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Evaluation of reversible contraceptive activities of Cuminum cyminum in male albino rats
Contraception 84 (2011) 98 – 107
Original research article
Evaluation of reversible contraceptive activities of Cuminum cyminum in male albino rats Radhey S. Gupta⁎, Poonam Saxena, Rajnish Gupta, Jai B.S. Kachhawa Reproductive Physiology Section, Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 8 March 2010; revised 25 October 2010; accepted 25 October 2010
Abstract Background: The aim of the present study was to evaluate the contraceptive efficacy of Cuminum cyminum (jeera) seeds in male albino rats. Study Design: C. cyminum methanol extract (CcMtE) at dose levels of 100 and 200 mg/rat/day was orally administered to male rats for 60 days. The effect of the treatment on reproductive organs and fertility was investigated. Recovery and toxicity studies were also carried out. Results: C. cyminum methanol extract fed to male rats for 60 days did not cause any alterations in the body weight, whereas the weight of testes, epididymides, seminal vesicles and ventral prostate were significantly reduced (p≤.001). Animals treated with CcMtE showed a marked reduction in sperm density in the cauda epididymis and testes and sperm motility in the cauda epididymis. Reduction in fertility was 69.0% and 76.0% in 100 and 200 mg/rat/day dose levels, respectively. The circulatory hormones were also reduced significantly. Testicular biochemical analysis of protein, sialic acid, glycogen, ascorbic acid and fructose indicated a marked decline, whereas testicular cholesterol content was significantly increased, which showed altered biochemistry of the reproductive organs. After CcMtE treatment, significant decreases (p≤.001) were observed in the number of testicular cells (i.e., spermatogonia, primary spermatocytes [preleptotene and pachytene], secondary spermatocytes and round spermatids); nonsignificant change was observed in the Sertoli cell count. The treatment had no effect on levels of serum protein, cholesterol, bilirubin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea and hematological indices. Conclusions: The present study shows that C. cyminum treatment resulted in the inhibition of spermatogenesis and fertility without producing apparent toxic effects. © 2011 Elsevier Inc. All rights reserved. Keywords: Cuminum cyminum; Testis; Spermatogenesis; Epididymides; Sperm; Fertility
1. Introduction Countless efforts have been made to obtain male antifertility agents from plants [1,2]. Cuminum cyminum (Apiaceae), an annual herb with brownish or grayish yellow color seeds, is widely used as an anticarcinogenic, stomachache and astringent agent [3]. Phytochemical studies revealed the presence of some terpenes, glycosides, cumaldehyde, myrcene, α-phellandrene, α-pinene, β-pinene, cyminal, α-terpene, y-terpinene and p-cymene in C. cyminum. A high level of cumaldehyde (4-propan-2ylbenzaldehyde) is found in C. cyminum [4]. In the present study, we investigated the effect of C. cyminum on the
reproductive system of the male albino rats in order to evaluate the potential of this plant as a male fertilityregulating agent. 2. Materials and methods Male albino rats of Wistar strain weighing 150–180 g were used for the present study. Animals were kept in standard conditions of 12-h light/12-h dark cycle and 22°C±2°C temperature. Standard pellet diet (Ashirvad Food Industries, Chandigarh, India) and tap water were provided ad libitum. 2.1. Ethical aspects
⁎ Corresponding author. Department of Zoology, University of Rajasthan, Jaipur 302004, India. Fax: +91 141 2701137. E-mail address: [email protected] (R.S. Gupta). 0010-7824/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2010.10.013
The study was approved by the Departmental Ethical Committee, University of Rajasthan, Jaipur, India. The
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Table 1 Effect of CcMtE on body and organ weights Treatment
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
Body weight (g)
Testes (mg/100 g body weight)
Cauda epididymides (mg/100 g body weight)
Seminal vesicle (mg/100 g body weight)
Ventral prostate (mg/100 g body weight)
Initial
Final
160.00±7.60 172.00±7.39 155.00±7.07
