Effects of Myristica fragrans oil on the reproductive organs of male albino rats

Abstract

This research aimed to study the effects of Myristica fragrans Houtt. (Myristicacae) oil on male reproductive functions in Wistar strain rats, the oil of Myristica fragrans at the dose levels of 0.2, 0.4, and 0.5 mL/kg body weight/day for 60 days. The oil reduced fertility to 0% by 60 days of treatment at the highest dose level. The results also revealed the oil caused significant reduction in the weight of testis, epididymis, seminal vesicle, ventral prostate and vas deferens. Sperm motility as well as sperm density were significantly reduced which resulted in the reduction of male fertility by 100% at the highest dose level tested. Leydig cell nuclear area and mature Leydig cell numbers were significantly reduced when compared with controls. The round spermatids were decreased by 72.71%. The hematological parameters show a non-significant change when compared with the control group. A recovery of 80% fertility was recorded after withdrawal of 120 days, proving that the drug is safe to use. These results suggest the antifertility and recovery effect from the oil of Myristica fragrans in male albino rats.

Keywords::

• Myristica fragrans Houtt.
• sperm motility
• Leydig cell
• recovery

Introduction

Plants that were considered of no value are now being investigated, evaluated, and developed into drugs with little or no side effects. Fertility regulation with plant preparations has been reported in ancient literature of indigenous systems of medicine. Myristica fragrans Houtt. (Myristicacae) is a tropical, dioecious evergreen tree native to the Molluccas, of the Spice Islands of Indonesia. Its seeds were reported to be useful in various ailments (Chopra et al., 1956). Dried kernels and oil of Myristica fragrans are used as aromatic, spasmolytic, and anxiogenic agents (Sonavane et al., 2002).

Pharmacologically and therapeutically, the value of Myristica fragrans seed oil is for the treatment of diarrhea and controlling blood pressure (Grover et al., 2002). The effect of the oil of nutmeg on the fertility and induction of meiotic chromosome rearrangements in mice and their first generation have been studied (Pecevski et al., 1981).

Since no attention has been drawn towards contraceptive effects, and the mode of action of Myrsitica fragrans has not been reported in detail, the present communication was undertaken to study the possible antifertility effects of Myristica fragrans oil at 0.5 mL/kg body weight (bw) for 60 days in male albino rats, and also to interpret the recovery after 120 days of withdrawal of the treatment.

Materials and methods

Plant material

The oil of Myristica fragrans was purchased from the Himalaya Drug Company, Banglore, India. Myristicin is the chief constituent previously isolated from the oil (Figure 1).

Figure 1.  Showing the Chemical structure of Myristicin.

Animals used

Thirty adult male Wistar strain rats weighing 180–200 g were used. They were maintained at standard conditions (12 h light/12 h night, 26° ± 2°C) in wire-bottomed polypropylene cages with free access to standard rat pellet diet (Aashirwad, Chandigarh) and provided water ad libitum. All the described experiments have been carried out under the approval of the departmental ethics committee. The Guidelines for the Care and Use of Animals for scientific Research (INSA, 2000) were strictly followed throughout the course of investigation.

Before starting the experiments, the male rats were kept for fertility tests, the number and size of litters were recorded.

Study protocol

The rats were divided into three groups containing ten animals in each.

• Group – I (Control)  Control or vehicle treated (1.0 mL olive oil)

• Group – II (Treatment)  0.2 mL Myristica fragrans oil/kg bw/day for 60 days

• Group – III (Treatment)  0.4 mL Myristica fragrans oil/kg bw/day for 60 days

• Group – IV (Treatment)  0.5 mL Myristica fragrans oil/kg bw/day for 60 days

• Group – V (Recovery)  0.5 mL Myristica fragrans oil/kg bw/day for 60 days and kept for 120 days for recovery.

Myristica fragrans oil was diluted in 0.5 mL olive oil and administered orally; the animals were weighed before and after the treatment.

