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Lantana camara (Family : Verbenaceae – commonly known as 'Kurri') is an exotic plant came from America years ago. It is a fast growing perennial herbaceous plant found abundantly in each and every corner of India. It is never consumed as a fodder by domestic and wild animals. It grows under varying conditions of climate and soil including gravel and laterite and in low lying areas and on hills up to 1800 m. It is a draught resistant, light loving and tolerate moderate shades. As far as human is concern, the leaf oil is used in treatment of itches of skin and antiseptic for wounds. The plant is considered vulnerary, diaphoretic, and antiseptic. It is useful for fistulae, pustules and tumours. A decoction of the plant is given in tetanus, rheumatism, malaria and atoxy of abdominal viscera. In Philippines, a decoction of fresh roots is used as gargle for toothache. Powdered leaves are applied to cuts, ulcers and swellings. A decoction of leaves and fruits is used as a lotion for wounds. It is thought that the plant may cause adverse effect on reproductive potential of animals.

Earlier Singh et al. (2000) reported antifertility effect of leaves of this plant on female albino rats but no such effect on male animals reported so far. Plants affecting reproductive function (anti-spermatogenic and anti-ovulatory etc.) have been already reported by Kirtikar and Basu, 1935; Nadkarni and Nadkarni, 1954; de Laszlo and Henshaw, 1954; Casey, 1960; Chaudhary, 1966; Malhi and Trivedi, 1972; Saxena, 1973; Farnsworth et al., 1975; Kamboj and Dhawan, 1982; Singh, 1985; Gupta et al., 2005; Singh and Singh 2017; Singh & Purohit. 2019 and Singh et al., 2021. Authors have conducted experimental study of leaf powder of Lantana camara in male albino rats and the results are being reported in this paper.

Observations

A.  Effect on body and organ weight

The result are presented in Table 1. The rats of control group did not show much change in the body weight. It is slightly increased after 30 days of experimental period. Experimental male rats administered with leaf powder of Lantana camara showed some reduction in te body weight. The doses of leaf powder at 40 and 60 mg/kg/day for 30 days caused significant reduction (P < 0.05) in the weight of testes and epididymes in comparison to control rats.

B. Effect on testes

Control : The testes of control male rats after 30 days showed normal histological features. The seminiferous tubules were lined with germinal epithelium, whose cells by a process of growth and division (Spermatogenesis) produced the spermatozoa. There were a kind of specialized and large sized cells, the sertoli cells, placed between the epithelial cells of seminiferous tubules. Leydig's cells were present in the interstitium which secrete the male hormone, testesterone largely responsible for the development of secondary sexual characters. Other spermatogenic cells, spermatocytes, spermatids and spermatozoa are visible in the normal sized seminiferous tubules (Fig. 1).

Treated with doses : Administration of Lantana camara leaves powder as aqueous solution at 20 mg/kg dose for 30 days caused no significant histological changes in the seminiferous tubules of testes. Various cell types including spermatozoa are normal. The administration of 40 mg/kg dose for 30 days caused mild degenerative changes in the testes. The spematogenesis was stopped at spermatid stage in majority of seminiferous tubules. Germinal epithelium appeared normal. The degenerated Leydig's cells were observed in the intertubular spaces (interstitium). The administration of 60 mg/kg dose for 30 days caused severe degenerative changes. The degeneration in the testes consisted of the damage of the spermatocytes, spermatids and spermatozoa. The seminiferous tubules were reduced in size, become deshaped and filled with debris of connective tissues. The intermediate contained atrophied Leydig's cells. The vascularity was also affected (Fig. 2).

C. Effect on Epididymes

Control : The epididymes of control rats showed a normal histological structure. The epithelium of caput and cauda parts were normal. The columnar epithelial cells of caput were tall with basal nuclei (Fig. 3). The epithelium cauda was lined with low cuboidal cells (Fig. 4). The lumen of caput was smaller than the cauda ductules. The pronounced stereocilia were present in the caput than in the cauda. The intertubular connective tissue with vascularity between the ductules of caput and cauda were normal. Experiments have shown that when spermatozoa passed through the epididymes, they undergone a physiological maturation. The epididymes both caput and cauda were full of mature spermatozoa.

