Therapeutic and fertility restoration effects of Ionidium suffruticosum on sub-fertile male albino Wistar rats: effects on testis and caudal spermatozoa

Abstract

Context: Ionidium suffruticosum (L.) Ging (Violaceae) is an important medicinal plant widely used as a herbal traditional medicine in Ayurveda for the treatment of infertility. Currently, little pharmacological information is available on its male fertility properties following prolonged use.

Objective: To investigate I. suffruticosum leaf extracts for male fertility parameters.

Materials and methods: The ethanol lyophilized fraction was administered orally on carbendazim-induced sub-fertility rats (250 mg/kg body weight for 28 days). The effects of fractions on rat’s fertility parameters i.e., body and testes weight, sperm motility, sperm vitality, epididymal sperm counts, its morphology, enzyme and antioxidant stress and histopathology were studied and compared with clomiphene citrate.

Results: The sub-fertile male rats treated with I. suffruticosum leaf extract increased the body weight of 7 g, testis weight of 97 mg, increased cauda epididymal sperm counts of 34.2 × 10⁶ sperm/mL, motility of sperm 46% and vitality 28% also increased and normal sperm morphology also improved up to 32%. The carbendazim-treated group showed loss in body weight of 33 g, testis weight of 851 mg, decreased epididymal sperm counts of 15 × 10⁶ sperm/mL, with sluggish motility and a highly significant fall in the live sperms of about 57%.

Discussion and conclusion: The leaf fraction of I. suffructicosum increased the testicular weight, spermatogenesis, sperm counts, lessened sperm agglutination, and increased testicular oxidative biomarkers, SOD, and CAT. This study therefore supports the usage of I. suffructicosum in traditional medicine for infertility.

Keywords:

• Male reproduction
• sperm motility
• spermatogenesis
• epididymal sperm
• malondialdehyde
• catalase
• superoxide dismutase

Introduction

In the recent times, there has been a serious concern about the deterioration of human semen quality. It is generally believed that the counts of viable sperm in the normal human ejaculate have decreased to half over a period of about 50 years (Carlsen et al. 1992; Bradbury 1997) and the causes of male infertility due to oligospermia or azoospermia are also on the increase (Jager et al. 1999), causing a long queue in centres providing assisted reproductive technologies. Among the major causative factors of reduced semen quality and male infertility are environmental chemicals such as toxic chemical wastes from various industries and agricultural systems. These toxic substances appear to be carried into the food chain resulting in their bioaccumulation in the food chain. Exposure to such toxic substances either causes the death of the organisms directly or alters the ability of the organisms to grow, develop, and reproduce (Cooney 1995). Such chemicals have been shown to cause subtle to severe changes in several aspects of male reproduction particularly on spermatogenesis and androgenesis. Reproductive toxicities mostly remain undetected and thus, affect the very begetting.

Carbendazim is a fungicide widely in application in agricultural practices and preservation and storage of agricultural produces. Chemically, it is methyl-2-benzimidazole carbamate (MBC). It is derived from yet another benzimidazole compound namely benomyl (Teubert & Stringham 1984). The fungicidal property of the compound lies in its ability to dissociate the tubulin of fungal mycelium, thereby prevent their propagation. On access into non-target organisms including man, it can manifest effects disrupting the microtubules. As microtubules constitute an important aspect of male reproduction, namely cytoskeletal support of the Sertoli cell (Nakai et al. 1995) and other epithelial cells, the ectoplasmic specialization of the sertoli cell into germ cell associations (Nakai et al. 1995) and the cell division apparatus in male germ cells (Nakai & Hess 1997), carbendazim could be a serious male reproductive toxicant.

Traditional knowledge related to the use of natural resources including medicinal plants has been recognized as one of the important assets inherited through generations by the local communities. The tribal communities such as Gowlis Kaani, Kunabis and Siddis inhabiting the Western Ghats region are one of the richest knowledge systems on tribal medicines in India; the treatment of their diseases is almost entirely confined to herbal medicines. Several herbals are known to potentiate the male reproductive function and increase the sperm counts.

Eagle marmelos (L) Correa (Rutaceae) is a popular plant, with rich medicinal properties (Bhattacharya 1986), the leaves of this plant are used to treat spermatorrhoea (Bhattacharya 1986). Eurycoma longifolia Jack (Simaroubaceae) root extract treated rats retained a higher level of sexual activity and also showed proandrogenic activity (Anga & Cheanga 2000) The aqueous extracts of Cynomorium coccineum (L) (Cynomoriaceae) and Withania somnifera (L) Dunal (Solanaceae) showed direct spermatogenic influence on the seminiferous tubules of the immature rats as well as serum levels of testosterone. Withania somnifera is recommended and used to treat for impotence or seminal debility, as an aphrodisiac and as an invigorator (Sankaran 1984). Withania somnifera roots have many steroidal lactones, which have structural similarities with that of androgens (Tripathy et al. 1996). Yohimbine a drug obtained from Rauvolfia species is also reported as having a stimulatory effect on spermatogenesis in rats (Rastogi & Malhotra 1990).

Ionidium suffruticosum Ging (Violaceae) is a sporadic, rare, ephemeral, ethnomedicinal herb (Deshpande 2006) widely used in traditional healers to treat the diseases such as jaundice, male sterility (Kheraro & Bouquet 1950; Senthil Kumar et al. 2013), diabetes (Sarita et al. 2004), malaria (Soh & Benoit-Vical 2007), urinary tract infections and water retention (Pushpangadan & Atal 1984) and also used as a tonic. The fruits are antidote for scorpion-sting. The leaves are sub-sessile, liner to oblanceolate, 1.5–2.0 × 0.08–0.3 inches, entire or serrate, flowers solitary, axillary, red, spurred, fruit a small sub globose capsule containing ellipsoid, longitudinally striate, yellowish white seeds. The leaves and tender stalks are demulcent and used as a decoction or electuary, in conjunction with oil, employed in preparing cooling liniment for the head. The plant is a seasonal perennial herb and is widely distributed in Africa, Madagascar, Srilanka, China, New Guinea, tropical Australia and India. In nature, the plants appear for only few months from June to September (monsoon season). The roots and few basal stem stocks remaining in the soil regenerate only during rainy seasons and soon after the rainy season the plants dry and disappear. As less research was carried out on male fertility, an attempt was made in search for the traditionally used medicinal plants i.e., I. suffruticosum, to cure male impotency. It would be important to confirm the fertility effects of I. suffruticosum in carbendazim-induced sub-fertile male albino rats and compared with that of standard fertility drug clomiphene citrate.