Fertility test

The mating exposure tests of control and treated groups were performed on day 55 using the method of WHO (1983). After mating the males were removed for autopsy.

The animals were weighed and autopsied under light ether anesthesia 24 h after the last dose of the treatment. Blood samples were collected by cardiac puncture in heparinized tubes for hematological studies.

Body and organ weight measurements

Initial and final body weights of the animals were recorded. At autopsy, the reproductive and accessory sex organs (testes, epididymis, seminal vesicle, ventral prostate and vas deferens) along with the liver were dissected out, freed from adherent tissues and weighed, slices of tissues were fixed in Bouin’s fluid, paraffin sections were made and stained with Ehrlich hematoxylin and eosin (Humason, 1979).

Sperm motility and density

After anesthetizing the rat, the epididymis was exposed by scrotal incision, and spermatozoa were expressed out by cutting the distal end of the cauda epididymal tubule.

The motility of cauda epididymal spermatozoa was recorded and the percentage of motile sperms was calculated per unit area (Srikanth et al., 1999). Cauda epididymal sperm density was assessed using a Neubauer’s hemocytometer (Zaneveld & Polakoski, 1977).

Assay of serum testosterone concentration

Serum concentration of testosterone was measured following an immunoenzymatic method with a ELISA reader (Merck, Japan), according to the standard protocol given by the National Institute of Health and Family Welfare (Shrivastava, 2002).

Hematology

Blood was collected and the values of red blood cell (RBC) and white blood cell (WBC) counts (Lynch et al., 1969), hematocrit (Strumia et al., 1954), hemoglobin percentage (Crossby et al., 1954), and blood sugar (Astoor & King, 1954) were estimated.

Histological studies of testes

The testes of the animals were processed for histopathological evaluation. For histology the tissues were fixed in Bouin’s fluid, dehydrated, and embedded in paraffin for sections at 5 μm. These sections were stained in Harris hematoxylene and eosin.

Histometry

The evaluation of cell population dynamics was based on the calculations made for each cell type per cross section of seminiferous tubule. Various cell components were quantitatively analyzed using spherical tubules and a correcting factor was introduced (Berndtson, 1977). Interstitial cell types such as fibroblast, mature and degenerating Leydig cells were estimated (Dixon & Massey, 1957).

Statistical analysis

All of the recorded values of body/organ weight, sperm dynamics, hematological parameters and testicular cell dynamics were expressed in terms of mean ± SEM. The treated groups were compared to control using the Student’s t-test (Ipstein & Poly, 1970).

Ethical aspects

The study was approved by the ethical committee of the University, Department of Zoology, Jaipur, India. Indian National Science Academy, New Delhi (INSA, 2000) guidelines were followed for maintenance and use of the experimental animals.

Results

Weight response

The oral treatment of Myristica fragrans oil did not cause any significant change in the body weight of the treated rats with respect to the initial body weight (Table 1). However, the weight of testes, epididymis, seminal vesicle, ventral prostate and vas deferens decreased significantly (P < 0.001) with respect to the weights of the control group in all the treatment groups, while a non-significant change in the weight of liver was observed after the treatment. A non-significant change in the above-mentioned parameters was observed in Group V of the experiment after 120 days of recovery.

Table 1.  Body and organs weight of Myristica fragrans oil treated male albino rats.