225.00±14.14 220.00±7.80, ns 230.00±4.90, ns
1376.98±13.49 962.89±7.34⁎⁎ 1321.37±16.44‡, ns
460.22±9.54 319.53±1.35⁎⁎ 437.02±6.80‡, ns
456.26±7.46 361.64±6.68⁎⁎ 431.19±5.24‡, ns
364.13±7.89 283.44±1.67⁎⁎ 337.68±6.57‡, ns
165.00±4.53 170.00±5.00
210.00±5.50, ns 235.00±5.38, ns
849.88±5.23⁎⁎,†† 1308.24±16.76§, ns
297.99±2.39⁎⁎,†† 426.80±6.39§, ns
313.12±2.12⁎⁎,†† 422.02±4.56§, ns
253.30±1.56⁎⁎,†† 326.53±9.61§, ns
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01 and ⁎⁎p≤.001 compared with Group I (control); †p≤.01 and †† p≤.001 compared with Group IIA; ‡p≤0.001 comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
Table 2 Effect of CcMtE on sperm dynamics and fertility Treatment
Sperm motility (%)
Sperm density (million/mL)
Cauda epididymides
Testes
Cauda epididymides
Fertility (%)
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
76.21±1.96 33.19±0.28⁎⁎ 67.05±2.46‡, ns 30.67±0.59⁎⁎,† 65.89±2.74§, ns
4.65±0.25 1.85±0.10⁎⁎ 4.37±0.15‡, ns 1.32±0.12⁎⁎,† 3.97±0.40§, ns
44.70±2.60 10.70±1.21⁎⁎ 41.85±1.20‡, ns 9.15±0.10⁎⁎,†† 40.40±2.77§, ns
100 69.0 91.3 76.0 86.7
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01 and ⁎⁎ p≤.001 compared with Group I (control); †p≤.01 and ††p≤.001 compared with Group IIA; ‡p≤.001 comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
guidelines of the Indian National Science Academy of New Delhi [5] for the use of experimental animals were followed. 2.2. Plant material Fresh seeds of C. cyminum were collected from the local market of Jaipur and were identified and authenticated in the
Department of Botany, University of Rajasthan, Jaipur. One hundred percent methanol extract was prepared by Soxhlet extraction for 72 h. Methanol was evaporated under reduced pressure to obtain the concentrated oily extract (15 g), which was then suspended in olive oil just before experimentation (C. cyminum methanol extract [CcMtE]).
Fig. 1. Effect of CcMtE on serum testosterone.
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Fig. 2. Effect of CcMtE on serum estradiol.
2.3. Acute toxicity studies The study of acute toxicity of the CcMtE was performed according to the method of Turner [6]. The LD50 value of the experimental drug was 1500 mg/kg body weight of male albino rats. 2.4. Treatment protocol and autopsy schedule Male albino rats of proven fertility were grouped as follows: Group I: control rats received 0.5 mL/day of the vehicle (i.e., olive oil).
Group IIA: rats treated with CcMtE at 100 mg/rat/day dose level. Group IIB: recovery after treatment of CcMtE at the 100 mg/rat/day dose level from Group IIA. Group IIIA: rats treated with CcMtE at 200 mg/rat/day dose level. Group IIIB: recovery after treatment of CcMtE at 200 mg/rat/day dose level from Group IIIA. An olive oil suspension of extract was prepared and administered daily by oral gavage for 60 days. Twenty-four hours after the last dose at Day 61, the animals were autopsied under light ether anesthesia.
Fig. 3. Effect of CcMtE on serum luteinizing hormone.
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Fig. 4. Effect of CcMtE on serum follicle-stimulating hormone.
2.5.2. Sperm parameters Sperm motility in the cauda epididymis and sperm density in the epididymis and testes were assessed by the method of Prasad et al. [7].
2.5.4. Serum hormonal assay The concentrations of serum testosterone, luteinizing hormone, follicle-stimulating hormone and estradiol levels were assayed from samples according to the standard protocol using radioimmunoassay. The serum concentration of testosterone was measured according to standard methods [8]; the sensitivity of the assay was 10 pg/mL. Luteinizing hormone and follicle-stimulating hormone were measured by the method of Moudgal and Madhwaraj [9], and serum levels of estradiol were analyzed using 125 I radioimmunoassay kit, which has been previously tested in rat sera against other radioimmunoassays [10].
2.5.3. Fertility test The mating test was performed from Days 55 to 60. The male rats were cohabited with proestrous female rats at a ratio of 1:3. Successful mating was confirmed by the presence of spermatozoa in the vaginal smear. The mated females were separated to note the implantation site on Day 16 of pregnancy through laparotomy.