Materials and methods

Plant extracts preparation

The leaves of I. suffruticosum were collected locally from the foothills of the Western Ghat’s area adjacent to Bharathiar University, Coimbatore, Tamil Nadu, India in mid-2008. Botanic identification was first conducted in the field and further confirmed by P. Mahendran, Scientist, the Botanical Survey of India, Tamilnadu Agricultural University Campus, Coimbatore, Tamilnadu, India. The voucher herbarium specimen was deposited at the departmental General Library, School of Life Sciences, Bharathiar University, Coimbatore, India. The leaves were washed with double-distilled water and were shade dried at room temperature. The dried parts were powdered with the help of an electric blender. Crude extractions from the plants were performed in the medicinal chemistry laboratory of the School of Life Sciences, Bharathiar University, Coimbatore, India. The powdered dried leaves (50 g) were subjected to ethanol in a Soxhlet apparatus (Borasil, Mumbai, India) for 72 h (Saxena et al. 1994), and the solvents were removed from the plant extracts in a vacuum rotary evaporator at 80 °C under reduced pressure yielded a semi-solid materials of 11 g. Chloroform and diethyl ether (1:3, v/v) were added to semisolid materials. The resulting mixture was stirred using a rotary (120 rpm) shaker (Gerhardt, Königswinte, Germany) at 20 °C for 5 h. The content was later filtered through Whatman grade 1 filter paper. The filtrate was again passed through a round filter paper of 90 mm diameter with the help of mini filter pump machine. Fraction of organic filtrate collected this way were pooled together, kept in wide mouth amber bottles. The organic solvent was then removed with the help of a rotary evaporator and afterwards subjected to lyophilization procedure. The lyophilization was done using a freeze dryer (Labconco Corporation, Kansas City, MO).

Experimental animals

Healthy adult male albino rats (Wistar strain) weighing 150–180 g, procured from Tamil Nadu Veterinary and Animal Sciences University Chennai, were used in this investigation. The animals were housed in well-ventilated polyurethane cages at normal room temperature, fed with standard rat pellet diet and clean drinking water ad libitum. Handling of the animals was done in accordance with the Guide for Care and Use of Laboratory Animals, Bharathiar University; this work was approved by the ethical committee for using animals (no. 721/02/R/CPCS EA).

Clomiphene citrate

The fertility effects of I. suffruticosum leaf extracts were compared with a minimal dose (0.03 mg/kg) of clomiphene citrate 2-[p-(2-chloro-1, 2-diphenylvinyl) phenoxy] triethylamine dihydrogen citrate. clomiphene citrate 25 mg Fertyl-M Tablets, Ar-Ex Laboratories Pvt Ltd, Goregaon (E), Mumbai, India. Stock solutions of 25 mg/mL is prepared and test solutions are prepared accordingly by adopting V₁C₁= V₂C₂ approach.

Carbendazim

Carbendazim (methyl 2-benzimidazole carbamate) (Bavistin, BASF-India Ltd, Mumbai) was purchased from a local agrochemical supplier. The product powder contains carbendazim at 50% strength (w/w).

Carbendazim treatment

Bavistin (carbendazim) was suspended quantitatively in refined sunflower oil in such a way as to prepare 100 mg powder in 1 mL oil (50 mg carbendazim active compound in 1 mL). Each animal weighing about 150 g received Bavistin at a single bolus dose of 800 mg/kg body weight (i.e., 400 mg carbendazim active compound per kg body weight) and the control rat received equal quantity of the sunflower oil only.

Experimental design was done as below. The albino rats were divided into four groups and each groups comprising of five animals.

Group-1: Served as control. One group received 1 mL of water orally as vehicle and another group received plant extract of I. suffruticosum by using an intragastric catheter (IGC) for 28 days.

Group-2: Carbendazim-induced sub-fertile male rats. They received Bavistin (carbendazim) at a single bolus dose of 800 mg/kg (i.e., 400 mg carbendazim active compound per kg body weight through oral route by using an IGC

Group-3: Carbendazim-induced sub-fertile male rats treated with the leaf extracts of I. suffruticosum orally by using an IGC (250 mg/kg/day) for 28 days.

Group-4: Carbendazim-induced sub-fertile male rats treated with clomiphene citrate (0.3 mg/kg/day) orally by using an intragastric catheter for 28 days.

All groups are maintained for 28 days with food and water ad libitum.

Harvest and preparation for analysis

The rats were weighted before and after the treatment. At the end of the treatment, the rats were subjected to anaesthesia and the testes were removed and washed in physiological saline. The right testis was weighted in an electronic balance and data for each group were used to calculate the respective means and the standard deviation. Slices of left testis were fixed in Bouin’s fluid and used for histological analysis. The cauda epididymis from the right side was transferred on to a cavity slide. After washing in physiological saline of 0.9% NaCl, the cauda was incised at two or more places allowing the semen to ooze out. The semen was used for the several spermatological analyses.

Spermatological studies

The cauda epididymal duct on one side was exposed and incised. The sperm oozing out from the incision was quickly sucked into a capillary tube up to 0.05 μL mark. It was diluted 200 times using phosphate buffer saline. After thorough mixing, the dilute semen was used for the spermatological analysis.

Sperm motility assessment

A drop of dilute sperm was transferred with help of Pasteur pipette on to a cover glass. The cover glass was inverted over the cavity slide to obtain a hanging drop. The edges of the cover slip were sealed using Vaseline. The hanging drop preparation was observed under a compound microscope at 450 × magnification. The preparation was observed at regular intervals in such a way as to find the duration, in minutes, of the motility of the last motile sperm. For each animal, two separate hanging drop preparations were made, and motility was assessed by two independent observers. The data for each animal were used to obtain the average.

Sperm vitality

Sperm vitality is assessed by adding one drop of eosin stain Y and one drop of Nigrosine in an Eppendorf tube. To this, one drop of semen was added and mixed using a Pasteur pipette. Drop of the mixture was placed on a micro slide and covered with coverslip and observed for at least 200 spermatozoa under microscope and considered as stained spermatozoa as dead and unstained as alive.

Sperm counts and morphology

Sperm counts were made according to Gopal and Shah (1985), briefly diluted and thoroughly mixed semen was transferred to improved Neubauer counting chamber and a cover slip was overlaid. The Neubauer chamber was observed under a compound microscope and sperm in Central Square were counted. The central square has 25 large squares. The volume of each of the 25 squares is 0.1 mL. The sperm counts were calculated using the following formula: $=(\mathrm{Number}\mathrm{of}\mathrm{sperm}\mathrm{in}25\mathrm{squares}/25)\times 10\times \text{dilution factor}\mathrm{\hspace{1em}\hspace{1em}\hspace{1em}}\times 1000\hspace{1em}\mathrm{sperm}/\mathrm{mL}$

Sperm morphology was observed adopting Eosin–Nigrosin methods. One or two drops of Eosin–Nigrosin stain and a drop of semen were separately placed on the one end of a clean warm microscopic glass slide. The semen and the stain were mixed well and drawn out with the edge of another slide which served as a spreader so that, a thin film was and air dried and was observed under microscope. At least 200 spermatozoa from different fields of the slide were examined for their morphological features.

