimplantation losses. There was no value for resorption index at the doses of 25 and 50 mg/kg body weight of the extract, whereas the 100 mg/kg body weight produced twice the control value (Table 5). 4. Discussion The importance of reproductive health in society cannot be overemphasized as evidence on the deteriora-
Fig. 1. Effect of Bulbine natalensis stem extract on serum reproductive hormones of female rats. Bars for each hormone carrying superscripts as that of the control are not significantly different (P > 0.05).
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during the 7-day treatment period could be due to the inability of the male rats to make sexual advances toward the females and or sedative action rather than the adverse effect on the fertilizing ability of the spermatozoa. The similar number and average weight of the litters as well as gestation period in the control and all the extract-treated groups suggest that this aspect of reproductive function was not hindered. There was also no super-fecundation in the distilled water and extract-treated groups as all the litters were delivered within similar time frames after their normal gestation periods. The weight of testis, epididymis, and accessory organs as well as sperm parameters (sperm count, morphology, and motility) are sensitive end points that can be used to assess the direct effect of a compound on testicular cells and accessory organs [26]. The increase
Fig. 2. Photomicrographs of the cross section of testis of male rat orally administered distilled water for 7 days (control): (a) magnification 100; (b) magnification 400. The circled spot shows wellpreserved seminiferous tubules with normal amount of stroma and normal population of spermatogenic and supporting cells. The spermatocytes within the lumen are also intact (H & E).
tion of male and female fertility is accumulating [24]. Therefore, there is the need to screen more natural resources such as botanicals for their effect on reproductive function in rats and humans. The increase in the success rates of mating and fertility as well as quantal frequency by the doses of 25 and 50 mg/kg body weight of the extract in this study is an indication that sexual desire and reproductive function in male rats at these doses were not impaired while the experiment lasted. However, the reduction in these parameters by the dose of 100 mg/kg body weight of the extract may be adduced to the inability of the animals to initiate copulation as there was profound increase in the hesitation time of the male rats toward the receptive females. These findings complement that of Yakubu and Afolayan [10] who reported unwillingness and hesitation on the part of the males in making sexual advances toward the receptive females. Similarly, Ratnasooriya and Dharmasiri [25] have also attributed inhibition of sexual parameters to a sedative effect by the extract of Terminalia catappa seeds at a dose of 3000 mg/kg body weight. Therefore, the reduction in the success of mating and fertility observed in the highest dose group (100 mg/kg body weight)
Fig. 3. Photomicrographs of the cross section of testis of male rat orally administered B. natalensis stem extract at a dose of 25 mg/kg body weight for 7 days: (a) magnification 100; (b) magnification 400. The circled spot shows intact seminiferous tubules. The amount of stroma and population of spermatogenic and supporting cells are normal. The spermatocytes within the lumen are also normal (H & E).
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in the testicular body weight at all the extract doses (25, 50, and 100 mg/kg body weight) investigated could be due to increase in the metabolic activity, secretory ability, or an indication of organ swelling. Because the histologic examination did not reveal any inflammation, the increase in absolute testicular weight at doses of 25 and 50 mg/kg body weight of the extract can be attributed to increased metabolic activity, leading to increased testosterone production. The increase in the weight of the testes at the highest dose (100 mg/kg body weight) despite the non-increase in testosterone level could possibly be due to a generalized effect of enhanced testicular weight of the extract. This contrasts with the findings of Gupta et al. [27] on the reduction in testicular weight of male rats after the administration of saponins of Al-bizia lebbeck. However, the nonsignificant effect on the absolute body weights of the cauda epididymis, ventral prostate, and seminal vesicles may be an indication that the general metabolic conditions of these accessory sex organs were within the normal range [28]. Alterations in the sperm motility, viability, and morphology are an indication of a disturbed testicular and epididymal microenvironment [29]. Therefore, the absence of significant effect at all the doses investigated on the epididymal sperm count, motility, and morphology could possibly imply that the extract did not produce disturbance in the microenvironment of the epididymides. This may rule out impairment/hindrance in the normal functioning of the epididymal sperm cells during the experimental period. It is also possible that the extract was unable to cross the blood-testes barrier and thus could not interfere with the normal functioning of the testicular epithelium [30]. The fact that viscosity of the semen was not affected also indicated that liquefaction was within the normal range. All these showed that the plant extract may not hamper sperm function in male rats if administered repeatedly for 7 days. This further confirms that the reduction in the mating and fertility successes by the dose of 100 mg/kg body weight of the extract is not associated with the fertilizing capacity of the spermatozoa. Testosterone is the main male gonadal hormone produced by the interstitial Leydig cells [31]. It is required for the maintenance of normal sexual desire, nocturnal penile tumescence, and nonerotic penile erections in most men. The increase in testosterone level by the doses of 25 and 50 mg/kg body weight of the extract may be adduced to induction of the hormone synthesis by the Leydig cells, as the cells are the main source of testosterone [32]. This implies that the extract at these doses stimulated the mechanism intervening in
the process of the hormone synthesis in the Leydig cells [33]. Such increase will enhance androgen-dependent parameters like mating behaviour and maintenance of spermatogenesis. This may account for the increased libido in male rats observed in the 25 and 50 mg/kg body weight extract-treated groups in this study. The reduced testosterone content observed with the dose of 100 mg/kg body weight might imply that the extract at this dose reached a critical level to start exerting a negative effect on the hormone. The reduction in the testosterone concentration may explain the reduced mating success observed with the highest dose as testosterone is considered to contribute to improvement in sexual function, libido, and penile erection [34]. This is an indication of a potential negative effect of the extract on the male hormone at this dose. Normal male reproductive function depends on the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the pituitary gland under the influence of hypothalamic gonadotropin-releasing hormone (GnRH). LH in males stimulates the testicular Leydig cells to secrete testosterone, and FSH induces spermatogenesis in the seminiferous tubules of the testes [35,36]. The elevated levels of LH and FSH by the doses of 25 and 50 mg/kg body weight of the extract may be adduced to a stimulatory effect on the hypothalamic-pituitary axis and suggests a progonadotropic effect at the given doses and time points [31]. Such elevation in the gonadotropins may account for the observed increase in testosterone concentration in this study. In contrast, the absence of significant effect on LH and FSH at the highest dose may imply nonstimulation of the GnRH-LH signaling. In males, progesterone influences spermiogenesis, sperm capacitation, and testosterone biosynthesis in Leydig cells [36]. The increase in serum progesterone by the doses of 25 and 50 mg/kg body weight of the extract may have stimulated the biosynthesis of testosterone leading to its elevated levels in this study. The decrease in progesterone concentration by the dose of 100 mg/kg body weight of the extract may account for the reduced levels of testosterone observed in this study. Estradiol has an androgen-independent inhibitory effect on gonadotropin release [37]. The reduction in estradiol levels in the male rats may imply reduced inhibitory effect on the release of gonadotropins that were raised in this study. Interestingly, it is not clear why the dose of 100 mg/kg body weight of the extract also reduced the levels of estradiol as well despite nonelevation of the levels of FSH and LH in this study. The alterations in the hormonal profile of males at the doses of 25 and 50 mg/ kg body weight of the extract may account for the
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Fig. 4. Photomicrographs of the cross section of testis of male rat orally administered B. natalensis stem extract at a dose of 50 mg/kg body weight for 7 days: (a) magnification 100; (b) magnification 400. The circled spots depicts well preserved seminiferous tubules with normal amount of stroma and spermatogenic and supporting cells. The spermatocytes within the lumen are also normal (H & E).