Treated with doses : Administration of Lanatana camara leaves powder as aqueous solution of 20 mg/kg dose for 30 days caused no histological changes in both caputa and cauda epididymes. The doses 40 and 60 mg/kg for 30 days caused marked changes in the histology of both caput and cauda epididymes. The epithelium of caput was slightly hypertrophied thereby showing the increased height of epithelial cells and wide lumen. The nuclear dysplasia was observed greatly in the epithelial cells of caput than in the cauda part. Sperm concentration became very less in the lumen of both caput and cauda ductules. Stereocilia were severely affected in both parts of epididymes (Fig. 5 and 6). 

Discussion

The leaf powder of L. camara as aqueous solution administration orally to adult male albino rats caused marked inhibitory effects of spermatogenesis at the doses of 40 and 60 mg/kg for 30 days. The dose 20 mg/kg was ineffective. The effect on body weight was negligible but the higher doses for 30 days reduced the weight of male reproductive organs i.e. testes and epididymes significantly. Not only weight reduction was noticed in the reproductive organs but, feeding of L. camara leaf powder, caused histopathological changes in testicular elements. Leydig's cells were mostly atrophied which are the main testosterone producing cells.

The Follicle Stimulating Hormone (F.S.H.) secretion is directly related to the weight of testes. Heavier will be the testes more will be secretion of F.S.H. Testosterone is an androgenic hormone essential for sexual development. Chemically, it is a steroidal hormone. It is confirmed by studies of Dorfman et al. (1963) that both non-steroidal (i.e. plant product etc.) and steroidal agents inhibits pituitary gonadotrophins either acting directly on pituitary or through the hypothalamo-hypophyseal axis. The reduced F.S.H. production causes significant decrease in the weight of the testes and accessory reproductive organs of male rats. Paul et al., (1953) had demonstrated the reduction of weight of testes and accessory organs in absence or presence of spermatids and spermatozoa in the testes of male animals. According to Nelson and Patanelli (1965), the change in weight (reduction) of testes also corresponds to the presence or absence of post meiotic cells in the testes. The physiology of male reproductive (genital) organs are dependent on androgen hormone. Findings of the present study are in agreement with the above stated studied of male reproductive physiology with the following references. The aqueous leaf extract of Vinca rosea caused significant histological changes in testes and epididymes and the weight reduction also (Chinoy and Geetha Ranga, 1983). The results of anti-spermatogenic effects of leaf extracts of Aristolochia indica in male rats by Pakrashi and Pakrashi (1977) may be mentioned for the support of present studies. Administration of Ocimum sanctum L. leaves by Kashinathan et al., 1972; Khanna et al., 1986 and Cannabis sativa L. leaves by Dixit et al. (1974) to male rats and mice respectively concerning with degenerative changes in the testes can be corroborated. Ocimum sanctum caused adherence of spermatological cells and regression of Leydig's cells. Cannabis sativa also induced Leydig's cells regression and reduction in size and spermatocytes population of seminiferous tubules in the testes of mice.

Singh (1985) reported degenerative changes at higher doses of Semicarpus anacardium Linn. Spermatogenic cells were degenerated. In some of the germ cells, the nuclei were swollen. Seminiferous tubules were deshaped and distorted. Sperm concentration in the ductules of epididymes were greatly reduced. Chaudhary et al. (1990) reported effects of leaf extracts of some plants in male rats which caused antifertility in them. Administration of these plants caused mass atrophy of the spermatogenic elements. The germ cells were left 1 to 2  layers only. Epididymal epithelium was regressed and the lumen was devoid of spermatozoa.

Table 1 : Effect of leaf powder of Lantana camara on body wt. (gms) and wt. of Reproductive organs (mgs) of male albino rats administered for 30 days with doses of 20, 40 and 60 mg/kg/day. Five rats were used in each group. Values are mean ± S.E.

