Effect of Alcohol Extracts of the Ruta graveolens L. On the Count, Motility and in vitro Fertilization Capacity of Rat's Sperm

INTRODUCTION

One of the important concerns is the problem of the overpopulation. The population of the world is growing faster than the supplies of food, shelter and fuel. A lot of efforts have made to find a safe and effective method with the least side effects but beyond condom and vasectomy there is no other choices for male contraception (Nieschlag and Henke, 2005). Many hormonal, immunological and chemical substances are being investigated but none of them is completely desirable and without side effects (Jensen, 2002; Rahim et al., 2009). Looking through history, ancient people were also interested in contraception and had their own way of controlling their family size. Medicinal plants were one of those methods. Ruta graveolens L. is a member of Rutaceae family. It is a hardy, evergreen shrub of up to one meter tall, with characteristic grayish green color and a sharp unpleasant odor. This plant have different established effects like antimicrobial, cytotoxic (Ivanova et al., 2005), fungicide (Oliva et al., 2003), herbicide (Hale et al., 2004) and anti-inflammatory (Raghav et al., 2006, 2007).

Ruta graveolens L. is one of the most ancient and effective contraceptive plants (Maurya et al., 2004). It's potent female anti-fertility and abortive effects have been reported from countries like Brazil (De Freitas et al., 2005), Indian (Gandhi et al., 1991), Peru (Gutiérrez-Pajares et al., 2003) and Mexico (Conway and Slocumb, 1979) both according to their traditional usage and in animal studies. In addition to its potent abortive effects, it has been mentioned by folk and traditional medicine to have a male contraceptive effect. There are a few reports about effect of this plant on count, motility and in vitro fertilization capacity of sperm (Harat et al., 2008; Khouri and El-Akawi, 2005). The present study is an attempt to evaluate the effect of alcohol extracts of the Ruta graveolens L. on the count, motility and in vitro fertilization capacity of Wistar rat's sperm.

MATERIALS AND METHODS

Animals: This experimental study was performed in physiology research center of Ahwaz Jondishapour University of Medical Sciences (AJUMS) from March 2008 to August 2009. Total 24 adult male Wister rats, 90-day-old and weighing 210±10.6 g were used in this study. The animals were purchased from Laboratory Animals Care and Breeding Center of Ahwaz Jondishapour University of Medical Sciences, Ahwaz, Iran. The fertilizing ability of male mice was proved by selecting post first wave of spermatogenesis that mate and observed positive pregnancy at the beginning of experiment. All animals were housed individually per cage under a 12 h light/dark cycle, 20±2°C temperature and 60-65% humidity-controlled room with food and water ad libitum. All procedures were approved by international guidelines and by the Institute Research Ethics and Animal Care and Use Committee of Ahwaz Jondishapour University of Medical Sciences. Every effort was made to minimize the number of animals used and their suffering.

Preparation of extract: Ruta graveolens L. aerial parts were collected from an Iranian plants species and dried in a ventilated place at room temperature for 15 days. The aerial parts were grinded in an electric grinder and the powder (72.3 g) was extracted in 1500 mL of 70% ethanol under stirring for 72 h. The extract was filtered, concentrated in a vacuum evaporator at 50°C and then lyophilized. The yield of the lyophilized extract was stored in the dark, at room temperature, in desiccators with silica gel (Harat et al., 2008).

Experimental setup: Twenty-four adult male rats were subdivided randomly to 3 groups: group 1: serve as untreated controls, group 2: received 1 mL day^-1 of 5% ethanol and group 3: received 1 mL day^-1 of 5% ethanol extract of Ruta graveolens L. (20 mg day^-1) for 50 consecutive days by intraperitoneal injection. Twenty-four hours after last treatment, rats in three groups were killed and their epididymis were excised and weighed.

Sperm motility analysis: Sperm motility of three groups was determined using Makler chamber. All counts were performed at 37°C in T6 media. The sperm motility was assessed and classified as progressive, no progressive. Initial sperm motility was manually assessed by a single individual in duplicate for each sample by evaluating 100 sperms. Total motility was defined as any movement of the sperm head and progressive motility was defined as the count of those spermatozoa that moved in a forward direction (Movassaghi et al., 2009).

Measurements of sperm count: Sperms were collected from the epididymis of each rat by flushing with the same volume (about 8 mL) of T6 medium. Collected samples were centrifuged at 100 g for 2 min and the precipitate portion was resuspended in 10 mL of fresh T6 medium. A fraction of suspension (100 μL) was mixed with an equal volume of 1% Trypan blue in the same medium and numbers of sperms were counted in four chambers of hemocytomere slide (Pook et al., 2005). The sperm number was expressed mL^-1 of suspension.