increased libido and enhanced reproductive function of the animals, which contrasts to the effect of the dose of 100 mg/kg body weight of the extract on these indices. The adverse effect of the highest dose of the extract (100 mg/kg body weight) after repeated administration for 7 days could adversely affect reproductive capacity of the male rats. In this study, the histologic examination of the cross section of rat testes treated with the various doses of the extract revealed well-preserved seminiferous tubules with normal amount of stroma. These are indications that the extract did not adversely affect the histoarchitecture of the organs. The normal population of spermatogenic and supporting cells as well as spermatids within the lumen could possibly imply that the morphologic characteristics and the abundance of spermatozoa seen within the lumen of the seminiferous tubules were not adversely affected by the extract in this study. It also shows that the exocrine function of the testes was not impaired. This agrees with the findings of Moundipa et al. [33] who observed similar features after
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the administration of aqueous extract of Hibiscus macranthus and Basella alba to Wistar rats. The increase in the number of pregnant females is an indication of sex-enhancing properties of the extract at the doses of 25 and 50 mg/kg body weight. The reduction in the number of pregnant females by the dose of 100 mg/kg body weight of the extract may be attributed to either deficit in sexual arousal or the inability to initiate copulation [38,39]. Maternal parameters (body weight changes, vaginal bleeding, food and water intake) can be used to evaluate the integrity of maternal homeostasis [40]. The similarity in the maternal parameters as well as the absence of clinical signs of toxicity (salivation, diarrhea, changes in behavioural signs, and vaginal bleeding) in all the experimental groups suggest that the extract was not toxic to the mother [41]. The observation that the full-term sacrifice parameters (number of corpora lutea, implantation sites, live fetuses and masses of fetus, placenta, and ovary) were not different from the control values is an indication that the extract did not cause any malformation in the fetuses and that reproductive performance of the mothers was normal. This agreed with the findings of Guerra et al. [42]. The implantation index and preimplantation loss rate evaluate blastocyst implantation in the uterus, and
Fig. 5. Photomicrographs of the cross section of testis of male rat orally administered B. natalensis stem extract at a dose of 100 mg/kg body weight for 7 days: (a) magnification 100; (b) magnification 400. The circled spot shows intact seminiferous tubules with normal amount of stroma. The population of the spermatogenic and supporting cells as well as the spermatocytes within the lumen are normal (H & E).
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Table 5 Effect of Bulbine natalensis stem extract on female rat reproductive parameters. Parameters
Number of female rats paired Number pregnant Index of pregnancy, n (%) Mass gain in pregnancy (g) Mass gain in the organogenic period (g) Food intake (g/day per rat) Water intake (mL/day per rat) Number of live fetuses Number of dead fetuses Mass of fetuses (g) Mass of placenta (g) Mass of ovary (g/100 g) Number of implantation sites Number of resorption sites Number of corpora lutea Implantation index (%) Resorption index (%) Preimplantation loss (%) Postimplantation loss (%)
Control
10 6 6 of 10 (60%) 114.30 8.21a 30.10 1.50a 17.10 2.14a 24.88 3.14a 12.24 0.30a 0 3.90 0.05a 0.35 0.04a 0.015 0.002a 13.40 0.03a 1.00 0.01a 14.20 0.40a 94.37 3.61a 7.46 0.21a 5.63 0.12a 8.66 0.22a
B. natalensis extract dose (mg/kg body weight) 25
50
100
10 10 10 of 10 (100%) 115.10 7.42a 30.26 1.70a 18.21 1.50a 25.31 2.63a 12.40 0.20a 0 4.00 0.03a 0.35 0.02a 0.016 0.002a 13.60 0.01a 0.00 0.00b 14.40 0.50a 94.44 4.01a 0 0.00b 5.56 0.21a 8.82 0.11a
10 10 10 of 10 (100%) 115.30 6.83a 30.44 1.08a 18.40 1.07a 26.41 1.55a 12.61 0.20a 0 3.97 0.02a 0.35 0.04a 0.016 0.001a 13.70 0.01a 0.00 0.00b 14.51 0.04a 94.42 4.07a 0 0.00b 5.58 0.16a 7.96 0.74a
10 4 4 of 10 (40%) 114.60 7.08a 30.05 1.21a 17.69 1.79a 25.72 1.82a 12.28 0.14a 0 4.01 0.06a 0.35 0.05a 0.015 0.001a 13.30 0.05a 2.00 0.01c 14.30 0.03a 93.01 4.26a 15.04 1.16c 6.99 0.08a 7.67 0.86a
n = 10, mean SD. a–c Parameters with different superscripts are significantly different.
