Abstract
The current study was undertaken to observe the effect of oral administration of Neem seed oil and its various chromatographic fractions on sperm dynamics and testicular cell population dynamics of male albino rats. Significant reduction in sperm density in testis and cauda epididymis and sperm motility in cauda epididymis was observed in male rats treated with neem seed oil and its various chromatographic fractions for 60 days. The maximum reduction in sperm motility and sperm density was observed in rats treated with neem seed oil fraction III and fraction IV. Results of testicular cell population dynamics showed the arrest of spermatogenesis at spermatid stage after treatment with neem seed oil and its various chromatographic fractions suggesting the antiandrogenic nature of neem seed oil fractions. It is suggested that the antifertility effect of neem seed oil and its fraction in male albino rats was through inhibition of FSH and LH secretion and steroidogenesis via hypothalamo-hypophyseal gonadal axis. The neem seed oil fractions III and IV were shown to be the most potent contraceptive agents among all the fractions.
Introduction
Neem (Azadirachta indica A. Juss; Meliaceae) is one of the very few trees known on the Indian subcontinent since antiquity. In time, neem was found to be a multipurpose tree (CitationAhmed & Grainage, 1986; CitationRiar, 1996; CitationPuri, 1999).Various researchers have shown the antifertility properties of neem seeds, leaf, and flower extracts in male and female rats and mice (CitationJoshi et al., 1996; CitationPurohit, 1998; CitationMishra & Singh, 2005). Neem seed oil is known to have antifertility properties (CitationLal et al., 1986; CitationRiar et al., 1988). But the earlier preparations of neem seed oil were observed to be toxic, which discouraged further research into its male contraceptive effects. Recent scientific investigation on its male contraceptive activities yielded very positive results. The hexane extract of neem seed and preliminary results on corresponding column fractions showed potent and reproducible antifertility results (CitationGarg, 1994). Keeping this view in mind, the current study was undertaken to evaluate the effect of hexane-extracted pure neem seed oil and various chromatographic active fractions on sperm dynamics and testicular cell population dynamics in male albino rats.
Materials and Methods
Preparation of neem seed oil
The ripe fruits of neem were collected in and around New Campus of Jai Narain Vyas University, Jodhpur. The plant material was identified by Dr. S. R. Rao, Assisstant Professor, Department of Botany, Jai Narain Vyas University, Jodhpur, and a voucher specimen was preserved in the departmental herbarium. After cleaning and depulping, the seeds were dried in bright sunlight. The neem kernel is the richest source of neem seed oil. Therefore, instead of crushing the whole seed, the seeds were decorticated mechanically removing the husk. The kernels were used for solvent extraction after de-hulling. The crushed kernels were initially blended with hexane in the ratio 1:5 then were packed into a cellulose thimble. The thimble was placed in the extractor. Hexane (AR grade) was successively refluxed on the top of the ground kernels placed in the thimble and percolated through the mass by gravity until removal of desired constituents was completed. The miscella after extraction was filtered. Hexane was recovered from the miscella by distillation. The residue left after distillation was collected. The resulting yield was 20% of material taken. To prevent oxidation, the neem seed oil was stored under refrigeration at −20°C
Fractionation of neem seed oil
Neem seed oil (50 g) was chromatographed over deactivated silica gel (60–120 mesh) in a glass column. Six major fractions were eluted with EtOH/hexane mixture in six different proportions ().
Table 1 Proportion of ethyl alcohol and hexane in different chromatographic fractions of neem seed oil.
These fractions were eluted separately and dried under reduced pressure. They were diluted as per required doses in olive oil and used for treatment. In the current study, all the fractions were used to see the effect on sperm dynamics and testicular population dynamics.
The dosages were decided after careful consideration of the available literature of earlier workers (CitationTalwar et al., 1997; CitationGarg et al., 1998) and on the basis of analysis of the ratio of neem seed oil charged in the column and elute extracted as chromatographic fraction.
Animals and treatments
Healthy adult male albino rats of the Sprague-Dawley strain, aged between 3 and 5 months and weighing 150 to 250 g with proven fertility, were selected for the current investigation. Animals were kept under controlled environment conditions and provided commercial diet and water ad libitum. This study was approved by the departmental ethical committee. They were divided into eight groups of 10 animals each as follows:
Group 1: Vehicle-treated control: The group received drug vehicle only i.e., olive oil (1 ML/kg body wt per day) for 60 days orally.
Group 2: Neem seed oil (500 mg/kg body wt per day) for 60 days orally.
Group 3: Neem seed oil fraction I (9 mg/kg body wt per day) for 60 days orally.
Group 4: Neem seed oil fraction II (10 mg/kg body wt per day) for 60 days orally.
Group 5: Neem seed oil fraction III (19 mg/kg body wt per day) for 60 days orally.
Group 6: Neem seed oil fraction IV (28 mg/kg body wt per day) for 60 days orally.
Group 7: Neem seed oil fraction V (10 mg/kg body wt per day) for 60 days orally.
Group 8: Neem seed oil fraction VI (7 mg/kg body wt per day) for 60 days orally.
After 55 days of treatment, the fertility test was performed. Male rats were cohabited with proestrous females in the ratio 1:2. The presence of sperm in the morning vaginal smear was evidence of mating. On the 61st day i.e., 24 h after the last dose, animals were sacrificed using light ether anesthesia. Blood was collected through cardiac puncture and serum was separated. The RBC and WBC count, glucose, hemoglobin, and hematocrit were estimated with routine methods. No changes were observed in routine hemotological parameter suggesting the nontoxic nature of the fractions. The testis and accessory sex organs (epididymis, seminal vesicle, and ventral prostate) were dissected, freed from fat and connective tissue, and weighed.