2.5.5. Tissue biochemistry After autopsy, reproductive organs (i.e., testes, epididymis, seminal vesicles and ventral prostate) were removed, cleared of fat and connective tissue, weighed on electronic balance and kept at −4°C until assayed for various biochemical parameters. The estimation of protein [11] and sialic acid [12] were performed in the testis, epididymis, seminal vesicle and ventral prostate. Concentrations of
2.5. Parameters 2.5.1. Body weight and organ weights Initial and final body weights were recorded. The testes, epididymis, seminal vesicle and ventral prostate were excised, carefully removed from the surrounding fat and connective tissue and weighed on an electronic balance.
Table 3 Effect of CcMtE on tissue biochemistry Treatment
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
Testis Cholesterol (mg/g)
Protein (mg/g)
Glycogen (mg/g)
Sialic acid (mg/g)
9.43±0.31 12.74±0.17⁎⁎ 8.60±0.07‡, ns 13.92±0.10⁎⁎,†† 10.18±0.09§, ns
213.75±5.62 167.09±2.51⁎⁎ 198.64±4.89‡, ns 153.30±1.33⁎⁎,†† 192.86±3.99§, ns
3.56±0.25 2.21±0.03⁎⁎ 2.85±0.12‡, ns 2.10±0.02⁎⁎,† 2.92±0.09§, ns
5.10±0.16 3.69±0.03⁎⁎ 4.86±0.08‡, ns 3.57±0.02⁎⁎,† 4.81±0.05§, ns
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01; ⁎⁎ p≤.001 compared with Group I (control); †p≤.01 and comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
††
p≤.001 compared with Group IIA; ‡p≤.001
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Table 4 Effect of CcMtE on tissue biochemistry Treatment
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
Protein (mg/g)
Sialic acid (mg/g)
Fructose (mg/g)
Cauda epididymides
Seminal vesicle
Ventral prostate
Cauda epididymides
Seminal vesicle
Ventral prostate
Seminal vesicle
238.41±2.19
208.64±4.08
189.86±1.52
5.64±0.25
5.22±0.21
5.31±0.07
4.90±0.18
200.42±2.00⁎⁎
183.31±2.22⁎⁎
162.20±1.33⁎⁎
3.75±0.04⁎⁎
4.05±0.08⁎⁎
4.26±0.06⁎⁎
3.72±0.07⁎⁎
229.97±5.11‡,ns
201.75±3.10‡,ns
180.20±2.94‡,ns
5.46±0.04‡,ns
5.07±0.06‡,ns
5.19±0.30‡,ns
4.83±0.20‡,ns
189.31±1.78⁎⁎,††
174.64±0.44⁎⁎,†
150.87±1.25⁎⁎,††
3.28±0.05⁎⁎,††
3.72±0.05†
3.96±0.06⁎⁎,†
3.18±0.09⁎⁎,††
226.86±6.59§,ns
199.75±2.31§,ns
179.19±2.62§,ns
5.28±0.12§,ns
4.98±0.12§,ns
5.10±0.02§,ns
4.80±0.14§,ns
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01 and ⁎⁎ p≤.001 compared with Group I (control); †p≤.01 and ††p≤.001 compared with Group IIA; ‡p≤.001 comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
glycogen [13] and cholesterol [14] in the testes and fructose [15] in the seminal vesicle were also estimated. 2.5.6. Blood and serum biochemistry Blood from each animal was collected by cardiac puncture. Total red blood cell (RBC) and white blood cell (WBC) count [16], hemoglobin [17] and hematocrit [18] values were recorded. Blood urea, sugar [19], serum protein, cholesterol, phospholipid, bilirubin, glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were also recorded simultaneously using reagent kits (SPAN Diagnostics, Surat, India). 2.5.7. Histopathology and testicular cell dynamics Tissues were fixed in Bouin's fluid, sectioned at thickness of 5 μm and stained with hematoxylin and eosin for histopathological studies and discrimination of the stages of spermatogenesis by the method of Leblond and Clermont [20]. The Sertoli cells, spermatogonia, primary spermatocytes (preleptotene and pachytene), secondary spermatocytes and round spermatids were counted under magnification of ×800 [21]. Interstitial cell types such as mature, degenerating and fibroblast were also estimated using a differential cell count.