Sperm agglutination

To find the agglutination, a drop of diluted semen was smeared on a clean slide, fixed in ethanol and stained with the Leishman stain. Then, the slide was examined under 450 × to find the incidence of sperm agglutination.

Oxidative stress and antioxidant enzyme assays

The antioxidant and oxidative stress of carbendazim-induced sub-fertile rats were measured. The lipid peroxidation (LPO) was estimated by adopting methodology of Ohkawa et al. (1979). Superoxide dismutase (SOD) activity was measured using methods of Rest et al. (1977). Catalase (CAT) activity was determined from the method of Aebi et al. (1974) by measuring the rate of decomposition of hydrogen peroxide. Briefly, the testis and tissues were homogenized in 0.1 M phosphate buffer and centrifuged at 18,000 RPM for 30 min and the supernatant was utilized for biochemical analysis.

Histological analysis

The histology of the testis was studied adopting the paraffin method by Humason (1979) and sections were observed under microscope and photographed at 100×, 200× and 450 × magnification.

Statistical analysis

The statistical data were analyzed using Student’s t-test (Olsen et al. 1995) and expresses as mean ± Standard Error of Mean (SEM) and calculated as: $\mathrm{SEM}=\frac{\sqrt{\sum ^{}{x}^2-(\sum \hspace{1em}x{}^2/n)}}{n(n-1)}$ where x = individual observations, n = number of observationt value was calculated by the following formula and compared by the table value of 5% and1% levels of significance. $T\hspace{1em}=\frac{X_1-X_{{}^{{}_2}}}{S\surd (1/n_1+1/n_2)}$ where $S=\frac{\surd \sum x_1^2\hspace{1em}-\hspace{1em}(\sum x_1^2/n_1)+\sum x_2^2\hspace{1em}-(\sum x_1^2/n_1)}{n_1+n_2-2}$ n₁ and n₂ denotes the number of observed value in the classes being compared (Olsen et al. 1995).

The following levels of significance were used. The values falling in p < 0.01 levels were considered significant data and p > 0.01 are non-significant data. One-way ANOVA with post hoc Tukey’s HSD Test Calculator with the Scheffé multiple comparison tests was used.

Results

Effect of I. suffruticosum on body and testis weight in carbendazim-induced sub-fertile male rats after 28 days of the treatment

Body weight

The results of the body and testicular weight of the experimental animals are shown in Table 1. The body weight of the control rats (group 1) were 164 g. After 28 days, there was a normal weight gain in the body weight of 8–9 g leading to 172 g (saline) to 174 g (extract). But, in the carbendazim induced sub-fertile rat in the group 2, there was a significant loss in the body weight of about 33 g when compared to its initial weight of 158 g. In group 3, rats treated with I. suffruticosum have shown a loss in the body weight of about 24 g from the initial weight of 156 g to the final weight of 132 g. In group 4, the rats treated with clomiphene citrate had a loss in the body weight of about 30 g from the initial weight, when compared to carbendazim-induced sub-fertile group 2 rats, the remaining treated groups 3 gained a slight gain in the body weight and are statistically significant (p < 0.01).

Table 1. Effect of I. suffruticosum on body and testis weight and epididymal spermcounts in Carbendazim-induced subfertile male rats after 28 days of treatment.

	Body weight (g)
Experimental group	Initial ± SEM	Final ± SEM	Testis weight(mg/kg body wt) ± SEM	Caudal epididymalsperm counts × 10^6 sperm/ml ± SEM
Group 1: Control (Saline only)	164 ± 0.6	172 ± 0.7	1248 ± 1.4	40.50 ± 0.5
Control (extract only)	164 ± 0.7	174 ± 0.4	1258 ± 1.2	49.0 ± 0.7
Group 2: Carbendazim-induced subfertile male rats	158 ± 0.7	125 ± 0.7	851 ± 0.3	15.40 ± 0.3
Group 3: Carbendazim-induced subfertile male rats treated with I. suffruticosum	156 ± 0.4*	132 ± 0.6*	948 ± 0.4*	49.60 ± 0.5*
Group 4: Carbendazim-induced subfertile male rats treated with Clomiphene citrate	150 ± 0.7	120 ± 0.5	906 ± 0.3	45.50 ± 0.4

Each value is of Mean ± SEM of five animals.

* Significantly different (p < 0.05) from the control group by Dunnett’s test.

Scheffé multiple comparison tests used to identify the pairs of treatments are significantly different from each other. Control saline vs control extract, Group 2 vs Group 3 in initial body weight, control saline vs control extract in final body weight and in caudal epididymal sperm counts Group extract vs Group 3 are statistically insignificant (p > 0.01) and in testis weight all groups are statistically significant (p < 0.01) .

Weight of the testis

The weights of the right testis of the group 1 rats were 1248–1258 mg/100 g of body weight. In group 2 rats treated with carbendazim, the testicular weight was 851 mg/100 g of body wt. and in group 3 rats treated with I. suffruticosum was 948 mg/100 g body wt. In group 4 rats treated with clomiphene citrate, the testicular weight was 906 mg/100 g body wt. When compared to group 2 treated animals, the remaining group 3 rats have gained testicular weight of about 97 mg, and are statistically significant.

Effect of I. suffruticosum on cauda epididymal sperm counts in carbendazim-induced sub-fertile male rats after 28 days of treatment

The caudal epidermal sperm counts of the experimental animals are shown in Table 1. The group 1 control rats having normal epidermal sperm counts of about 40.50–49.0 × 10⁶ sperm/mL. But, in group 2 carbendazim-induced sub-fertile rats, the counts significantly decreased to about 15 × 10⁶ sperm/mL and the difference compared with the control group was highly significant. In the group 3 carbendazim-induced sub-fertile rats treated with I. suffruticosum have observed a tremendous increase in the sperm count of about 49 × 10⁶ sperm/mL. In group 3 rats, epidermal sperm count was comparatively higher than that of other groups. In group 4 rats treated with clomiphene citrate the epididymal sperm counts were 45 × 10⁶ sperm/mL. The increased sperm counts were observed in group 3 than that of group 1 and group 2 rats and are statistically significant.