Criteria of observation
Sperm dynamics
Sperm motility in cauda epididymis and sperm density in cauda epididymis and testis was estimated (CitationPrasad et al., 1972).
Testicular cell population dynamics
Spermatogenic elements (spermatogonia, spermatocytes, and spermatids) were counted in 5-μ-m thick sections of 10 seminiferous tubules in 5 animals of each group.
All raw counts were transformed to true counts by adaptation of the Abercrombie formula (CitationAbercrombie, 1946) for cell diameter measurements. Interstitial cell types (such as fibroblasts, immature and mature Leydig cells, and degenerating cells) were estimated, applying a differential count over 200 cells of this cell population, and statistically verified by the binomial distribution (CitationDixon & Massey, 1957).
All the values of the testicular cell population count were expressed in terms of mean values ± standard error. The different experimental groups were compared with vehicle-treated control groups using Student's t-test.
Results
Non-significant changes occurred in body weight of experimental animals of all treatment groups. Administration of neem seed oil (Gr. 2) and its chromatographic fraction (Gr. 3–8) brought about significant reduction in the weight of the testis and accessory sex organs in relation to control. However, there was no significant reduction in weight of seminal vesicle and ventral prostate in rats treated with neem seed oil fraction V and VI (Gr. 7,8) ().
Table 2 Body and organ weights of rats treated with neem seed oil and its various chromatographic fractions (mean ± SEM).
The neem seed oil (Gr. 2), fraction I (Gr. 3), fraction II (Gr. 4), fraction III (Gr. 5), and fraction IV (Gr. 6) treated groups exhibited 100% negative fertility whereas fraction V (Gr. 7) and fraction VI (Gr. 8) treated groups showed 70% and 80% negative fertility respectively ().
Table 3 Sperm dynamics and fertility test of rats treated with neem seed oil and its chromatographic fractions (mean ± SEM).
The sperm motility in cauda epididymis was decreased by 84%, 88%, 55%, and 65% in neem seed oil, fraction III, fraction V, and fraction VI treated rats, respectively. No sperm motility was observed in fraction IV treated rats, whereas non-significant changes in sperm motility were observed in fraction I and fraction II treated groups. The sperm density in testis and cauda epididymis showed reduction in all treatment groups, but maximum reduction was observed in neem seed oil (Gr. 2), fraction III (Gr. 5) and fraction IV (Gr. 6) treated rats. The number of spermatogonia in neem seed oil (Gr. 2), fraction III (Gr. 5), fraction IV (Gr. 6) treated rats displayed noticeable decline. However, the fall in the number of spermatogonia in the fraction I (Gr. 3), fraction II (Gr. 4), fraction V (Gr. 7), and fraction VI (Gr. 8) treated rats, in relation to mean control value, was insignificant.
The number of primary and secondary spermatocytes after administration of neem seed oil and all the chromatographic fraction but maximum reduction was observed in Neem seed oil, fraction III and fraction IV treated rats. The number of spermatids displayed in all of the experimental groups demonstrated a striking significant decrease in comparison with the control ().
Table 4 Testicular cell population dynamics of rats treated with neem seed oil and its chromatographic fractions (mean ± SEM).
A significant increase in number of degenerating Leydig cells was observed in neem seed oil, fraction II, fraction III, and fraction IV treated rats, whereas non-significant changes in number of degenerating leydig cells were observed in fraction I, fraction V, and fraction VI treated rats. The number of mature Leydig cells after administration of neem seed oil (Gr. 2), fraction II (Gr. 3), fraction III (Gr. 5), fraction IV (Gr. 6) for 60 days exhibited a remarkable decline, but the reductions in mature Leydig cells of fraction I (Gr. 3) and fraction V (Gr. 7) treated rats were insignificant compared with control mean value ().
Discussion
A decrease in the weight of testis and accessory sex structure suggested a decreased androgen level as these organs are androgen-dependent. An antiandrogenic effect of neem seed oil and its chromatographic fractions is supported by reduced sperm density in testes and cauda epididymis and sperm motility in cauda epididymis.
This effect is possibly due to the antiandrogenic nature of the fractions. It may cause selective androgen deprivation. The selective androgen deprivation is brought about by competition between drug and androgen for binding sites in the androgen dependent tissue (CitationChinoy et al., 1985), which is responsible for the testicular dysfunction (CitationJha & Dixit, 1986).
The absence of spermatids in the seminiferous tubules was conspicuous. The process of spermatogenesis is androgen dependent (CitationChaudhary & Steinberger, 1975). Spermatogenesis is activated by testosterone, which is synthesized in Leydig cells. It seems that the arrest of spermatogenesis is due to the reduced activity of Leydig cells (CitationKasturi et al., 1995). FSH plays a major role in transformation and maturation of round to elongated spermatid (CitationMoudgal & Suresh, 1995; CitationShetty et al., 1996).
In conclusion, it is suggested that the antifertility effect of neem seed oil and its fractions in male albino rats is due to the arrest of spermatogenesis and by inhibiting FSH and LH secretion and steroidogenesis via the hypothalamo-hypophyseal gonadal axis. The reduction in sperm motility, sperm density, and testicular cell population were more pronounced in rats treated with neem seed oil fractions III and IV. The neem seed oil fractions III and IV were shown to be the most potent contraceptive agents among all the fractions.
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