2.5.8. Statistical analysis Data are expressed as mean±SEM and analyzed for statistical significance using one-way ANOVA. Results were significant at p≤.001. 3. Results 3.1. Body and organ weights No significant changes were observed in the body weight of CcMtE-treated rats, whereas the weights of the testes, epididymides, seminal vesicles and ventral prostate of the CcMtE-treated group decreased significantly (p≤.001) but were restored after another 60 days of recovery period (Table 1). 3.2. Sperm dynamics and fertility index The sperm density in the testis and cauda epididymides and sperm motility in the cauda epididymis were found to be significantly reduced (p≤.001) after CcMtE administration (Table 2). Decline in fertility was 69.0% and 76.0% at dose levels of 100 and 200 mg/rat/day, respectively, in the extracttreated groups. The number of implantation sites and viable fetuses were markedly decreased (p≤.001) in Group II,
Table 5 Effect of CcMtE on blood analysis Treatment
RBC (million/mm3)
WBC (per mm3)
Hematocrit (%)
Hemoglobin (g/100mL)
Blood sugar (mg/dL)
Blood urea (mg/dL)
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
5.26±0.25 5.05±0.13, 5.32±0.18, 4.95±0.15, 5.21±0.14,
8548.75±50.00 8440.00±60.00, ns 8525.00±65.00, ns 8400.00±80.00, ns 8515.00±50.00, ns
45.60±1.92 44.05±1.51, ns 45.20±1.40 43.07±1.75, ns 45.00±0.99, ns
14.25±0.75 13.80±0.15, ns 14.15±0.08, ns 13.70±0.20, ns 14.05±0.10, ns
90.54±2.98 84.04±1.69, ns 90.05±1.12, ns 85.67±1.86, ns 88.27±0.98, ns
44.21±3.90 43.78±0.92, ns 44.11±0.85, ns 45.30±0.76, ns 42.20±1.21, ns
Values are mean±SEM (n=10). ns, nonsignificant.
ns ns ns ns
37.10±0.66, ns 35.10±1.85, ns 0.74±0.05, ns 45.26±1.02, ns
108.56±2.85, ns
104.21±2.06, ns
15,788.87±122.22, ns
16,133.31±177.82, ns
Values are mean±SEM (n=10). HDL, high-density lipoprotein; LDL; low-density lipoprotein; VLDL, very low density lipoprotein; ns, nonsignificant.
17.00±0.33, ns 41.80±0.56, ns 85.01±1.65, ns
105.55±3.33, ns 15,988.86±166.66, ns
93.41±2.60, ns
35.77±0.90, ns 32.00±2.10, ns 0.69±0.085, ns 53.95±0.58, ns 16.43±0.45, ns 38.18±1.82, ns 82.16±2.25, ns
107.27±1.82, ns 15,888.87±88.89, ns
91.44±1.97, ns
36.33±0.26, ns 36.70±2.10, ns 0.76±0.02, ns 46.38±1.16, ns 17.50±0.50, ns 41.66±1.66, ns 87.50±2.50, ns
34.44±0.26, ns 33.50±1.45, ns 0.68±0.06, ns 49.03±1.57, ns 17.33±0.50, ns 40.90±2.72, ns
38.77±1.17 0.78±0.05 44.19±3.25 104.44±4.44 16,188.86±144.44
Control rat testes show different stages of spermatogenesis in the seminiferous tubule. Spermatids are located peripherally near the lumen of the seminiferous tubule prior
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
3.7. Histological studies
Total protein (mg/dL)
C. cyminum extract brought about a significant reduction in most of the germinal cell types. Spermatogonia decreased significantly at both dose levels of 100 and 200 mg/rat/day. The preleptotene and pachytene spermatocytes were also reduced significantly (pb.01) (Fig. 5). The number of mature Leydig cells decreased significantly (pb.01), whereas significant elevation was seen in degenerating Leydig cells at the 100 and 200 mg/rat/day dose levels (Fig. 6).
Table 6 Effect of CcMtE on serum analysis
3.6. Testicular cell dynamics
Treatment
Total cholesterol (mg/dL)
Blood estimation (i.e., RBC, WBC, hemoglobin, hematocrit, blood sugar and blood urea) was found to be within the normal range as with control animals (Table 5). Serum protein, cholesterol, phospholipid, bilirubin, GOT and GPT contents of serum did not change significantly (Table 6).