Effect of I. suffruticosum on cauda epididymal sperm motility and pattern in carbendazim-induced sub-fertile male rats after 28 days of the treatment

Cauda epididymal sperm motility and pattern are shown in Table 2. In the control rats, the cauda epididymal sperms exhibited rapid and progressive motility and it lasted for about 55.17 to 60.12 min. The group 2 rats treated with carbendazim alone, the sperms exhibited very sluggish motility for about 10.16 min. During investigations, we have observed a tremendous change in the pattern and duration of motility in group 3 I. suffruticosum-treated rats which last for about 56.20 and the pattern was rapid and progressive. Group 3 rats treated with I. suffruticosum, the duration of the motility and the pattern was highly significant when compared to group 2 treated animals. In group 4 animals treated with clomiphene citrate sperms exhibited rapid and progressive motility with duration of about 57.11 min. The group 3 experimental animals’ sperm motility and pattern was highly significant compared with that of group 2 animals treated with carbendazim.

Table 2. Effect of I. suffruticosum on Cauda epididymal sperm motility and sperm pattern in Carbendazim-induced subfertile male rats after 28 days of treatment.

Experimental groups	Cauda epididymalsperm motilityand pattern (Min)
Group 1: Control (saline only)	55.17 ± 0.4
Control (extract only)	60.12 ± 0.7
Group 2: Carbendazim-induced subfertile male rats	10.16 ± 1.8
Group 3: Carbendazim-induced subfertile male rats treated with I. suffruticosum	56.20 ± 0.8*
Group 4: Carbendazim-induced subfertile male rats treated with Clomiphene citrate	57.11 ± 0.2

Each value is Mean ± SEM of five animals.

* Significantly different (p < 0.05) from the control group by Dunnett’s test.

Scheffé multiple comparison tests used to identify the pairs of treatments are significantly different from each other. Control saline vs Group 3, control saline vs Group 4, Group 3 vs Group 4 are statistically insignificant p > 0.01.

Effect of I. suffruticosum on cauda epididymal sperm vitality in carbendazim-induced sub-fertile male rats after 28 days of treatment

The results of the sperm vitality in experimental animals are shown in Table 3. In group 1 control rats, the live sperms were about 83–91% and the dead sperms were 16–9%. After carbendazim treatment in group 2 rats, a highly significant fall in the live sperms of about 57% and a highly significant rise in the number of dead sperms of about 43%. In group 3 rats treated with I. suffruticosum, a highly significant rise in the percentage of live sperms to about 85% when compared to group 2 rats. In group 4 rats treated with clomiphene citrate, the percentage of live sperms were 83%. In group 3 and 4, the sperm vitality was superior to that of group 2-treated animals and are statistically significant

Table 3. Effect of Ionidium suffruticosum on Cauda epididymal sperm vitality and spermmorphology in Carbendazim induced subfertile male rats after 28 days of treatment.

Experimental groups	Sperm vitality		Sperm morphology
	Live sperms (%)	Dead sperms (%)	Normal sperms (%)	Abnormal sperms (%)
Group-1: Control (saline only)	83 ± 0.6	17 ± 0.5	93 ± 0.5	7 ± 0.8
Control (extract only)	91 ± 0.5	9 ± 0.3	97 ± 0.5	3 ± 0.3
Group 2: Carbendazim inducedsubfertile male rats	57 ± 0.4	43 ± 0.4	58 ± 0.5	42 ± 0.5
Group 3: Carbendazim induced subfertile male rats treated with Ionidium suffruticosum	85 ± 0.4*	15 ± 0.4*	90 ± 0.7*	10 ± 0.3*
Group 4: Carbendazim induced subfertile male rats treated with clomiphene citrate	83 ± 0.4	17 ± 0.3	87 ± 0.4	13 ± 0.7

Each value is of Mean ± SEM of 5 animals.

* Significantly different (p < 0.05) from the control group by Dunnett’s test.

Scheffé multiple comparison tests used to identify which of the pairs of treatments are significantly different from each other. Control saline Vs Group 4 in live sperms, Control saline Vs Group 3, Control saline Vs Group 4, Group 3 Vs Group 4, in dead sperms are statistically insignificant p > 0.01 and in normal and abnormal sperms counts all groups are statistically significant p < 0.01.

Effect of I. suffruticosum on cauda epididymal sperm morphology carbendazim-induced sub-fertile male rats after 28 days of the treatment

The sperm morphology in the treated groups is shown in Table 3. A typical sperm of rat measures a length of about 180 μm, and consisted of two parts namely head and flagellum. The head was sickle-shaped with a curved and hooked tip and almost tapering caudal end. The posterior end of the head projected a short distance on top of the early part of the flagellum. The tail was long and differentiated into middle piece, main piece and end piece.

In the control group 1 rats, about 93–97% of the sperms possessed normal morphology and 5–3% sperms possessed abnormalities of which head alone, curved tail and attached sperm predominant. In the group 2 rats treated with carbendazim after 28 days of treatment, only about 58% of sperms were in normal morphology and the remaining 42% sperms showing abnormalities of different kinds (Table 3). Highly significant rises in the percentage of normal sperms were observed in group 3 experimental rats, treated with I. suffruticosum when compared with groups 2 and 4 experimental rats treated with clomiphene citrate.

The major abnormalities observed were flexed head in which the tip of the head facing away from the flagellum, detached head, attached sperm-two or more sperm attached among themselves for short or long distances, broken middle piece and detached tail (Table 4).

Table 4. Percentage of predominant abnormalities of sperm observed in different experimentalgroups.

Experimental Groups	Predominant abnormalities (%)
	TFFAF	DTH	FAS	BMP	DTT	COF	TCOF
Group-1
Saline only	2 ± 0.3	3 ± 0.3	1 ± 0.3	0 ± 0	1 ± 0.4	0 ± 0	0 ± 0
Extract only	1 ± 0.3	0 ± 0	0.5 ± 0.1	0 ± 0	1 ± 0.3	0 ± 0	0.5 ± 0.1
Group-2	10 ± 0.7	13 ±o.7	9 ± 0.3	6 ± 0.5	2 ± 0.2	1 ± 0.3	1 ± 0.3
Group-3	2 ± 0.3*	4 ± 0.3*	0 ± 0*	1 ± 0.3*	1 ± 0.4*	0 ± 0*	1 ± 0.3*
Group-4	3 ± 0.4	5 ± 0.3	1 ± 0.3	2 ± 0.3	2 ± 0.3	0 ± 0	0 ± 0

Each value is of Mean ± SEM of 5 animals.*Significantly different (p < 0.05) from the control group by Dunnett’s test.

TFFAF: Sperm with head flexed, tip of the head facing away from the flagellum; DTH: Detached Head; FAS: Fusion/attachment of sperm at one or more points between two or more sperm. BMP: Broken middle piece; DTT: Detached Tail; COF: Coiling of the flagellum; TCOF: Twisting and coiling of the flagellum.