the resorption index and postimplantation loss rate establishes correlation between the number of implanted blastocysts and those that have not developed [40,43]. In this study, the absence of statistical difference in the implantation index, preimplantation and postimplantation losses between the distilled water–treated control and the extract-dosed groups (25, 50, and 100 mg/kg body weight) is an indication that the implantation of the blastocysts was within normal range. This complements the findings of CostaSilva et al. [16] on the effect of administration of Carapa quianensis seed oil during pregnancy in female Wistar rats. However, the nil value of resorption index observed with the 25 and 50 mg/kg body weight extracttreated groups suggests normal development of the implanted blastocysts and normal reproductive capacity of the animals. In contrast, the higher value of resorption index after the administration of 100 mg/ kg body weight of the extract connotes increased failure rate of embryo development and antipregnancy [43]. This is an indication that the extract at the highest dose (100 mg/kg body weight) investigated has the potential of being embryotoxic and may not be completely safe as an oral remedy during pregnancy at the given dose. The absence of significant effect on the serum prolactin concentration in female rats observed with the extract at all the doses investigated was an indication that the production of dopamine was not affected, as
prolactin secretion is controlled by the hypothalamic hormone. FSH and LH enhance the growth and maturation of ovarian follicles by acting directly on the receptors located on the granulosa cells [44]. Therefore, the similarity in the values of FSH and LH in all the experimental groups was an indication that folliculogenesis was not hindered. Similarly, the pattern obtained for progesterone and estradiol are indications that the various functions associated with conception, pregnancy, and growth of uterine lining were not affected [45,46]. The pattern exhibited by the extract at doses of 25, 50, and 100 mg/kg body weight on the female reproductive hormones suggests that the extract did not generally affect the reproductive hormones. It further implies that the potential of embryo lethality at the dose of 100 mg/kg body weight of the extract may not be mediated by hormonal changes but rather a direct effect on the embryo. In this study, the enhanced success rate of mating and fertility at the doses of 25 and 50 mg/kg body weight of the extract suggests that it was more efficiently active within the target tissues in the male animals at these doses than at the highest dose (100 mg/kg body weight). The results of the current study revealed that B. natalensis extract at the doses of 25 and 50 mg/kg body weight has the tendency of enhancing reproductive function in male rats and may not adversely affect pregnancy and embryo development in the female rats.
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The highest dose (100 mg/kg body weight) has demonstrated selective toxicity on some of the reproductive function indices investigated. The extract at this dose will hinder normal reproductive performance in male rats and has the tendency to be embryotoxic in pregnant female rats. However, further studies on the effect of long-term administration of the extract on spermatogenesis and, consequently, fertility in male rats is necessary. Acknowledgments This research was supported by grants from Govan Mbeki Research and Development Centre, University of Fort Hare, and the National Research Foundation, South Africa. The authors are also grateful to the University of Ilorin, Nigeria, for the postdoctoral fellowship support of Dr. M.T. Yakubu. References [1] Zheng BL, He K, Kim CH, Rogers L, Shao Y, Huang ZY, et al. Effect of a lipidic extract from Lepidium meyenii on sexual behavior in mice and rats. Urology 2000;55:598–602. [2] Kamtchouing P, Mbongue GY, Dimo T, Watcho P, Jatsa HB, Sokeng SD. Effect of Aframomum melegueta and Piper guineense on sexual behaviour of male rats. Behav Pharmacol 2002;13:243–7. [3] Gonzales GF, Rubio J, Chung A, Gasco M, Villegas L. Effect of alcoholic extract of Lepidium meyenii (Maca) on testicular function in male rats. Asian J Androl 2003;5:349–52. [4] Yakubu MT, Akanji MA, Oladiji AT. Aphrodisiac potentials of aqueous extract of Fadogia agrestis (Schweinf, Ex Heirn) stem in male albino rats. Asian J Androl 2005;7:399–404. [5] Hadley MA, Lin YC, Dym M. Effects of gossypol on the reproductive systems of male rats. J Androl 1981;2:190–9. [6] Gandhi M, Lal R, Sankaranarayanan A, Sharma PL. Post-coital antifertility action of Ruta graveolens in female rats and hamsters. J Ethnopharmacol 1991;34:49–59. [7] Elbetieha A, Oran SA, Alkofahi A, Darmani H, Raies AM. Fetotoxic potentials of Globularia arabica and Globularia alypum (Globulariaceae) in rats. J Ethnopharmacol 2000;72: 215–9. [8] Van Wyk BE, Van Oudtshoorn B, Gericke N. Medicinal plants of South Africa. Pretoria, South Africa: Briza Publications; 1997. p. 64–5. [9] Pujol J. Naturafrica—The Herbalist Handbook. Jean Pujol Natural Healers’ Foundation; 1990. [10] Yakubu MT, Afolayan AJ. Effect of aqueous extract of Bulbine natalensis (Baker) stem on the sexual behaviour of male rats. Int J Androl 2009, doi:10.1111/j.1365-2605.2008.00910.x, epub ahead of print. [11] Amelar RD, Dublin L, Schoenfeld C. Serum analysis: an office technique. Urology 1973;2:606–11. [12] Wang C, Sinha-Hikim AP, Leung A. The anti-progestin CDB2914 has no antifertility effect in male rats. Contraception 1995;136:215–8.
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[33] Moundipa FP, Kamtchouing P, Koueta N, Tantchou J, Foyang NPR, Mbiapo FT. Effects of aqueous extracts of Hibiscus macranthus and Basella alba in mature rat testis function. J Ethnopharmacol 1999;65:133–9. [34] Gauthaman K, Adaikan PG, Prasada RNV. Aphrodisiac properties of Tribulus terrestis extract (Protodioscin) in normal and castrated rats. Life Sci 2002;71:1385–96. [35] Bano A, Tahir F, Subhan F, Sultan S, Deepa F, Shakeel S, et al. A preliminary study of gonadotropin ratios among infertile Pakistani men. Pakistan J Med Res 2003;42:185–7. [36] Andersen ML, Tufik S. Does male sexual behavior require progesterone? Brain Res Rev 2006;51:136–43. [37] de Ronde W, Ols HAP, van Leeuwen JPTM, de Jong FH. The importanceofoestrogensinmales.ClinEndocrinol2003;58:529–42. [38] Crowley WR, Popolow HB, Ward Jr OB. From dud to stud: copulatory behavior elicited through conditioned arousal in sexually inactive male rats. Physiol Behav 1973;10:391–4. [39] Stefanick ML, Davidson J. Genital responses in noncopulators and rats with lesion in the medial preoptic area of midthoracic spinal cord. Physiol Behav 1987;41:439–44. [40] Almeida FCG, Lemonica IP. The toxic effects of Coleus barbatus B. on the different periods of pregnancy in rats. J Ethnopharmacol 2007;73:53–60.