90.47±1.97, ns
Phospholipids (mg/dL)
3.5. Hematological and serological parameters
86.66±3.30, ns
Triglyceride (mg/dL)
3.4.2. Epididymis, seminal vesicle and ventral prostate In response to CcMtE treatment, a significant reduction in the sialic acid and total protein contents of the epididymis, seminal vesicle and ventral prostate was observed in all the animals in Groups II and III (Tables 3 and 4). A marked reduction (p≤.01) in the fructose of seminal vesicle was also observed (Table 4). All the parameters were found to be within the normal range after the 60-day recovery period.
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89.29±2.81, ns
HDL-cholesterol (mg/dL)
VLDL-cholesterol (mg/dL)
3.4.1. Testis Following CcMtE administration to rats, testicular cholesterol content increased, whereas protein content decreased significantly (p≤.001). The levels of sialic acid and glycogen of CcMtE-treated rats decreased significantly (p≤.001) (Table 3).
17.75±0.25
3.4. Tissue biochemistry of reproductive organs
42.49±0.83
LDL-cholesterol (mg/dL)
Bilirubin (%)
Serum testosterone level of CcMtE-treated rats decreased highly significantly (p≤.001) with respect to the control group. The decrease in serum testosterone concentration recovered to normal values following the withdrawal period of 60 days. Contrary to this, no significant differences were found in the level of serum estradiol, follicle-stimulating hormone and luteinizing hormone between the controls and treated rats in all the treatment as well as in the recovery groups (Figs. 1–4).
88.75±1.25
3.3. Serum hormonal concentration
95.82±1.78
GOT (U/L)
GPT (U/L)
whereas no implantation sites or viable fetuses were recorded in the female rats that cohabited with Group III-treated male rats; the placebo group was found to be normal. After the recovery period, fertility was 91.3% and 86.7% in rats treated with CcMtE 100 and 200 mg/rat/day, respectively.
38.66±1.27
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Fig. 5. Effect of CcMtEs on testicular cell dynamics.
to being released, and the lumen contains sperm (Fig. 7). Administration of C. cyminum extract at 100 and 200 mg/ rat/day dose levels for 60 days produced histoarchitectural alterations in the seminiferous tubules and arrested spermatogenesis (Figs. 8 and 9). However, the alterations recovered after 60 days of recovery period (Figs. 10 and 11).
4. Discussion The body weight of CcMtE-treated rats remained unchanged throughout the experiment. Regular body weight
examination was done to obtain the necessary health information of the animals for interpretation of reproductive effects [22]. The weight of reproductive organs, specifically the testes, is important to confirm reproductive competence in male animals [23]. The results of the present study showed that CcMtE treatment did not cause alterations in the weight of the vital organs but caused marked decrease in testicular weight, which may be attributed to the alterations in kinetics of spermatogenesis due to the loss of germinal cell [24]. Decreased weight of the accessory sex gland indicates glandular tissue atrophy [25]. Reduced protein content may be another reason for the diminutive growth rate of any
Fig. 6. Effect of CcMtEs on Leydig cell counts.
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Fig. 7. Section of control rat testis. Different stages of spermatogenesis can be seen in the seminiferous tubule. Spermatids are located peripherally near the lumen of the seminiferous tubule prior to being released, and the lumen contains sperm.
organ, which is proportional to its protein content [26] and also due to the absence of the spermatogenic elements in the testes. The accessory sex organs (i.e., epididymis, seminal vesicle and ventral prostate) are androgen-dependent, relying on testosterone for their growth and function [27]. Oral CcMtE administration reduces the level of serum testosterone, testicular weight and other accessory sex organ weights of the experimental rats. Their weight loss reflects a decline in bioavailability and production of androgens [28]. Treatment with CcMtE was efficient in suppressing the male reproductive potency because it reduces sperm dynamics. Inadequate sperm concentrations with sluggish or immotile sperm tend not to penetrate the cervical mucosa. Thus, sterility results from the failed fertilization of the ova [2]. A significant decrease in serum testosterone concentration might be due to the adverse effect of CcMtE treatment on the testicular hormonal milieu [29]. C. cyminum extract directly affects the testicular hormonal system without changing the levels of other endocrine hormones [2], which is also supported by the decreased number of mature Leydig cells, the main source of testosterone. Significantly decreased glycogen content may be due to the inhibition of glycolysis during spermatogenesis [1]. The developing spermatozoa consume glucose as well as fructose
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Fig. 8. Rat testis treated with 100 mg/rat/day for 60 days. The reduced size of seminiferous tubule and inhibition of spermatogenesis can be seen. Degenerated Leydig cells are also seen, and the lumen contains cellular debris.