[Scheffé multiple comparison tests used to identify which of the pairs of treatments are significantly different from each other. Control saline Vs extract, Control saline Vs Group 3, Control saline Vs Group 4, Control extract Vs Group 3 in TFFAF, Control saline Vs Group 3, Group 3 Vs Group 4 in DTH, Control saline Vs extract, Control saline Vs Group 3, Control extract Vs Group 3, Group 3 Vs Group 4 BMB, Control saline Vs Group 2, Control extract Vs Group 2, Group 2 Vs Group 3, Group 2 Vs Group 4 in COF and all groups from DTT are statistically insignificant p > 0.01 and Control saline Vs Group 2, Control saline Vs Group 3, Control saline Vs Group 2, Control extract Vs Group 2, Group 2 Vs Group 3, Group 2 Vs Group 4 in FAS groups are statistically significant p < 0.01.

Further, germinal epithelial cell masses in a scattered manner admixture with the sperms. In group 3 rats treated with I. suffruticosum, a significant rise in the percentage of normal sperms to about 90% (Figure 1(A)) and the improvement over group 2 carbendazim-treated rats were significant. In group 4 rats treated with clomiphene citrate, the percentage of normal morphology was 90% as shown in Figure 1(B). The groups treated with I. suffruticosum and clomiphene citrate, the percentage of normal sperms was higher than that of group 2 carbendazim-induced untreated rats.

Figure 1. Effect of I. suffruticosum on epididymal sperm agglutination in Carbendazim-induced subfertile male rats after 28 days of treatment. (A) Sperms of Group of rats treated with I. suffruticosum (450×). (B) Sperms of Group of rats treated with Clomiphene citrate (450×). (C) Agglutination of sperms in carbendazim-treated group (450×). (D) Agglutination of sperms of group 3 rat treated with I. suffruticosum (450×). (E) Seminiferous tubule of a carbendazim-treated rat showing specific depletion of pachytenespermatocyte and total seminiferous tubular atrophy (450×). (F) Seminiferous tubules of a carbendazim-induced subfertile male rats treated with I. suffruticosum showing less damage and with a normal histology (100×). (G) Seminiferous epithelium of a carbendazim-treated rats showing sloughing off germinalepithelium (100×). (H) Single seminiferous tubules, of a group 3 rat treated with I. suffruticosum (450×) (I) Seminiferous tubules of a carbendazim-treated rat showing fibrosis (450×). (J) Single seminiferous tubules treated with I. suffruticosum showing normal histological restoration (450×) 1: Control; 2: Carbendazim-Treated; 3: I. suffruticosum treated; 4: Standard fertility drug Clomiphene citrate treated+

Figure 1. Continued

Effect of I. suffruticosum on cauda epididymal sperm agglutination in carbendazim-induced sub-fertile male rats after 28 days of treatment

Sperm agglutination in experimental animals treated with carbendazim and I. suffruticosum is carefully observed. The rate of sperm agglutination in the group 1 control was very minimal with 1–5%. In the carbendazim-treated group 2 rats, there was a significant rise in the sperm agglutination of about 32% and is shown in Figure 1(C). In the I. suffruticosum treated group 3 rats; there was a significant fall in the level of sperm agglutination of about 9% as shown in Figure 1 when compared to untreated group 2 rats. In the clomiphene citrate-treated group 4 rats, the sperm agglutination was 11%.

Effect of I. suffruticosum on malondialdehyde and antioxidant enzyme levels in carbendazim-induced sub-fertile male rats after 28 days of treatment

The results are presented in Table 5; there was a significant increase in malondialdehyde (MDA) levels of testes about 42.3 nmol/mL in animals treated with carbendazim for 28 days, which is significantly higher compared to control groups, which showed 27.9 nmol/mL. In the three group treated with I. suffructicosum showed decreased levels of MDA in testes of about 33.6 nmol/mL compared with carbendazim-induced untreated group rats. The clomiphenes citrate treated rat groups also showed decreased levels of MDA of 32.8 nmol/mL but not significant with that of I. suffruticosum-treated rat groups.

Table 5. Effect of Ionidium suffruticosum on malondialdehyde and antioxidant enzymelevels in Carbendazim induced subfertile male rats after 28 days of treatment.

Experimental groups	Enzyme levels
	MAD nmol/ml	CAT μmol/S/ml	SOD μ/ml
Group-1: Control (saline only)	27.9 ± 0.5	4.2 ± 0.6	136.2 ± 0.4
Control group (extract only)	28.2 ± 0.3	4.4 ± 0.3	138.3 ± 0.5
Group 2: Carbendazim induced sub fertilemale rats	42.3 ± 0.5	2.1 ± 0.1	88.7 ± 0.3
Group 3: Carbendazim induced subfertile male rats treated with Ionidium suffruticosum	33.6 ± 0.2*	3.8 ± 0.2*	131.3 ± 0.5*
Group 4: Carbendazim induced subfertile male rats treated with Clomiphene citrate	32.83 ± 0.3	3.6 ± 0.3	133.1 ± 0.5

MAD: Malondialdehyde; CAD: Catalase; SOD: Superoxide dismutase.

Each value is of Mean ± SEM of 5 animals.

*Significantly different (p < 0.05) from the control group by Dunnett’s test.

Scheffé multiple comparison tests used to identify which of the pairs of treatments are significantly different from each other. Control saline Vs Control extract, Group 3 Vs Group 4 in MAD, SOD are statistically insignificant p > 0.01 and in CAT Control saline Vs Group 2 and Control extracts Vs Group 2 are statistically significant p < 0.01.

The carbendazim-treated animals showed decreased testicular catalase (CAT) activity of 2.1 μmol/S/mL and superoxide dismutase (SOD) activities of 88.7 μ/mL compared with control groups of 4.2 μmol/S/mL and 136.2 μ/mL for CAT and SOD, respectively. The experimental rat group 2 treated with I. suffruticosum showed significant increased activity of CAT 3.8 μmol/S/mL and SOD 131.3 μ/mL, which is greater than the standard fertility drug clomiphene citrate CAD and SOD activities of 3.6 μmol/S/mL and 133.1 μ/mL, respectively.

Effects of I. suffruticosum on the histopathological changes of the testes in the carbendazim-induced sub-fertile male rats after 28 days of the treatment

In the control rats, the seminiferous tubules of the testis were compactly arranged, with tall seminiferous epithelium and a narrow lumen. The seminiferous epithelium consisted of tall and branched sertoli cells and germ cells belonging to different generation of the lineage spermatogonia, primary spermatocytes, secondary spermatocytes, round spermatid and elongating spermatids. The spermatids in the different steps in spermiogenesis, spermiated spermatozoa were present in the lumen. No atrophied tubules were seen in the control testes. The tubules of control rat spermatocytes were engaged in the meiotic division and cells in all the divisional stages including anaphase and telophase were noticed, and seminiferous epithelium above the level of spermatocytes was abundant with spermatids.