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Reproductive toxicologic evaluations of Bulbine natalensis Baker stem extract in albino rats M.T. Yakubu, A.J. Afolayan * Phytomedicine Research Centre, Department of Botany, University of Fort Hare, Alice 5700, South Africa Received 24 September 2008; received in revised form 28 January 2009; accepted 31 January 2009
Abstract The effects of oral administration of aqueous extract of Bulbine natalensis Baker stem at daily doses of 25, 50, and 100 mg/kg body weight on the reproductive function of Wistar rats were evaluated. The indices of mating and fertility success as well as quantal frequency increased after 7 days of treatment in all the dose groups except the 100 mg/kg body weight group. The number of litters was not statistically different (P > 0.05) from the control. Whereas the absolute weights of the epididymis, seminal vesicle, and prostate were not affected, that of the testes was significantly increased. The epididymal sperm count, motility, morphology, and viscosity were not different from the control after 7 days of treatment. The male rat serum testosterone, progesterone, luteinizing hormone, and follicle-stimulating hormone significantly increased in the 25 and 50 mg/kg body weight groups, whereas the estradiol concentration decreased significantly at all the doses. The extract dose of 100 mg/kg body weight decreased the serum testosterone and progesterone levels in male rats. The prolactin concentration was not affected by all the doses. All the indices of reproduction, maternal, embryo/fetotoxic, teratogenic, and reproductive hormones in the female rats were not statistically different from that of their control except the resorption index, which increased at the dose of 100 mg/kg body weight of the extract. Histologic examination of the cross section of rat testes that received the extract at all the doses investigated revealed well-preserved seminiferous tubules with normal amount of stroma, normal population of spermatogenic and supporting cells, as well as normal spermatocytes within the lumen. The results revealed that the aqueous extract of Bulbine natalensis stem at doses of 25 and 50 mg/ kg body weight enhanced the success rate of mating and fertility due to increased libido as well as the levels of reproductive hormones in male rats. The absence of alterations in the reproductive parameters of female rats at doses of 25 and 50 mg/kg body weight of Bulbine natalensis stem extract suggest that the extract is ‘‘safe’’ for use at these doses by females during the organogenic period of pregnancy, whereas the extract dose of 100 mg/kg body weight portends a negative effect on some reproductive functions of male and female rats. # 2009 Elsevier Inc. All rights reserved. Keywords: Bulbine natalensis; Embryo; Fertility; Pregnancy; Reproductive hormones; Reproductive functions
1. Introduction One of the fundamental areas of human life is fertility and conception. Therefore, attention is being
* Corresponding author. Tel.: +27 82 202 2167; fax: +27 866 282 295. E-mail address: [email protected] (A.J. Afolayan). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.01.026
given to plants with antifertility properties. These may act through effects upon sperm motility and viability, modification of uterine lining functions, implantation of fertilized egg, or a rejection effect within the uterus. The aphrodisiac, fetotoxic, antifertility, and fertility-enhancing properties of some plant extracts have been reported in previous studies. Fadogia agrestis, Piper guineense, Aframomum melegueta, and Lepidium meyenii have an aphrodisiac effect, and the root of L.
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meyenii has been shown to have fertility-enhancing properties [1–4]. The fertility-reducing effects of Ruta graveolens and Gossypol as well as the fetotoxic potential of Globularia arabica and Globularia alypum have also been reported [5–7]. However, one plant that is claimed to be used as an aphrodisiac in the folklore medicine of South Africa without information on its effect on fertility and pregnancy in animals is Bulbine natalensis. Bulbine natalensis Baker (Asphodelaceae) is locally known as ibhucu (Zulu), rooiwortel (Afrikaans), and ingcelwane (Xhosa). It is widely distributed in the eastern and northern parts of South Africa [8]. The leaf sap is widely used in the management of wounds, burns, rashes, itches, ringworm, and cracked lips. The infusion of the roots is taken orally to quell vomiting, diarrhea, convulsion, venereal diseases, diabetes, and rheumatism [9]. The aqueous extract of the stem contains alkaloids (0.200%), tannins (0.481%), saponins (1.970%), cardiac glycosides (0.887%), and anthraquinones (0.152%) [10]. It has been reported that the administration of aqueous extract of B. natalensis stem to male Wistar rats (Rattus norvegicus) significantly increased penile erection indices, frequencies of mount, intromission, and ejaculation, as well as postejaculatory interval [10]. This thus justified the acclaimed folkloric use of the stem as an aphrodisiac and sexual invigorator. However, studies have shown that plants with aphrodisiac potential could have fertility-enhancing properties [1,2]. Therefore, there is the need to provide information on the effects of the stem extract on the reproductive function in animals, which to the best of our knowledge has not been documented in scientific literature. The current investigation was designed to evaluate the effect of the plant stem extract on reproductive function indices such as success of mating and fertility, reproductive hormones, pregnancy and embryo lethality using albino rats as model.
2. Materials and methods 2.1. Plant material and authentication Samples of B. natalensis, collected from a single population in the Eastern Cape, were authenticated by Prof. D.S. Grierson of the Department of Botany, University of Fort Hare, South Africa. A voucher specimen of the plant (Yakmed. 2008/1) was deposited at the Giffen Herbarium of the university.