as energy source. Fructose is the main source of energy of spermatozoa. [30]. The results from this study indicate that CcMtE caused a decrease in the fructose level because the inhibition of fructose and the decrease in sperm motility are always correlated [31]. The reduced content of sialic acid might alter the acrosomal membrane structural integrity, which eventually affects the metabolism, motility and fertilizing capacity of spermatozoa [32]. Testicular cholesterol plays a pivotal role as a precursor molecule in steroid hormone synthesis [33]. Increased accumulation of the testicular cholesterol suggests that it is not used in the testosterone biosynthesis, which corroborates that CcMtE inhibits steroidogenesis [29]. The nontoxic behavior of the C. cyminum was confirmed by unaltered hematological indices and clinical parameters (i.e., serum protein, cholesterol, phospholipids, triglyceride, high-density lipoprotein cholesterol, very low density lipoprotein cholesterol, low-density lipoprotein cholesterol, bilirubin, GOT and GPT), which indicate normal functioning of vital organs. From the present study, it can be concluded that C. cyminum is capable of suppressing male fertility that can be reversed after the cessation of treatment without altering the general metabolism. Hence, the possible contraceptive efficacy of C. cyminum seed extract should be considered in the development of a potent herbal male contraceptive.
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Fig. 9. Rat testis treated with 200 mg/rat/day for 60 days. Increased interstitial space with reduced amount of connective tissue and decreased diameter of seminiferous tubule can be seen. Degenerative Leydig cells can be seen as well.
Acknowledgments The authors are thankful to the head of the Department of Zoology and the coordinator of the Center of Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur, for providing necessary facilities and to the University Grant Commission, Regional Office, Bhopal, Madhya Pradesh, India, for financial support.
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Fig. 10. Recovered rat testis after treatment with 100 mg/rat/day for 60 days. The seminiferous tubule show normal diameter with well-developed spermatogenesis and spermatozoa can be seen in the lumen. [6] Turner RA. Screening methods in pharmacology. New York: Academic Press; 1965. [7] Prasad MRN, Chinoy NJ, Kadam KM. Changes in succinate dehydrogenase level in rat epididymis under normal and altered physiologic conditions. Fertil Steril 1972;23:180–90. [8] Belanger A, Carnons RV. Simultaneous radioimmunoassay of progestins, androgens and estrogens in rat testis. J Steroid Biochem 1980;13:185–92. [9] Moudgal NR, Madhwaraj HG. Pituitary gonadotropin. In: Jaffe BM, Behrman HR, editors. Methods of hormone radioimmunoassay. New York: Academic Press; 1974, pp. 57–85. [10] Strom JO, Theodorsson A, Theodorsson E. Substantial discrepancies in 17beta-oestradiol concentrations obtained with three different commercial direct radioimmunoassay kits in rat sera. Scand J Clin Lab Invest 2008;68:806–13. [11] Lowry OH, Rosenburg NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:265–75. [12] Warren L. A thiobarbituric assay of sialic acid. J Biol Chem 1959;234:1971–5. [13] Montgomery R. Determination of glycogen. Arch Biochem Biophys 1957;67:378–81. [14] Zlatkis A, Zak B, Boyle AJ. A new method for the direct determination of serum cholesterol. J Lab Clin Med 1953;41:486–92. [15] Mann T. Fructose, polyols and organic acids. In: Nam T, editor. The biochemistry of semen and of the male reproductive tract. London. Methuen and Company; 1964, pp. 273–344. [16] Lynch JM, Raphel SS, Melier LD, Spare PD. In: Wood MJH, editor. Collection of blood sample and haemocytometry. Red cell count, white cell count. Medicinal laboratory technology and clinical pathology. Tokyo: WB Saunders Company Igakar Sohim; 1969, pp. 626–62. [17] Crossby WH, Munn JI, Furth FW. Standardizing a method for clinical hemoglobinometry. J US Armed Force Med 1954;5:695–703.