In the carbendazim-treated group 2 rats, the seminiferous tubules were disorganized. The loss of several pachytene spermatocytes in the stage 3 tubules and the corresponding places appear empty are shown in Figure 1(E). In tubules, the entire mass of cells beyond spermatocytes (i.e., the round and elongating spermatids) appeared as detached and sloughing off germinal epithelium including Sertoli cells at a level from round spermatids are shown in Figure 1(G). In several tubules aggregations of sloughed material were present in the lumen. In the carbendazim-treated rats, the spermatocytes appeared in arrested cell division and cells in anaphase and telophase were totally missing, also the layer appeared as depleted. An extreme manifestation of carbendazim treatment was fibrosis of several seminiferous tubules and depletion of germinal layers in several other tubules are shown in Figure 1. The 30% atrophied tubules in the carbendazim-treated group 2 rats were observed.

In group 3 rats, the testes consisted of seminiferous tubules in a compact arrangement with tall seminiferous epithelium and a narrow lumen. The seminiferous tubules were in an organized manner and histologically were much better than the carbendazim-treated group 2 rats as shown in Figure 1(H). The seminiferous tubules invariably contained all the germ cell types in the prescribed order and in a manner exactly comparable with that of a control rats. In I. suffruticosum treated rats, the seminiferous tubules recovered to a very extent and looks like the seminiferous tubule of a control rats as shown in Figure 1(J). In the seminiferous tubules, carbendazim-induced histopathological changes such as atrophy of the seminiferous tubules, degenerative epithelium, cavity formation due to killing of spermatocytes, sloughed of germinal epithelium and fibrosis were completely recovered in group 3 rats treated with I. suffruticosum after 28 days of treatment are shown in Figure 1(F).

We have observed seminiferous tubules as normal like control groups without any signs of atrophy condition in clomiphene citrate-treated rats are shown in Figure 1(B). Treatment with leaf extracts of I. suffruticosum for 28 days resulted in normal histological features of the seminiferous tubules with compact cells arranged in an orderly manner, showing all stages of meiotic division. Clomiphene citrate completely reversed the carbendazim-induced changes in the seminiferous tubules. The histological picture of group 4 rats was better than group 2 carbendazim-treated rats by reducing atrophy of the seminiferous tubules and inhibiting the sloughing of germinal epithelium and improving the cell divisions of spermatogenesis.

Discussion

A lack of pharmacological and its toxicological information on medicinal plants considerably restrict their therapeutic application in ethnomedicine and ethnoveterinary medicine. Consequently, experimental pharmacokinetic and toxicological studies are paramount in the development of botanical products as safe and efficacious drugs (Wu et al. 2000). Changes in body weight and testicular weight rate are important parameters in prolonged experimental toxicity studies on effects of drugs and chemicals on laboratory animals. The results suggested that the untreated control group rats had a significant normal body weight gain, but the carbendazim-administered male albino rats showed a significant decrease in the body weight by inhibiting mitotic division. Edward and Keith (1986) reported that these fungicides are able to bind microtubules and there by inhibit mitosis. Such reduction in the mitotic division caused reduction in the body weight of our experimental animals treated with carbendazim.

The carbendazim-induced sub-fertility rats treated with traditional medicinal plant I. suffruticosum showed a little or marginal body weight gain after 28 days of treatment, compared with the groups treated with carbendazim alone. This may be attributed to the curative effect of Ionidium sp. by reducing the damage caused by carbendazim and improving the mitotic division. The treatment of carbendazim-induced sub-fertility rats with I. suffruticosum, improved the testicular damage caused by carbendazim, further it induced the significant decrease in LPO and increased the testicular oxidative biomarkers, SOD and CAT. We found that carbendazim treatment caused a significant decrease in the testicular weight of about 400 mg/100 g body weight. Nakai et al. (1995) observed that the carbendazim exposure caused such decrease in the testicular weight of about 490 mg after 70 days of treatment. Gray et al. (1990) reported that these fungicides cause long-term atrophy in the testis. When carbendazim is administered orally to mammals, produces various adverse effects on male reproduction, such as sloughing off germ cells (Gray et al. 1990), inhibition of germ cell division (Tyrkiel 1984), seminiferous tubular atrophy (Gray et al. 1990), and alterations in hormone concentrations (Goldman et al. 1989). The decrease in the testicular weight in our studies may be due to the effect of carbendazim on the inhibition of germ cell division, seminiferous tubule atrophy, alteration in hormone concentration and sloughing off germ cells.

The carbendazim-induced sub-fertile male rats treated with leaf extract of I. suffruticosum showed a significant testicular weight gain of about 100 mg after 28 days of treatment. The gain in the testicular weight may be due to the effect of curative property of the I. suffruticosum on carbendazim-induced male testicular dysfunction by improving the mitotic division thereby increasing the spermatogenesis by preventing the sloughing off the germ cells and by enhancing the secretion of male sex hormone. One of the testicular effects of the carbendazim is the reduction of the testicular blood flow (Nakai et al. 1995). Tripathy et al. (1996) reported that Withania somnifera accustomed to remove blocks in the blood flow to the testis. Our experimental plant I. suffruticosum may also have such property by enhancing the blood flow to the testis and thereby increasing the testicular weight. We have observed that the gain in the testicular weight was more in I. suffruticosum-treated animals than that of clomiphene citrate-treated rats. So, the I. suffruticosum leaf extract showed a superior effect on male reproductive organs than the clomiphene citrate drugs. The experimental rat groups treated with standard male fertility drug clomiphene citrate, showed a slight gain in the testicular weight, compared to the group 2 carbendazim--treated rats. The gain may be due to the fact that by enhancing male sex hormone secretions. Weniger et al. (2004) reported that low plasma level of testosterone in rats caused decreases in the testicular weight. Clomiphene citrate stimulates the production of LH and thereby stimulating the production of male sex hormone testosterone.

The cauda epididymal sperm counts in control group rats were normal, but in group 2 carbendazim-treated rats, the sperm counts were significantly decreased. We have observed in group 3 carbendazim-induced sub-fertile male rats treated with I. suffruticosum, the cauda epididymal sperm counts were tremendously increased. Such a tremendous increase of sperm counts was due to the curative effect of I. suffruticosum leaf extract on carbendazim-induced damages in relation to spermatogenesis as well as increased the activities of acid and alkaline phosphatases and LDH of cauda epididymis, thereby increased the epididymal sperm counts. In the Indian traditional systems of medicine such as Siddha, Ayurveda and Unani, I. suffruticosum is used as tonic, diuretic, demulcent and aphrodisiac (Arun & Sonappanavar 2011). The leaves and roots of I. suffruticosum are used in bowel complaints of children and fruits are used to treat scorpion sting. This plant shows a great medicinal value on male fertility by increasing the sperm counts.