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2.2. Experimental animals Male albino rats (Rattus norvegicus) of Wistar strain weighing 270.00 5.21 g and females weighing 252.08 4.37 g were obtained from the Animal House of the Agricultural and Rural Development Research Institute (ARDRI), University of Fort Hare. The animals were housed individually in separate clean metabolic cages of dimension 48.9 cm 33.5 cm 22.5 cm, placed in well-ventilated house conditions. They were also allowed free access to Balanced Trusty Chunks (Pioneer Foods (Pty) Ltd, Huguenot, South Africa) and tap water. The cleaning of the cages was done on a daily basis. 2.3. Hormonal assay kits and other reagents The assay kits for testosterone, prolactin, progesterone, luteinizing hormone, and follicle-stimulating hormone were obtained from Roche Diagnostic GmbH (Mannheim, Germany). All other reagents used were of analytical grade and were supplied by Merck Chemicals (Pty) Ltd. (Bellville, South Africa). 2.4. Preparation of extract The plant stem was carefully washed in tap water, oven-dried at 40 8C for 48 h, and pulverized with an electric blender. Twenty grams of the powder was extracted in 1000 mL distilled water for 48 h at room temperature with constant shaking on an Orbital Shaker (Stuart Scientific Co. Ltd., Essex, United Kingdom, model no: SO1). The extract was suction filtered with Whatman No. 1 (Maidstone, UK) filter paper, and the resulting filtrate was freeze-dried (Savant Refrigerated Vapor Trap, RV T4104, California, USA) to give a yield of 5.81 g. This was reconstituted separately in distilled water to give the required doses for each experiment. 2.5. Animal grouping and extract administration Eighty albino rats (40 each of males and females) were completely randomized into 4 groups of 10 each and orally administered with the extracts as follows: On a daily basis, Group A (control) was repeatedly administered 1 mL distilled water, and Groups B, C, and D were treated like the control except they received doses of 25, 50, and 100 mg/kg body weight of the extract, respectively. In the male rat fertility study, the administration was done orally on a daily basis for 7 days using metal oropharyngeal cannula, whereas the administration was done during the organogenic period
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(between Days 7 and 14 of pregnancy) in the female rat fertility and embryonic study. The dose typically used as obtained from our ethnobotanical survey of the plant is 50 mg/kg body weight. The study was carried out after approval from the Ethical Committee on Animal Care and Use of the University of Fort Hare, South Africa. 2.6. Male rat fertility study The effect of the extract on the fertility of male rats was assessed for 7 days. Ten animals in each treatment group, housed individually in metabolic cages, were paired with a receptive female after their treatment doses in a ratio of 1:1. The precoital sexual behaviour was then observed for 60 min. Successful mating was confirmed by the presence of sperm in the vaginal smear the following morning. The gestation period and number of litters were noted. The following reproductive parameters were then computed: percent mating success ([number mated/number paired] 100); quantal frequency ([number pregnant/ number mated] 100); percent fertility success ([number pregnant/number paired] 100). 2.7. Epididymal semen analysis The procedure described by Amelar et al. [11] was used for the epididymal semen analysis. Briefly, cauda epididymides were removed from the freezer after storing it for 12 h at 20 8C to maintain the longevity of the sperm cells. This was allowed to thaw slowly in a refrigerator at 4 8C for one-half hour. The distal cauda epididymides were thereafter minced with scissors to release the epididymal content into a 35-mm Petri dish containing 2 mL phosphate buffer (0.1 M, pH 7.4). The samples were maintained at 37 8C for 30 min before determining the sperm parameters with a Neubauer Improved Haemocytometer (Marienfeld Laboratory Glassware, Lauda-Ko¨nigshofen, Germany) as described for sperm count [12], motility [13], morphology [14], and turbidity [15]. 2.8. Female rat fertility and embryonic study The procedure described by Costa-Silva et al. [16] was used. The extract at doses of 25, 50, and 100 mg/kg body weight was administered during the organogenic period (Days 7 to 14 of pregnancy) [17]. The rats (n = 10) in each treatment group, housed individually in metabolic cages, were observed for survival, changes in behaviour, and signs of vaginal bleeding. The maternal weights were also recorded on Days 1 and 20.
The animals were laparotomized under ether anesthesia on Day 20 of pregnancy and their uterine horns removed. The number of implants, resorptions, corpora lutea, and live and dead fetuses was recorded. The fetuses, placentas, and ovaries were observed for any abnormality and their weights noted. From these data, the implantation index ([total number of implantation sites/number corpora lutea] 100), resorption index ([total number of resorption sites/total number of implantation sites] 100), preimplantation loss ([number of corpora lutea – number of implantations/ number of corpora lutea] 100), and postimplantation loss ([number of implantations number of live fetuses/number of implantations] 100) were calculated. The serum was also prepared for the determination of reproductive hormone levels. 2.9. Preparation of serum The procedure described by Yakubu et al. [4] was employed. Briefly, under ether anesthesia, rats were made to bleed through their cut jugular veins, which were slightly displaced (to prevent blood contamination by interstitial fluid) into centrifuge tubes. These were left to clot for 10 min at room temperature. The tubes were centrifuged at 1282 g for 5 min using a Hermle Bench Top Centrifuge (model Z300; Hermle, Hamburg, Germany). The sera were stored frozen and used within 12 h of preparation for the hormonal assay. Immediately thereafter, the testes were excised from the animals, freed of surrounding tissues, and prepared for the histopathologic analysis. 2.10. Determination of serum hormone concentration The serum hormone concentration was carried out using a Roche E170 modular analytics immunoassay analyzer (Roche Diagnostic GmbH) as described for testosterone and follicle-stimulating hormone [18], luteinizing hormone [19], progesterone, prolactin, and oestradiol [20]. The Roche modular analyzer was calibrated for the animal blood. 2.11. Histopathologic examination The left testes were fixed in 10% (v/v) formaldehyde, dehydrated through ascending grades of ethanol (70%, 90%, and 95%, v/v), cleaned in xylene, and embedded in paraffin wax (melting point 56 8C) [21]. Tissue sections were prepared according to the method described by Drury and Wallington [22] and stained with hematoxylin
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Table 1 Effect of Bulbine natalensis stem extract on the fertility of male rats. Parameters
Control
Mating success,* n (%) Quantal frequency,* n (%) Fertility success,* n (%) Number of litters,y n (Mean SD) Average weight of litters,y g (Mean SD) Gestation period,* d (Mean SD)
B. natalensis extract dose (mg/kg body weight)
6 of 10 (60%) 4 of 6 (67%) 4 of 10 (40%) 12.20 1.01a 5.20 0.21a 21.20 0.44a
25
50
100
10 of 10 (100%) 8 of 10 (80%) 8 of 10 (80%) 12.40 1.11a 5.25 0.14a 21.20 0.44a
10 of 10 (100%) 10 of 10 (100%) 10 of 10 (100%) 12.30 1.05a 5.30 0.11a 21.40 0.55a
4 of 10 (40%) 2 of 4 (50%) 2 of 10 (20%) 12.25 2.00a 5.22 0.27a 21.40 0.55a
*n = 10. y n = 10 to 13. a Parameters with different superscripts are significantly different.
and eosin (H & E). The photomicrographs were taken at 100 and 400 with a Canon PowerShot G2 digital camera (Canon Inc., Melville, NY, USA). 2.12. Statistical analysis Results were expressed as the mean SD of 10 replicates except as otherwise stated. One-way ANOVA complemented with Student’s t-tests were used to evaluate significant differences between the extracttreated groups and distilled water control group. Percentage data were transformed to arcsine before analysis. Differences within values at P < 0.05 were considered statistically significant [23]. 3. Results The aqueous extract of Bulbine natalensis at doses of 25 and 50 mg/kg body weight increased the success rate of mating, fertility, and quantal frequency in male rats, whereas the dose of 100 mg/kg body weight reduced these parameters (Table 1). The average weight and number of litters as well as gestation period were not significantly affected in the animals. All the litters were delivered within a few hours after their normal gestation period.