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Fig. 11. Recovered rat testis after treatment with 200 mg/rat/day for 60 days. The seminiferous tubule shows active spermatogenesis, with all successive spermatogenic cells; the lumen is filled with spermatozoa.
[18] Strumia MM, Sample AB, Hart ED. An improved microhematocrit method. Am J Clin Pathol 1954;24:1016–24. [19] Astoor AM, King EJ. Simplified colorimetric blood sugar method. Biochem J 1954;56:44–7.
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[20] Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 1952;55:548–73. [21] Abercrombie M. Estimation of nuclear population from microtome sections. Anat Rec 1946;19:238–41. [22] U.S. Environmental Pollution Agency. Health effects test guidelines OPPTS 870.3800. Reproduction and fertility effects. U.S. Environmental Protection Agency; 1998, pp. 1–12. [23] Ong CN, Chia HMS. Biomarkers for male reproductive health hazards: Are they available? Toxicol Lett 2002;134:17–30. [24] Shaha C. Advances in experimental medicine and biology. In: Cheng CY, editor. Molecular mechanisms in spermatogenesis. New York: Springer; 2009, pp. 42–64. [25] Reiter E, McNamara M, Cosset J, Hennen G. Expression and functionality of leuteinising hormone/chorions. Gonadotropin receptor in rat the prostate. Endocrinol 1995;136:917–23. [26] Gupta RS, Kachhawa JBS, Sharma A. Effect of methanolic extract of Dendrophthoe falcata stem on reproductive function of male albino rats. J Herb Pharmacother 2008;7:1–13. [27] Das R, Roy S, Bandopadhyaya G. Effect of castration and testosterone treatment on spermatozoa and accessory genital organs of rats. Contraception 1973;8:471–84. [28] Gupta RS, Rehwani H, Khushalini V, Tanwar K, Joshi YC. Antispermatogenic effects of Parkinsonia aculeata stem-bark in male rats. Pharm Biol 2007;45:1–8. [29] Das KK, Dasgupta S. Effect of nickel sulfate on testicular steroidogenesis in rats during protein restriction. Environ Health Perspect 2002;110:923–6. [30] Ponglowhapan S, Birgitta EG, Catharina LF. Influence of glucose and fructose in the extender during long-term storage of chilled canine semen. Theriogenology 2004;62:1498–517. [31] Jadot-Van de Casseye M, Schoysman R, Smets G, Gepts W. Ultrastructural aspects of asthenospermia. Int J Androl 1980;3:15–22. [32] Chinoy NJ, Bhattacharya S. Effect of chronic administration of aluminium chloride on reproductive function of testes and some accessory sex organs of male mice. Ind J Environ Toxicol 1997;35:133–8. [33] Gupta SK. Current status of development and clinical testing of male intravasal contraceptives. International Symposium on Male Contraception: Present and Future. New Delhi (India); 1995.
Original research article
Evaluation of reversible contraceptive activities of Cuminum cyminum in male albino rats Radhey S. Gupta⁎, Poonam Saxena, Rajnish Gupta, Jai B.S. Kachhawa Reproductive Physiology Section, Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 8 March 2010; revised 25 October 2010; accepted 25 October 2010
Abstract Background: The aim of the present study was to evaluate the contraceptive efficacy of Cuminum cyminum (jeera) seeds in male albino rats. Study Design: C. cyminum methanol extract (CcMtE) at dose levels of 100 and 200 mg/rat/day was orally administered to male rats for 60 days. The effect of the treatment on reproductive organs and fertility was investigated. Recovery and toxicity studies were also carried out. Results: C. cyminum methanol extract fed to male rats for 60 days did not cause any alterations in the body weight, whereas the weight of testes, epididymides, seminal vesicles and ventral prostate were significantly reduced (p≤.001). Animals treated with CcMtE showed a marked reduction in sperm density in the cauda epididymis and testes and sperm motility in the cauda epididymis. Reduction in fertility was 69.0% and 76.0% in 100 and 200 mg/rat/day dose levels, respectively. The circulatory hormones were also reduced significantly. Testicular biochemical analysis of protein, sialic