After 28 days of treatment with I. suffruticosum leaf extract on carbendazim-induced sub-fertility group 3 rats, increased the cauda epididymal sperm counts which can be attributed to an increasing spermatogenesis by preventing the sloughing off germ cells and by enhancing the secretion of male sex hormone testosterone levels. Similarly, Nakai et al. (2002) reported that the presence of alkaloids, steroidal lactones and flavonoids are directly influenced on the fertility enhancing property of I. suffruticosum. These alkaloids, flavonoids and steroidal lactones have an influencing role on spermatogenesis and increased production of male sex hormone testosterone. From our results, we have inferred that the alkaloids and flavonoids present in the leaf extract of I. suffruticosum may have a stimulating role on microtubule polymerization and increasing the formation of mitotic spindle by binding to tubulin, which thereby increases the spermatogenesis. Our results are similar to Tripathy et al. (1996) who reported that Withania somnifera has certain steroidal lactones with structural similarities to androgens, which stimulated the enzymes related to steroidogenesis, such as glucose 6-phosphate dehydrogenases (Yu et al. 2009). Our experimental plant I. suffruticosum also has steroidal lactones which behaved the same as androgens, thereby increased spermatogenesis, which ultimately led to tremendous increase of cauda epididymal sperm counts.

Rastogi and Malhotra (1990) reported that the phytochemical drug yohimbine obtained from the plant Rauvolfia stimulated spermatogenesis by increasing the mitotic activity of spermatogonial cells. From the results, it is evident that the standard fertility drug clomiphene citrate on carbendazim-induced sub-fertility group 4 rats enhanced the epididymal sperm counts significantly. Many previous reports suggested that clomiphene citrate has a strong influence on the hypothalamus hypo physical testicular axis. It stimulated the hypophysis for releasing more gonadotropin by which there is faster and higher release of FSH and LH occurs, results in an elevated endogenous testosterone level. Clomiphene citrate normalized the testosterone level and the spermatogenesis process within 10–14 days of treatment, and had a direct influence on the hypothalamus and hypophysis, thus regenerating the entire regulating cycle and shows a pronounced ability to stimulate ovulation.

The untreated control group on cauda epididymal sperm motility was normal and the pattern was very rapid and progressive, but the carbendazim-treated rats showed very poor sluggish motility. In group 3 rats treated with I. suffruticosum, the duration of motility and progressive pattern were highly significant. Such an improved motility was due to conducive effect of I. suffruticosum by providing normal microenvironment in the cauda epididymis. Further attributed to the fact that the presence of alkaloids, steroidal lactones and flavones which acted on micro-tubular apparatus of the flagellum. The group 4 rats treated with clomiphene citrate showed the sperm motility was normal with a rapid and progressive pattern. Such improved motility of the sperms was due to its action over hypothalamo-hypophysical gonadal axis and by increasing the secretion of male sex hormone.

Our investigations on morphology of sperms showed that the group1control rats showed 92% of sperms with normal morphology. The group 2 carbendazim-induced untreated rats showed a very abnormal cauda epididymal sperm morphology with different kinds. The major abnormalities were flexed head, detached head, attached sperms, broken tails, curved or coiled tail etc., further germinal epithelial cell masses containing cells in a scattered manner or as a compact mass in admixture with sperms. Various abnormalities of the cauda epididymal spermatozoa strongly indicated the impact of carbendazim through an epididymal route as the spermatozoa were complete as far as the developmental changes acquired in testis are concerned. The origin of the abnormalities can be explained following Nakai et al. (2002), which reported that treatment of rats with a single bolus dose of 400 mg/kg weight of carbendazim resulted in decrease in testicular sperm head counts between days 8 through 16, and increase in the incidence of abnormal sperm in the epidymis with 10% of the spermatozoa having the head separated from flagellum.

Treatment of carbendazim resulted in significant decrease in the epididymal sperm counts (control, 21.88 ± 0.78 × 10⁶ per mL; treated 13.14 ± 0.11 × 10⁶ per mL), and inhibition of motility of the spermatozoa. In the control rats 93–95% of the spermatozoa possessed normal morphology, where as in carbendazim-treated rats only 55–61% had normal morphology and in about 15% spermatozoa, the head was flexed in such a way that the pointed tip of the head was either facing away from the flagellum or facing the flagellum. About 10% of the spermatozoa had the head detached from the flagellum (Akbarsha et al. 2001).

The breaking away of head from flagellum and flexion of head of the sperm appear to occur due to impact of the chemical at the neck or connecting piece of the flagellum. The main components of the connecting piece are the basal plate (the point at which implantation fossa lies), capitulum and the segmented columns. Fine filaments traversing the narrow region between the capitulum and basal plate presumably are responsible for attaching the capitulum of the flagellum to the basal plate of the head (Baccetti 1984). It has been reported that the critical protein at the connecting piece is Ankyrin (Hecht et al. 1984). This could be perceived that carbendazim disrupts the protein also, as much as disrupting tubulin, causing the breaking away of the head from the flagellum. A lesser impact at this point would cause head to flex or flexion itself may be a step towards the breaking away.

The curved or coiled nature of the flagellum may be sought in the microtubule support of the axoneme of the sperm. The microtubules are composed of α- and β-tubulins (Hecht et al. 1984). Microtubules are undoubtedly the established targets for carbendazim action, any disruption caused to microtubules of the sperm axoneme, and curvature/coiling of the flagellum. Agglutination or fusion or attachment of sperm may be explained in the light of imminent changes in the surface proteins. It has been conclusively showed that the spermatozoa, during their epididymal maturation are altered with respect to the surface proteins. The epithelium of the epididymis, particularly the principal cells of the initial segment and caput, secrete several proteins some of which get translocated on to the spermatozoa. Also, the changing luminal microenvironment along the ductuli efferentes and ductus epididymidis contributes to the change in the sperm surface proteins (Robaire et al. 1988).

So changes in the sperm surface proteins and pH of the medium are causative of sperm agglutination (Baccetti 1984). Cytoplasmic droplet (CD) is a small mass of cytoplasm which the spermatozoa carry while leaving the seminiferous tubules (Hermo et al. 1988; Robaire et al. 1988). This droplet is shed when the spermatozoa leave the corpus epidymidis and spermatozoa in storage at the cauda are devoid of the droplet. Spermatozoa that retain extra cytoplasm are inhibited motility (Akbarsha et al. 2003). The retention of the CD by cauda epididymis sperm of carbendazim-treated rats could be impairing of the process of shedding of cytoplasmic droplet and subsequently inhibited motility (Akbarsha et al. 2001).

In group 3 rats treated with Ionidium sp., the normal sperm morphology was significantly increased. The 90% increase of normal sperm morphology was due to the curative property of our experimental plant I. suffruticosum on increased sperm counts with improved morphological features. The occurrence of normal sperm morphology in clomiphene citrate-treated groups was observed. The treatment of clomiphene citrate clearly prevents the toxic effects of carbendazim-induced damages in testis and sperms to a great extent.