Although the significant increase in the absolute weight of the testes was dose-dependent, the epididymis, seminal vesicle, and prostate were not significantly affected (Table 2). Similarly, the count, motility, morphology, and viscosity of the sperm were not significantly altered by the dose levels of the plant extract (Table 3). The extract doses of 25 and 50 mg/kg body weight significantly increased the serum concentrations of testosterone, progesterone, luteinizing hormone, and follicle-stimulating hormone in male rats. The extract dose of 100 mg/kg body weight, however, decreased the serum testosterone concentration, whereas the values of luteinizing and follicle-stimulating hormones were not significantly altered (Table 4). The serum estradiol levels were significantly reduced in a dose-dependent manner, whereas that of prolactin was not significantly affected. Generally, the concentrations of the female reproductive hormones (luteinizing hormone, folliclestimulating hormone, progesterone, and estradiol) compared favorably with those of the control at all the doses (Fig. 1). Histologic examination of the cross section of the testes of extract-treated animals when compared with that of the control did not show any abnormality. The seminiferous tubules were well persevered. The amount
Table 2 Effect of Bulbine natalensis stem extract on the absolute weight of sex organs of male rats. Organs
Control
Weight (g) B. natalensis extract dose (mg/kg body weight) 25
Testes Cauda epididymis Seminal vesicles Ventral prostate
a
3.43 0.17 0.53 0.13a 0.25 0.004a 0.18 0.003a
50 b
3.50 0.14 0.53 0.10a 0.25 0.007a 0.18 0.002a
n = 10, mean SD. a–c Parameters with different superscripts are significantly different.
100 b
3.53 0.19 0.53 0.10a 0.25 0.005a 0.19 0.004a
3.58 0.13c 0.53 0.15a 0.25 0.002a 0.19 0.003a
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Table 3 Effect of Bulbine natalensis stem extract on some characteristics of male rat sperm. Sperm parameters
Control
B. natalensis extract dose (mg/kg body weight) 25
6
Sperm count (10 ) Sperm motility (%) Sperm morphology (%) Viscosity
357.88 14.21 94.21 7.69a 93.42 5.81a Normal
a
50
360.30 15.94 94.48 8.20a 93.63 5.20a Normal
a
100
355.92 16.92 94.53 8.11a 92.94 6.11a Normal
a
359.60 13.20a 93.96 9.34a 91.03 5.14a Normal
n = 10, mean SD. a Parameters with different superscripts are significantly different. Table 4 Effect of Bulbine natalensis stem extract on serum reproductive hormones of male rats. Hormones
Control
B. natalensis extract dose (mg/kg body weight) 25
Testosterone (ng/mL serum) LH (ng/mL serum) FSH (ng/mL serum) Progesterone (ng/mL serum) Prolactin (ng/mL serum) Estradiol (pmol/L)
a
1.50 0.05 0.55 0.07a 7.01 0.20a 7.00 0.30a 0.50 0.02a 88.00 4.21a
50 b
3.90 0.02 1.16 0.03b 9.28 0.14b 9.20 0.11b 0.49 0.04a 70.20 3.80b
100 c
5.20 0.03 1.48 0.02c 11.22 0.08c 11.90 0.58c 0.51 0.02a 57.30 4.20c
1.00 0.02d 0.40 0.01a 6.95 0.05a 4.30 0.14d 0.50 0.03a 64.50 5.21d
n = 10, mean SD. a–d Parameters with different superscripts are significantly different.
of stroma, population of spermatogenic and supporting cells, as well as the spermatocytes within the lumen were normal (Figs. 2 to 5). The extract of Bulbine natalensis did not produce any deaths or clinical signs of toxicity (salivation, diarrhea, changes in behaviour, and signs of vaginal bleeding) in the pregnant rats. Weight gained by the mothers and in the organogenic period, daily food and water intake, number and mass of live fetuses, number of dead fetuses, masses of ovary and placenta were unaffected by the plant extract (Table 5). Similarly,
there was no significant change in the number of corpora lutea, sites of implantation and resorption, or pre- and postimplantation losses. There was no value for resorption index at the doses of 25 and 50 mg/kg body weight of the extract, whereas the 100 mg/kg body weight produced twice the control value (Table 5). 4. Discussion The importance of reproductive health in society cannot be overemphasized as evidence on the deteriora-
Fig. 1. Effect of Bulbine natalensis stem extract on serum reproductive hormones of female rats. Bars for each hormone carrying superscripts as that of the control are not significantly different (P > 0.05).
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during the 7-day treatment period could be due to the inability of the male rats to make sexual advances toward the females and or sedative action rather than the adverse effect on the fertilizing ability of the spermatozoa. The similar number and average weight of the litters as well as gestation period in the control and all the extract-treated groups suggest that this aspect of reproductive function was not hindered. There was also no super-fecundation in the distilled water and extract-treated groups as all the litters were delivered within similar time frames after their normal gestation periods. The weight of testis, epididymis, and accessory organs as well as sperm parameters (sperm count, morphology, and motility) are sensitive end points that can be used to assess the direct effect of a compound on testicular cells and accessory organs [26]. The increase
Fig. 2. Photomicrographs of the cross section of testis of male rat orally administered distilled water for 7 days (control): (a) magnification 100; (b) magnification 400. The circled spot shows wellpreserved seminiferous tubules with normal amount of stroma and normal population of spermatogenic and supporting cells. The spermatocytes within the lumen are also intact (H & E).
