Dried RGL herb was purchased from Zard-Band Herbal Medicine Factory. The plant was cultured in botanic farm of the factory in Zard-band village, north of Tehran, with these geometrical characteristics: Latitude 35° 47 north, longitude 51° 37 east, altitude 1548 m from sea. In the last 10 years, the maximum temperature has been 39 °C, and the minimum −6.8 °C and the average humidity has been 489.2 mm in this area. The plant was harvested before the seeds were fully developed (June 2009) and dried in dark room with free ventilation. The plant was approved in the herbarium of Pharmacognosy Department of the Faculty of Pharmacy affiliated to Shahid Beheshti University of Medical Sciences by a Botanist with herbarium number 066.

Ruta graveolens L. herb (100 gr) was immersed in 200 ml of distilled water, put on stirrer for 48 hours in room temperature and a dark place, and filtered through a paper filter. Afterwards, the container was placed into a water bath for evaporation. The brown jelly extract was kept in −4 °C for further experiments.

Adult Wistar male rats (42) were divided into six groups (n=7), the first group serving as the control group. Ruta graveolens L. aqueous extract (5 g/kg) was administered orally to all test groups and sperm motility was checked after half, one, two, four and six hours (groups 2 to 6). The rats were kept in standard conditions (light/dark cycles: 12/12 hr per day, temperature: 22–25 °C, humidity: 40%−50%) and received laboratory rat chow and water ad libitum. All experimental procedures were consistent with the guidelines of Ethical Committee of Tehran University of Medical Sciences about research on animals and humans.

According to Opdyke (24), the RGL lethal dose for rats is more than 5 g/kg. Hence, we considered this dose as the maximum safe dose and fed it to rats to evaluate if the recommended safe dose had any contraceptive effects. Each group received 5 g/kg RGL aqueous extract orally by intra-gastric gavage and were checked for sperm parameters after half, one, two, four, six hours. There control group received only the vehicle (distilled water). To determine sperm count and motility, one centimeter of cauda epididymis was resected and minced in 1 ml of 37 °C normal saline solution. Ten microliter (µl) of a mixed sample was placed on a pre-warmed slide and a minimum of 10 fields were viewed and 200 spermatozoa were assessed by high power microscopy (400× magnifications). Motility was assessed according to the WHO classification system (25). Briefly, the criteria were as follows: grade a: rapidly progressive spermatozoa, grade b: slowly progressive spermatozoa, grade c: no progressive motility, grade d: immotile spermatozoa. The group which showed the highest significant level compared to the control animals in this test was selected for more evaluations.

Sperm viability was asses, using eosin-nigrosin staining test ((25). Briefly, 25 µl of dye and the same amount of sample were mixed for 30 seconds. Then a smear was provided by a 15 µl droplet of the mixture. After drying the smear in room temperature, the slides were examined under a light microscope (1000× magnification). Live spermatozoa were unstained (white) but dead cells were stained red. After counting at least 200 sperm cells, the percentage of dead and alive spermatozoa was calculated.

Morphology assessment was done by Diff-Quick staining method (25). After mincing one centimeter of cauda epididymis in 1 ml of 37 °C normal saline solution, one drop was placed on microscope slide to prepare a smear and fixed for 15 seconds in fixative solution (1.8 mg/l triarylmethane in methyl alcohol) and stained in solution No. 1 (1 g/l xanthene in sodium azide-preserved buffer) for 10 seconds and then in solution No. 2 (1.25 g/l thiazine dye mixture, 0.625 g/l Azure A and 0.625 g/l methylene blue in buffer) for 5 seconds and then was dried in room temperature. After mounting the slides, they were seen by a light microscope (1000× magnification) (Figure 1). The slides were assessed for any abnormality in tail, neck or head.

The spermatozoa of treated rats and the control group were stained by acridine orange dye according to the modified method of Tejada et al. (26). Briefly, a smear was prepared from a 15 µl sample and then fixed in a 1:1 (v/v) ethanol–acetone solution for 30 min at 4 °C. After drying at room temperature, the slides were placed in a dark jar containing acridine orange dye for 5 min in the same temperature. After washing the slides with distilled water, they were immediately viewed and 200 cells were counted in each slide under 1000-fold magnification using a florescent microscope in 470 nm florescent beams. Red to yellow-stained cells indicated on a denatured chromatin with single-stranded DNA were considered abnormal while the green ones on an intact chromatin with double-stranded DNA were considered as normal spermatozoa DNA fragmentation index was calculated by dividing the abnormal to the total (normal and abnormal) spermatozoa and reported as percentage.

Blood testoster-one level was assessed in the controls and the 1 hour test groups. Blood sample was taken directly from heart 1 hour after gavage, under anesthesia. Hormonal analysis was done by ELIZA method using LIAISON^® testosterone (310410) assay kit (DiaSorin Inc., USA).

One-way ANOVA and Tukey's post-hoc tests were employed for compareson between groups. Student's t-test was applied for statistical comparisons between two groups. The significance level (p-value) was considered smaller than 0.05.

Thirty minutes after oral gavage of RGL extract in male rats, motility of the spermatozoa was reduced and one hour after gavage the percentile of immotile sperm increased significantly compared to the controls (64% and 31.85%, respectively; p <0.01). The motility gradually increased afterwards, and by 6 hours, it was the same as controls (65.43% and 68.15%, respectively). Thereafter, the immobilizing effect was reversed and no group was different from the control group (Figure 2). The group which showed the most significant difference in motility was called the “one-hour group” and was used for subsequent assessments. Regarding the progresssive and non-progressive motility, although the data showed a trend to decrease both parameters after administration of RGL till one hour and a trend to increase both parameters 1−6 hour after the intervention, but the differences were statistically insignificant for both parameters between different groups (For progressive motility, 48.1±4.3, 25±6.4, 36.3±6.5 and for non-progressive motility, 20.05±4.6, 11±6.5 and 19.13±6.5 for control, one-hour and six-hour groups, respectively).

Although a reduction about 9% in viability of spermatozoa was seen one hour after RGL gavage compared to the control group, but it was no statistically significant (65.85±3.8 and 74.28±2.72, respectively) (Table 1).

Diff-Quick staining revealed no significant differences in normal morphology of spermatozoa in the one-hour and control groups (72.71%±4.26 and 66.28%±3.18, respectively) (Table 1).

Measurement of blood testosterone level showed that RGL at the dose of 5 g/kg had no adverse effects on serum testoster-one levels in the treated rats and the reduction was not statistically significant compared to the control group (475±80 and 507.5±79 ng/ml, respectively).

Acridine orange staining technique did not show any significant difference for abnormal DNA status between RGL treated (one-hour) and control groups (12±1.39 and 10.42±1.55, respectively) (Table 1).