The results suggested that, the group 1 control rats showed very minimal sperm agglutination compared to group 2 carbendazim-induced untreated rats. The higher percentage of agglutination is the result of changes in the surface proteins. The spermatozoa are altered during their epididymis maturation in respect to surface proteins. The epithelium of the epididymis, particularly the principal cells of the initial segment and caput, secrete several proteins, some of which translocated on to the spermatozoa. Also, the changing luminal microenvironment along the ductuli efferentes and ductus epididymis contributes to the change in the sperm surface proteins (Robaire & Hermo 1988), therefore it is reasonable to speculate that carbendazim affects the epididymis epithelium towards secretion of proteins or luminal microenvironment, affecting change in the sperm surface proteins rendering them to stick together in small to large numbers and over short to long distances (Eddy & Brien 1994). The I. suffruticosum treated rats showed a minimal sperm agglutination because of the curative property on the epididymal epithelium, and regulating the secretion of epididymal cell protein. There by maintaining normal sperm surface proteins and maintaining a normal pH in the environment.

Carbendazim-treated animals showed an increased LPO, which can be mirrored by the increased level of MDA. Subsequently marked decrease in the level of testicular CAT and SOD activity. The results were further supported by Metwally et al. (2011) and reported that carbendazim-treated animals showed a significant change in MDA levels and significant decrease in the activity of SOD and glutathione peroxidase (GPx) in testis of rat. Similarly Rajeswary et al. (2007) reported that Leydig cellular activities of carbendazim-treated animals antioxidant enzymes SOD, GR (glutathione reductase), CAT, GPx, GST (glutathione-S-transferase), G-6-PDH (glucose-6-phosphate dehydrogenase), γ-GT and non-enzymatic antioxidants, such as GSH (reduced glutathione), and vitamins C, E were significantly diminished, whereas LPO and ROS were markedly elevated. Similarly, the results of the present work indicated that carbendazim induced oxidative stress in treated experimental animals. This can be evidenced by increased level of LPO and decreased activity levels of CAD and SOD. Hamdy et al. (2010) reported that carbendazim administration caused testicular dysfunction with an increase in MDA and reduction in SOD and GPx activity.

Treatment of carbendazim-induced sub-fertility rats with I. suffruticosum decreased the testicular damage caused by carbendazim, further it induced the significant decrease in LPO and increased the testicular oxidative biomarkers, SOD and CAT. The results can be comparable to Huo et al. (2011) reported that Glycyrrhiza glabra aqueous extracts had protective effects against CCl₄-induced toxicity in rats and returned the increased LPO and decreased GSH, and antioxidant enzymes levels bring back to their normal control levels. Ionidium suffructicosum leaf extracts have been shown to be a potent free radical scavenger and stimulating activities of antioxidant enzymes. This finding is in agreement with Al-Olayan et al. (2014) Punica granatum fruit juice showed significant elevation in testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), depleted by the injection of CCl₄. Activity levels of endogenous testicular antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) and glutathione (GSH) contents were increased while lipid peroxidation (LPO) and nitric oxide (NO) were decreased with Punica granatum fruit juice. Moreover, degeneration of germ and Leydig cells along with deformities in spermatogenesis induced after CCl₄ injections were restored with the treatment of pomegranate juice.

Histopathological changes

In the control group rats, the seminiferous tubules were compactly arranged. The seminiferous tubules consisted of tall seminiferous epithelium and a narrow lumen. The epithelium in the different tubules was at different stage of spermatogenic cycle and testes were without atrophied seminiferous tubule. In the group 2 carbendazim-treated rats showed significant histological changes such as loss of pachytene spermatocytes, sloughing off germinal epithelial cells, arrested cell division, spermatid and germinal layer depletion and fibrosis an atrophy of the seminiferous tubules. The selective loss of pachytene spermatocytes from the seminiferous epithelium probably reflects a direct effect on carbendazim toxicity to the germ cells. Breslin et al. (2013) reported that the carbendazim prevents polymerization of tubulin and prevents the formation of fresh microtubules, the metaphase chromosomes fail to separate and the cell division arrested and sloughing off the related cell types in the seminiferous epithelium and 30% of the atrophied seminiferous tubules were observed in the present study. Nakai et al. (1995) reported that carbendazim treatment in rats resulted in a 48% of atrophied seminiferous tubules after 70 days of exposure.

Further, carbendazim directly acts on the excurrent ducts of the testis, primarily in the ductuli efferentes and resulted in occlusion of these ductules. These occluded ductules produced a fluid pressure that causes seminiferous tubular swelling, loss of germ cells and eventual atrophy of the testis. In the group 3 treated with I. suffructicosum, the histological observations of testes were normal. After 28 days of treatment, the seminiferous epithelium and a narrow lumen are compactly arranged. Further, the epithelium in the different tubules was at different stage of spermatogenic cycle and the testis without atrophied seminiferous tubule. The pachytene spermatocytes were increased.

The presence of various compounds in I. suffructicosum such as flavonoids, steroidal lactones and alkaloids exerted a curative role on the carbendazim-damaged pachytene spermatocytes and regulating meiotic division. The extract had a role in enhancing polymerization of tubulin and had a role in the formation of fresh microtubules which resulted in enhanced normal cell division, subsequently prevented sloughing off seminiferous epithelium. Further, the treatment of I. suffructicosum decreased the percentage of atrophy on ductuli efferentes by preventing the occlusion and regulating the normal sperm transport and pressure in the seminiferous epithelium. In the group 5 rats, treated with clomiphene citrate, the histopathological observations were normal. The various histopathological damages caused by carbendazim treatment were minimized and showed a normal impression of the seminiferous tubules. An increase in the number of pachytene spermatocytes, a reduction in the rate of sloughing off germinal epithelial cells and a reduction in the percentage of atrophied seminiferous tubules were observed.

Conclusions

Our results demonstrate that the leaf extracts of I. suffructicosum possess potent therapeutic fertility properties as well as curative properties against the chemical-induced damages in male reproductive system. The extract having a role in enhancing the levels of male sex hormone testosterone, increasing the testicular weight, increasing spermatogenesis, increasing sperm, counts lessening sperm agglutination by maintaining normal pH in testicular micro environment and increasing the testicular oxidative biomarkers, SOD and CAT. This study therefore supports the usage of I. suffructicosum in traditional medicine for infertility.

Acknowledgements

The authors thank Dr K. Sridar Komuddy (Professor-Animal sciences) for his warm support and critical evaluation of the manuscript.

Disclosure statement

The authors have declared having no conflict of interest.

Additional information

Funding

This work was supported by the University Research Fellowship [No. 29614].