tion of male and female fertility is accumulating [24]. Therefore, there is the need to screen more natural resources such as botanicals for their effect on reproductive function in rats and humans. The increase in the success rates of mating and fertility as well as quantal frequency by the doses of 25 and 50 mg/kg body weight of the extract in this study is an indication that sexual desire and reproductive function in male rats at these doses were not impaired while the experiment lasted. However, the reduction in these parameters by the dose of 100 mg/kg body weight of the extract may be adduced to the inability of the animals to initiate copulation as there was profound increase in the hesitation time of the male rats toward the receptive females. These findings complement that of Yakubu and Afolayan [10] who reported unwillingness and hesitation on the part of the males in making sexual advances toward the receptive females. Similarly, Ratnasooriya and Dharmasiri [25] have also attributed inhibition of sexual parameters to a sedative effect by the extract of Terminalia catappa seeds at a dose of 3000 mg/kg body weight. Therefore, the reduction in the success of mating and fertility observed in the highest dose group (100 mg/kg body weight)
Fig. 3. Photomicrographs of the cross section of testis of male rat orally administered B. natalensis stem extract at a dose of 25 mg/kg body weight for 7 days: (a) magnification 100; (b) magnification 400. The circled spot shows intact seminiferous tubules. The amount of stroma and population of spermatogenic and supporting cells are normal. The spermatocytes within the lumen are also normal (H & E).
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in the testicular body weight at all the extract doses (25, 50, and 100 mg/kg body weight) investigated could be due to increase in the metabolic activity, secretory ability, or an indication of organ swelling. Because the histologic examination did not reveal any inflammation, the increase in absolute testicular weight at doses of 25 and 50 mg/kg body weight of the extract can be attributed to increased metabolic activity, leading to increased testosterone production. The increase in the weight of the testes at the highest dose (100 mg/kg body weight) despite the non-increase in testosterone level could possibly be due to a generalized effect of enhanced testicular weight of the extract. This contrasts with the findings of Gupta et al. [27] on the reduction in testicular weight of male rats after the administration of saponins of Al-bizia lebbeck. However, the nonsignificant effect on the absolute body weights of the cauda epididymis, ventral prostate, and seminal vesicles may be an indication that the general metabolic conditions of these accessory sex organs were within the normal range [28]. Alterations in the sperm motility, viability, and morphology are an indication of a disturbed testicular and epididymal microenvironment [29]. Therefore, the absence of significant effect at all the doses investigated on the epididymal sperm count, motility, and morphology could possibly imply that the extract did not produce disturbance in the microenvironment of the epididymides. This may rule out impairment/hindrance in the normal functioning of the epididymal sperm cells during the experimental period. It is also possible that the extract was unable to cross the blood-testes barrier and thus could not interfere with the normal functioning of the testicular epithelium [30]. The fact that viscosity of the semen was not affected also indicated that liquefaction was within the normal range. All these showed that the plant extract may not hamper sperm function in male rats if administered repeatedly for 7 days. This further confirms that the reduction in the mating and fertility successes by the dose of 100 mg/kg body weight of the extract is not associated with the fertilizing capacity of the spermatozoa. Testosterone is the main male gonadal hormone produced by the interstitial Leydig cells [31]. It is required for the maintenance of normal sexual desire, nocturnal penile tumescence, and nonerotic penile erections in most men. The increase in testosterone level by the doses of 25 and 50 mg/kg body weight of the extract may be adduced to induction of the hormone synthesis by the Leydig cells, as the cells are the main source of testosterone [32]. This implies that the extract at these doses stimulated the mechanism intervening in
the process of the hormone synthesis in the Leydig cells [33]. Such increase will enhance androgen-dependent parameters like mating behaviour and maintenance of spermatogenesis. This may account for the increased libido in male rats observed in the 25 and 50 mg/kg body weight extract-treated groups in this study. The reduced testosterone content observed with the dose of 100 mg/kg body weight might imply that the extract at this dose reached a critical level to start exerting a negative effect on the hormone. The reduction in the testosterone concentration may explain the reduced mating success observed with the highest dose as testosterone is considered to contribute to improvement in sexual function, libido, and penile erection [34]. This is an indication of a potential negative effect of the extract on the male hormone at this dose. Normal male reproductive function depends on the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the pituitary gland under the influence of hypothalamic gonadotropin-releasing hormone (GnRH). LH in males stimulates the testicular Leydig cells to secrete testosterone, and FSH induces spermatogenesis in the seminiferous tubules of the testes [35,36]. The elevated levels of LH and FSH by the doses of 25 and 50 mg/kg body weight of the extract may be adduced to a stimulatory effect on the hypothalamic-pituitary axis and suggests a progonadotropic effect at the given doses and time points [31]. Such elevation in the gonadotropins may account for the observed increase in testosterone concentration in this study. In contrast, the absence of significant effect on LH and FSH at the highest dose may imply nonstimulation of the GnRH-LH signaling. In males, progesterone influences spermiogenesis, sperm capacitation, and testosterone biosynthesis in Leydig cells [36]. The increase in serum progesterone by the doses of 25 and 50 mg/kg body weight of the extract may have stimulated the biosynthesis of testosterone leading to its elevated levels in this study. The decrease in progesterone concentration by the dose of 100 mg/kg body weight of the extract may account for the reduced levels of testosterone observed in this study. Estradiol has an androgen-independent inhibitory effect on gonadotropin release [37]. The reduction in estradiol levels in the male rats may imply reduced inhibitory effect on the release of gonadotropins that were raised in this study. Interestingly, it is not clear why the dose of 100 mg/kg body weight of the extract also reduced the levels of estradiol as well despite nonelevation of the levels of FSH and LH in this study. The alterations in the hormonal profile of males at the doses of 25 and 50 mg/ kg body weight of the extract may account for the
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Fig. 4. Photomicrographs of the cross section of testis of male rat orally administered B. natalensis stem extract at a dose of 50 mg/kg body weight for 7 days: (a) magnification 100; (b) magnification 400. The circled spots depicts well preserved seminiferous tubules with normal amount of stroma and spermatogenic and supporting cells. The spermatocytes within the lumen are also normal (H & E).