Treatment	Body weight (g)		Tissue weight (mg/100 g body weight)
	Initial	Final	Testes	Epididymis	Seminal vesicle	Ventral prostate	Vas deferens	Liver
Group I Control (vehicle treated)	180.00 ± 6.14	190.00 ± 7.96	1275.00 ± 42.53	402.00 ± 8.20	461.11 ± 9.12	271.20 ± 25.10	157.22 ± 13.33	3292.33 ± 27.13
Group II 0.2 mL oil/kg body weight for 60 days	186.62 ± 5.81	197.60 ± 8.42^ns	1064.76 ± 21.59*	397.44 ± 5.65*	418.89 ± 5.34*	218.70 ± 10.45*	142.89 ± 10.98*	3237.61 ± 22.96^ns
Group III 0.4 mL oil/kg body weight for 60 days	189.40 ± 6.43	198.76 ± 8.59^ns	902.67 ± 32.98**	353.27 ± 7.88**	387.09 ± 7.45**	184.32 ± 09.34**	139.56 ± 9.34**	3289.03 ± 30.90^ns
Group IV 0.5 mL oil/kg body weight for 60 days	190.00 ± 9.12	198.00 ± 6.16^ns	860.00 ± 69.20**	340.11 ± 3.66**	356.21 ± 3.66**	160.33 ± 16.33**	128.00 ± 10.10**	3267.11 ± 16.66^ns
Group V Recovery for 120 days after 60 days of treatment	185.00 ± 8.02	192.00 ± 9.62^ns	1185.00 ± 50.25^ns	390.00 ± 50.25^ns	436.66 ± 6.69^ns	256.80 ± 8.79^ns	152.38 ± 12.74^ns	3307.92 ± 22.24^ns

(Mean ± SEM of 10 animals). Group II, III, IV and V compared with Group I.

Ns, not significant; * P ≤ 0.01; **P ≤ 0.001.

Sperm dynamics and fertility test

Results given in Table 2 showed a significant reduction (P < 0.001) of sperm motility in cauda epididymis. The fertility test showed 55%, 85%, and 100% negative fertility in Group II, III, and IV, respectively. The number of implantation sites and the number of viable fetuses was recorded to be nil at the highest dose level tested. In Group V, sperm motility and sperm density showed non-significant changes and 80% positive fertility was observed after 120 days of recovery. In the recovery phase, the number of pregnant females was 16 out of the group of 20, and a non-significant change was observed in the values of the number of implantation sites and number of viable fetuses when compared with the normal group.

Table 2.  Effect of Myristica fragrans oil on male rat fertility.

Treatment	No. of male	No. of female	No. of pregnant females	No. of implantation sites	No. of viable fetuses	Sperm motility (%) (cauda epididymis)	Sperm density (million/mL) (cauda epididymis)	Fertility test
Group I Control (vehicle treated)	10	20	20/20 (100%)	10.50 ± 03.05	09.02 ± 01.75	75.23 ± 00.91	52.22 ± 00.62	100% (-ve)
Group II 0.2 mL oil/kg bw for 60 days	10	20	9/20 (45%)	7.41 ± 02.67*	06.22 ± 03.48*	54.67 ± 0.61*	41.65 ± 00.89*	55% (-ve)
Group III 0.4 mL oil/kg bw for 60 days	10	20	3/20 (15%)	3.89 ± 01.65**	02.95 ± 02.22**	41.76 ± 00.38**	24.01 ± 00.45**	85% (-ve)
Group IV 0.5 mL oil/kg bw for 60 days	10	20	00/20 (0%)	Nil	Nil	25.66 ± 00.72**	13.33 ± 00.72**	100% (-ve)
Group V Recovery for 120 days after 60 days of treatment	10	20	16/20 (80%)	09.20 ± 04.44^ns	08.60 ± 02.02^ns	69.24 ± 01.20^ns	48.65 ± 01.39^ns	80% (+ve)

(Mean ± SEM of 10 animals). Group II, III, IV and V compared with Group I.

Ns, not significant; *P ≤ 0.01; **P ≤ 0.001.

Serum testosterone levels

The concentration of serum testosterone was significantly decreased in all the treatment groups in a dose-dependent manner, while in the recovery group, the level of testosterone recovered to the control level (Table 3).

Table 3.  Effect of Myristica fragrans oil on the level of serum testosterone.