Doses (mg/kg)	Body weight (gms)		Reproductive organ weight (mgs)
	Initial	Final	Testes	Epididymes
Control	181.20 ± 2.70	200.80 ± 5.00	942.40 ± 4.20	365.40 ± 9.72
20	151.50 ± 1.80	157.20 ± 6.40	937.80 ± 3.60	337.45 ± 5.40
40	186.20 ± 2.50	188.50 ± 5.40	420.20 ± 3.00*	220.25 ± 5.20*
60	197.50 ± 2.80	198.30 ± 2.80	400.30 ± 10.00*	210.13 ± 3.45*

*  p < 0.05

 

Fig.1. Section of testes of control group for 30 days showing normal histological features in seminiferous tubules. Sprmatogenesis with germinal epithelium and layers of spermatogonial cells, spermatocyte, spermatogonia, spermatids and spermatozoa towards lumen. Leydig's cells are normal in the intertubular spaces´250

Fig.2. Section of testes of treated group with Lantana camara at 40 and 60 ml/kg dose for 30 days caused severe degenerative changes, deshaped seminiferous tubules, damaged spermatogonia, lumen without spermatozoa and atrophied Leydig's cells in the intertubular spaces 250.

Fig.3. Section of epididymes (caput) of control group of male rats for 30 days showing normal structure of tall columnar cells in the epithelium of ductules with spermatozoa in the lumen. Stereocilia are normal ´ 250.

Fig.4. Section of epididymes (cauda) of control group of male rats for 30 days showing normal structure of cuboidal cells in the epithelium of ductules with spermatozoa in the lumen. Stereocilia are normal ´ 250.

Fig.5. Section of epididymes (caput) of treated group with Lantana camara for 40 and 60 mg/kg doses for 30 days caused vacuole formation in epithelial cells of ductules, distorted stereocilia and absence of spermatozoa in lumen ´ 250.

Fig.6. Section of epididymes (cauda) of treated group with Lanata camara for 40 and 60 mg/kg doses for 30 days caused distortion of epithelial cells of deshaped ductules with vacuoles also, damaged stereocilia and absence of spermatozoa in the lumen ´ 250.

1. Albino Rats (Rattus rattus norvegicus) Healthy, adult, colony-breed, Swiss albino male rats, three-four months old weighing between 150-200 gms were selected and acclimatized to the laboratory condition for seven days prior to commencement of experiment. The rats were kept in cages (60cm  45cm  45cm) under normal conditions of photoperiod and at room temperature. Four groups of male rats each containing five albino rats were made. First group of rats were served as control while three groups of rats were treated as experimental. They were maintained on balanced diet containing soaked gram and food pallet (Hindustan Lever's Limited) and pure hygienic drinking water was allowed ad libitum. All the rats were cared as per U.G.C. guidelines and supervised by the members of Animal Ethical Committee appointed by the than Principal of the college. 2. Doses and Administration Fresh leaves of Lanatana camara were collected from Dehradun city barren lands, washed and dried in shade. They were crushed until change into fine powder. Three doses 20, 40 and 60 mg/kg body weight were prepared. Each dose was dissolved in distilled water until it changes into a homogenous mixture. The volume was adjusted in such a way that 01 ml of the solution containing 20 mg powder fed to second group of rats for 30 days. The rest of the doses 40 and 60 mg were administered in the 3rd and 4th group of rats respectively for 30 days. Gum-acacia powder as vehicle dissolved in water (05 mg/dose) was fed to control group (Ist) of rats for 30 days. The administration of doses were done orally with specially designed knobbed feeding needle fitted into a syringe. 3. Record of weight and Histology The initial and final (on day 31st) body weight of the experimental as well as control rats for 30 days of treatment were recorded. The rats from all groups of treated and control were sacrificed after 24 hours of the last dose under light either anaesthesia and dissected quickly. Testes and epididymes were taken out, freed from adjoining tissues and blood. Weight of testes and epididymes were recorded in a semi-micro balance. The testes and epididymes were washed thoroughly in 10% of saline solution, fixed with Bouin's fluid (for two days), washed in tap water, dehydrated in alcoholic series and embedded in paraffin wax at 60C. The blocks of reproductive tissues were sectioned at 5 (micron) using Rotary Microtome and stained with haemotoxylin and eosin for histological study. The slides were observed under microscope at high power and photographed at different magnification. 4. Statistical Analysis The body weight and organ weight (testes and epididymes) of both treated and control group of male rats were recorded in tabular form. The data were statistically analysed using student 't' test. The values were expressed as mean  standard error (S.E.). The significance of weight between treated and control rats were taken at P < 0.05 as significant.

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