increased libido and enhanced reproductive function of the animals, which contrasts to the effect of the dose of 100 mg/kg body weight of the extract on these indices. The adverse effect of the highest dose of the extract (100 mg/kg body weight) after repeated administration for 7 days could adversely affect reproductive capacity of the male rats. In this study, the histologic examination of the cross section of rat testes treated with the various doses of the extract revealed well-preserved seminiferous tubules with normal amount of stroma. These are indications that the extract did not adversely affect the histoarchitecture of the organs. The normal population of spermatogenic and supporting cells as well as spermatids within the lumen could possibly imply that the morphologic characteristics and the abundance of spermatozoa seen within the lumen of the seminiferous tubules were not adversely affected by the extract in this study. It also shows that the exocrine function of the testes was not impaired. This agrees with the findings of Moundipa et al. [33] who observed similar features after
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the administration of aqueous extract of Hibiscus macranthus and Basella alba to Wistar rats. The increase in the number of pregnant females is an indication of sex-enhancing properties of the extract at the doses of 25 and 50 mg/kg body weight. The reduction in the number of pregnant females by the dose of 100 mg/kg body weight of the extract may be attributed to either deficit in sexual arousal or the inability to initiate copulation [38,39]. Maternal parameters (body weight changes, vaginal bleeding, food and water intake) can be used to evaluate the integrity of maternal homeostasis [40]. The similarity in the maternal parameters as well as the absence of clinical signs of toxicity (salivation, diarrhea, changes in behavioural signs, and vaginal bleeding) in all the experimental groups suggest that the extract was not toxic to the mother [41]. The observation that the full-term sacrifice parameters (number of corpora lutea, implantation sites, live fetuses and masses of fetus, placenta, and ovary) were not different from the control values is an indication that the extract did not cause any malformation in the fetuses and that reproductive performance of the mothers was normal. This agreed with the findings of Guerra et al. [42]. The implantation index and preimplantation loss rate evaluate blastocyst implantation in the uterus, and
Fig. 5. Photomicrographs of the cross section of testis of male rat orally administered B. natalensis stem extract at a dose of 100 mg/kg body weight for 7 days: (a) magnification 100; (b) magnification 400. The circled spot shows intact seminiferous tubules with normal amount of stroma. The population of the spermatogenic and supporting cells as well as the spermatocytes within the lumen are normal (H & E).
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Table 5 Effect of Bulbine natalensis stem extract on female rat reproductive parameters. Parameters
Number of female rats paired Number pregnant Index of pregnancy, n (%) Mass gain in pregnancy (g) Mass gain in the organogenic period (g) Food intake (g/day per rat) Water intake (mL/day per rat) Number of live fetuses Number of dead fetuses Mass of fetuses (g) Mass of placenta (g) Mass of ovary (g/100 g) Number of implantation sites Number of resorption sites Number of corpora lutea Implantation index (%) Resorption index (%) Preimplantation loss (%) Postimplantation loss (%)
Control
10 6 6 of 10 (60%) 114.30 8.21a 30.10 1.50a 17.10 2.14a 24.88 3.14a 12.24 0.30a 0 3.90 0.05a 0.35 0.04a 0.015 0.002a 13.40 0.03a 1.00 0.01a 14.20 0.40a 94.37 3.61a 7.46 0.21a 5.63 0.12a 8.66 0.22a
B. natalensis extract dose (mg/kg body weight) 25
50
100
10 10 10 of 10 (100%) 115.10 7.42a 30.26 1.70a 18.21 1.50a 25.31 2.63a 12.40 0.20a 0 4.00 0.03a 0.35 0.02a 0.016 0.002a 13.60 0.01a 0.00 0.00b 14.40 0.50a 94.44 4.01a 0 0.00b 5.56 0.21a 8.82 0.11a
10 10 10 of 10 (100%) 115.30 6.83a 30.44 1.08a 18.40 1.07a 26.41 1.55a 12.61 0.20a 0 3.97 0.02a 0.35 0.04a 0.016 0.001a 13.70 0.01a 0.00 0.00b 14.51 0.04a 94.42 4.07a 0 0.00b 5.58 0.16a 7.96 0.74a
10 4 4 of 10 (40%) 114.60 7.08a 30.05 1.21a 17.69 1.79a 25.72 1.82a 12.28 0.14a 0 4.01 0.06a 0.35 0.05a 0.015 0.001a 13.30 0.05a 2.00 0.01c 14.30 0.03a 93.01 4.26a 15.04 1.16c 6.99 0.08a 7.67 0.86a
n = 10, mean SD. a–c Parameters with different superscripts are significantly different.
the resorption index and postimplantation loss rate establishes correlation between the number of implanted blastocysts and those that have not developed [40,43]. In this study, the absence of statistical difference in the implantation index, preimplantation and postimplantation losses between the distilled water–treated control and the extract-dosed groups (25, 50, and 100 mg/kg body weight) is an indication that the implantation of the blastocysts was within normal range. This complements the findings of CostaSilva et al. [16] on the effect of administration of Carapa quianensis seed oil during pregnancy in female Wistar rats. However, the nil value of resorption index observed with the 25 and 50 mg/kg body weight extracttreated groups suggests normal development of the implanted blastocysts and normal reproductive capacity of the animals. In contrast, the higher value of resorption index after the administration of 100 mg/ kg body weight of the extract connotes increased failure rate of embryo development and antipregnancy [43]. This is an indication that the extract at the highest dose (100 mg/kg body weight) investigated has the potential of being embryotoxic and may not be completely safe as an oral remedy during pregnancy at the given dose. The absence of significant effect on the serum prolactin concentration in female rats observed with the extract at all the doses investigated was an indication that the production of dopamine was not affected, as
prolactin secretion is controlled by the hypothalamic hormone. FSH and LH enhance the growth and maturation of ovarian follicles by acting directly on the receptors located on the granulosa cells [44]. Therefore, the similarity in the values of FSH and LH in all the experimental groups was an indication that folliculogenesis was not hindered. Similarly, the pattern obtained for progesterone and estradiol are indications that the various functions associated with conception, pregnancy, and growth of uterine lining were not affected [45,46]. The pattern exhibited by the extract at doses of 25, 50, and 100 mg/kg body weight on the female reproductive hormones suggests that the extract did not generally affect the reproductive hormones. It further implies that the potential of embryo lethality at the dose of 100 mg/kg body weight of the extract may not be mediated by hormonal changes but rather a direct effect on the embryo. In this study, the enhanced success rate of mating and fertility at the doses of 25 and 50 mg/kg body weight of the extract suggests that it was more efficiently active within the target tissues in the male animals at these doses than at the highest dose (100 mg/kg body weight). The results of the current study revealed that B. natalensis extract at the doses of 25 and 50 mg/kg body weight has the tendency of enhancing reproductive function in male rats and may not adversely affect pregnancy and embryo development in the female rats.
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