Treatment		Serum testosterone (ng/mL)
Group I	Control (vehicle treated)	4.85 ± 0.97
Group II	0.2 mL oil/kg body weight for 60 days	3.53 ± 1.20*
Group III	0.4 mL oil/kg body weight for 60 days	2.22 ± 1.03**
Group IV	0.5 mL oil/kg body weight for 60 days	1.73 ± 0.33**
Group V	Recovery for 120 days after 60 days of treatment	4.37 ± 0.16^ns

(Mean ± SEM of 10 animals). Group II, III, IV and V compared with Group I.

ns, not significant; *P ≤ 0.01; **P ≤ 0.001.

Hematology

The hematological parameters (RBC – Red Blood Cell Count, WBC – White Blood Cell Counts, hemoglobin, hematocrit, MCV – Mean Corpuscular Volume, MCH – Mean Corpuscular Hemoglobin, MCHC – Mean Corpuscular Hemoglobin Concentration and blood sugar) remained unaffected in all the treatment and recovery groups (Table 4).

Table 4.  Haematological changes of Myristica fragrans oil treated male rats.

Treatment	RBC counts (million/mm^3)	WBC counts (mm^3)	Hematocrit (%)	Hemoglobin (g%)	MCV μm^3	MCH Pg	MCHC %	Blood sugar (mg/100 mL)
Group I Control (vehicle treated)	5.02 ± 0.22	8817.77 ± 187.33	44.00 ± 0.82	13.96 ± 0.49	88.00 ± 0.20	27.92 ± 0.65	31.72 ± 0.95	90.10 ± 3.14
Group II 0.2 mL oil/kg bw for 60 days	5.18 ± 0.41^ns	8809.65 ± 115.42^ns	43.87 ± 0.90^ns	13.45 ± 0.74^ns	87.23 ± 0.67^ns	26.89 ± 0.50^ns	28.03 ± 0.63^ns	90.56 ± 2.76^ns
Group III 0.4 mL oil/kg bw for 60 days	5.72 ± 0.38^ns	8842.91 ± 184.01^ns	43.29 ± 0.73^ns	13.28 ± 0.88^ns	86.41 ± 0.42^ns	27.66 ± 0.10^ns	28.76 ± 0.80^ns	89.43 ± 3.87^ns
Group IV 0.5 mL oil/kg bw for 60 days	5.13 ± 0.33^ns	8802.00 ± 210.77^ns	43.66 ± 0.98^ns	12.21 ± 0.55^ns	85.60 ± 0.27^ns	23.94 ± 00.61^ns	27.96 ± 0.96^ns	91.43 ± 2.70^ns
Group V Recovery for 120 days after 60 days of treatment	5.60 ± 0.16^ns	8811.64 ± 192.66^ns	43.20 ± 0.86^ns	12.65 ± 0.69^ns	86.49 ± 0.18^ns	26.28 ± 0.63^ns	29.67 ± 0.84^ns	90.86 ± 2.94^ns

(Mean ± SEM of 10 animals). Group II, III, IV and V compared with Group I.

ns, not significant.

Histology

Histological studies of control rat testis show all successive stages of spermatogenesis, where the lumen was filled with sperm (Figure 2). The sections of testis of Group II fed rats show partial damage of spermatogenic elements (Figure 3). In Group III fed rats, the section of testis revealed a decrease in the diameter of seminiferous tubules and the lumen was devoid of spermatozoa while the Leydig cells were normal in appearance (Figure 4). Completely degenerating spermatogenesis could be seen with the Myristica fragrans oil treatment group (Figure 5), the main histological alterations observed were the decreased number of spermatogenic elements in the seminiferous tubule, cellular debris in lumen, increased interstitial space and damaged Leydig cells. The recovery group illustrated almost the control histoarchitecture of testis (Figure 6).

Figure 2.  Showing the section of a control Rat testis.

Figure 3.  Showing the section of Rat testis treated with Myristica fragrans Oil at the dose level of 0.2 ml/kg b. wt. for 60 days.

Figure 4.  Showing the section of Rat testis treated with Myristica fragrans Oil at the dose level of 0.4 ml/kg b. wt. for 60 days.