Oocyte collection: Adult female Wistar rats that were between 10 to 12 weeks old were administered intraperitoneally with 10 IU Pregnant Mare Serum Gonadotropine (PMSG) for superovulation; this was followed 46-48 h later by the intraperitoneal administration of 10 IU Human Chorionic Gonadotropine (HCG). Rats were killed 12-14 h after HCG injection by cervical dislocation method. After disinfection with 70% alcohol and opening the abdomen wall, the Y shaped uterus, ovaries and oviducts were identified. The oviducts were excised as follows: clamping cornuas, dissecting the peritoneum and fat between ovary and tube and then cutting the fallopian tube from the proximal end and cumulus- oocyte complexes were collected in T6 medium. The granulosa cells of oocytes were removed by pipetting in T6 medium containing 80 IU mL^-1 hyaluronidase and mature oocytes obtained and randomly divided into three groups (Hjollund et al., 2004).

In vitro fertilization: In vitro fertilization was carried out in drops of T6 medium plus 5 mg mL^-1 BSA under mineral oil. A pre-incubated capacitated sperm suspension of different groups as mentioned above was gently added to the freshly ovulated ova which divided in three groups to give a final motile sperm concentration on 100000 mL^-1. The combined sperm- oocyte suspension was incubated for 4-6 h. The ova were then washed through several changes of medium and finally incubated in drops of T6+5 mg mL^-1 BSA under mineral oil. Fertilization was assessed by recording the number of 2 cell embryos 24-26 h after completion of fertilization in vitro (Ozawa et al., 2002).

Statistical analysis: All the reported values except fertilization rate were reported as Mean±Standard Deviation (SD) and percentage. Fertilization rate reported as percentage (%). The statistical significance of difference between the control and experimental groups was determined by the unpaired t-test. Differences between the means were considered to be significant when p<0.05 was achieved.

Table 1:	Sperm count in 1 g of epididymis, different type of motility and in vitro fertilization capacity of sperm in three groups of study

Group 1: Untreated controls, Group 2: The adult male rats treated with the 5% ethanol. Group 3: The adult male rats treated with the extract of Ruta graveolens L. n: Number of replications in each group, The significant lower sperm count, the significant lower sperm motility*, the significant lower fertilization capacity (p<0.05)

RESULTS

In every group of this study 8 adult male rats with 12 weeks old were used. As shown in Table 1 the sperm count±SD in 1 g of epididymis was 2798.5±192.40 in group 1, 2801.8±418.67 in group 2 and 1017.4±820.69 in group 3. Therefore, group 3 has a significant lower sperm count in comparison with other groups. Sperm with progressive motility was 57.20±2.81 in group 1, 55.25±1.82 in group 2 and 19.26±3.17 in group 3 (Table 1). Statistical analysis show that rats in group 3 have significant lower sperm motility in comparison with other groups. There was no difference between other groups. The fertilization capacity of sperm of rats in group 3 was significantly lower than other groups.

DISCUSSION

The present study clearly shows that intra-peritoneal injection of Ruta graveolens L. extract decreased the epididymal sperm count of rats. This finding is in agreement with the previous studies (Harat et al., 2008; Khouri and El-Akawi, 2005). It is well known that the function of accessory reproductive organs and also process of spermatogenesis are androgen dependent (Choudhary and Steinberger, 1975) so the alcohol extract of Ruta graveolens L. may act directly or indirectly on the pituitary gland secretary function and cause decrease in the androgen. In this study, we observed that testosterone hormone level in treated rats significantly decreased but the level of Follicular Stimulating (FSH) and Leutinizing Hormone (LH) did not observed due to some limitation. In this study, we observed that the progress motility of sperm in treated rats significantly decreased comparing to other groups. This manifestation may due to an alteration in the microenvironment in the cauda epididymis, which also had a inhibition action on the metabolism of the treated rats as a result of androgen-inhibitory effect of the alcoholic extract of Ruta graveolens L. (Khouri and El-Akawi, 2005) It is well known that the production of spermatozoa able to fertilize and develop a normal progeny results from normal sperm maturation in the epididymis. The composition of the internal epididymal milieu, responsible for sperm maturation, is under androgen control (Almeida et al., 1998). In rats, an androgen-binding protein secreted by sertoli cells into the lumen of seminiferous tubules under follicle-stimulating hormone stimulation is transported to the epididymis, where it accumulates at concentrations higher than those found in the testes. This leads to a high local concentration of androgens, essential for maturation of epididymal spermatozoa (Almeida et al., 2000). The reduced fertilization capacity of treated rats is probably due to a decrease in the number and progress motility of sperm. As a conclusion, the alcohol extract of Ruta graveolens L. can be suggested as agent against male fertility but the exact mechanism of action is not understood yet.

ACKNOWLEDGMENTS

This project with grant No. u-22 was financially supported by the research deputy of Ahwaz Jondishapour University of Medical Sciences (AJUMS). We would like to express our great appreciation for their support.