Figure 5.  Showing the section of Rat testis treated with Myristica fragrans Oil at the dose level of 0.5 ml/kg b. wt. for 60 days.

Figure 6.  Showing the section of Rat testis after the recovery period of 120 days.

Histometry

Results presented in Figures 7–10 revealed that seminiferous tubule diameter and percentage of normal tubules decreased significantly (P <0.001) in all the treatment groups in a dose-dependent fashion, whereas the percentage of abnormal tubules increased significantly (P <0.001).

Figure 7.  Effect of administration of Myristica fragrans Oil on the seminiferous tubule count of male albino rats.

Figure 8.  Effect of administration of Myristica fragrans Oil on the seminiferous tubule diameter of male albino rats.

Figure 9.  Effect of administration of Myristica fragrans Oil on the Leydig cell area of male albino rats.

Figure 10.  Effect of administration of Myristica fragrans Oil on the Leydig cell count of male albino rats.

The cytoplasmic and nuclear area of Leydig cells showed a highly significant reduction (P <0.001). The percentage of differential count of mature Leydig cells was reduced significantly (P <0.01) and degenerating Leydig cells were increased significantly (P <0.001), whereas a non-significant decrease in the differential count of fibroblast like cells was recorded following the oral administration of Myristica fragrans oil (Figures 7–10).

In Group V of 120 days of recovery, a non-significant change was recorded in the values of all parameters mentioned above when compared with the control animals.

Discussion

The oral treatment of male rats with Myristica fragrans oil showed a significant reduction in testis, epididymides, seminal vesicles, ventral prostate, and vas deferens weights. This may be due to low plasma level of testosterone, as testicular size is the best primary assessment of spermatogenesis, since the tubules and germinal elements account for approximately 88% of the testicular mass (Sherines et al., 1978). Reduction in the weight of accessory reproductive organs directly supports the reduced availability of androgens (Sharma & Jacob, 2001). The recovery phase showed complete recovery from all the effects mentioned above.

The reduction in sperm motility in cauda epididymis is of importance with regard to fertilization; it may be due to androgen deficiency (Chitra et al., 2001). The 100% negative fertility test may be attributed to decreased spermatozoa density and motility of cauda epididymis. Moreover, the spermatozoa were sluggishly motile and, therefore, unable to fertilize the normal cycling females. Reduction in the progressive epididymal sperm motility of the treated rats may be responsible for the severe decrease in the average number of implantation sites, viable fetuses and number of litters born by the female rats cohabited with Myristica fragrans oil-treated male rats leading to the presence of sperm-toxic agents in the oil (Verma & Chinoy, 2001). The progressive mode in the recovery group suggested the deleterious effect was reversible.

A significant decline in serum testosterone might be due to the adverse effect of treatment on hormonal milieu of the testes. The reduced weights of seminal vesicle and ventral prostate further support the suppressed concentration of testosterone in circulation (Lohiya & Ansari, 1999).

No change in the values of hematological parameters in treated as well as in the recovery group showed that the drug did not affect the vital functioning of rats.

The seminiferous tubule diameters were decreased due to the absence of spermatogenic cells. The reduction in the number of spermatogenic cells may be due to the non-availability of testosterone, as spermatogenesis is activated by testosterone, which is synthesized by Leydig cells (Sharpe, 1987). The impairment of Leydig cell function was evident by its reduced nuclear area and lower number of mature Leydig cells. The number of mature Leydig cells had a direct effect on spermatogenesis. Deformation of Leydig cell and reduction in cauda epithelial cell height indicates the inefficiency of these cells to synthesize testosterone (Hulethal & Lunenfeld, 2004). The checking of treatment in the recovery group again normalized the process of spermatogenesis, thus maintaining the level of testosterone in the normal ranges.

These results suggested that oral administration of Myristica fragrans oil could lead to antifertile state in male rats due to the interference of the oil in the testicular androgen levels, which could recover after terminating the treatment.

Acknowledgements

The authors are thankful to the Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur for providing necessary facilities.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.