acid, glycogen, ascorbic acid and fructose indicated a marked decline, whereas testicular cholesterol content was significantly increased, which showed altered biochemistry of the reproductive organs. After CcMtE treatment, significant decreases (p≤.001) were observed in the number of testicular cells (i.e., spermatogonia, primary spermatocytes [preleptotene and pachytene], secondary spermatocytes and round spermatids); nonsignificant change was observed in the Sertoli cell count. The treatment had no effect on levels of serum protein, cholesterol, bilirubin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea and hematological indices. Conclusions: The present study shows that C. cyminum treatment resulted in the inhibition of spermatogenesis and fertility without producing apparent toxic effects. © 2011 Elsevier Inc. All rights reserved. Keywords: Cuminum cyminum; Testis; Spermatogenesis; Epididymides; Sperm; Fertility
1. Introduction Countless efforts have been made to obtain male antifertility agents from plants [1,2]. Cuminum cyminum (Apiaceae), an annual herb with brownish or grayish yellow color seeds, is widely used as an anticarcinogenic, stomachache and astringent agent [3]. Phytochemical studies revealed the presence of some terpenes, glycosides, cumaldehyde, myrcene, α-phellandrene, α-pinene, β-pinene, cyminal, α-terpene, y-terpinene and p-cymene in C. cyminum. A high level of cumaldehyde (4-propan-2ylbenzaldehyde) is found in C. cyminum [4]. In the present study, we investigated the effect of C. cyminum on the
reproductive system of the male albino rats in order to evaluate the potential of this plant as a male fertilityregulating agent. 2. Materials and methods Male albino rats of Wistar strain weighing 150–180 g were used for the present study. Animals were kept in standard conditions of 12-h light/12-h dark cycle and 22°C±2°C temperature. Standard pellet diet (Ashirvad Food Industries, Chandigarh, India) and tap water were provided ad libitum. 2.1. Ethical aspects
⁎ Corresponding author. Department of Zoology, University of Rajasthan, Jaipur 302004, India. Fax: +91 141 2701137. E-mail address: [email protected] (R.S. Gupta). 0010-7824/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2010.10.013
The study was approved by the Departmental Ethical Committee, University of Rajasthan, Jaipur, India. The
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Table 1 Effect of CcMtE on body and organ weights Treatment
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
Body weight (g)
Testes (mg/100 g body weight)
Cauda epididymides (mg/100 g body weight)
Seminal vesicle (mg/100 g body weight)
Ventral prostate (mg/100 g body weight)
Initial
Final
160.00±7.60 172.00±7.39 155.00±7.07
225.00±14.14 220.00±7.80, ns 230.00±4.90, ns
1376.98±13.49 962.89±7.34⁎⁎ 1321.37±16.44‡, ns
460.22±9.54 319.53±1.35⁎⁎ 437.02±6.80‡, ns
456.26±7.46 361.64±6.68⁎⁎ 431.19±5.24‡, ns
364.13±7.89 283.44±1.67⁎⁎ 337.68±6.57‡, ns
165.00±4.53 170.00±5.00
210.00±5.50, ns 235.00±5.38, ns
849.88±5.23⁎⁎,†† 1308.24±16.76§, ns
297.99±2.39⁎⁎,†† 426.80±6.39§, ns
313.12±2.12⁎⁎,†† 422.02±4.56§, ns
253.30±1.56⁎⁎,†† 326.53±9.61§, ns
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01 and ⁎⁎p≤.001 compared with Group I (control); †p≤.01 and †† p≤.001 compared with Group IIA; ‡p≤0.001 comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
Table 2 Effect of CcMtE on sperm dynamics and fertility Treatment
Sperm motility (%)
Sperm density (million/mL)
Cauda epididymides
Testes
Cauda epididymides
Fertility (%)
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
76.21±1.96 33.19±0.28⁎⁎ 67.05±2.46‡, ns 30.67±0.59⁎⁎,† 65.89±2.74§, ns
4.65±0.25 1.85±0.10⁎⁎ 4.37±0.15‡, ns 1.32±0.12⁎⁎,† 3.97±0.40§, ns
44.70±2.60 10.70±1.21⁎⁎ 41.85±1.20‡, ns 9.15±0.10⁎⁎,†† 40.40±2.77§, ns
100 69.0 91.3 76.0 86.7
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01 and ⁎⁎ p≤.001 compared with Group I (control); †p≤.01 and ††p≤.001 compared with Group IIA; ‡p≤.001 comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
guidelines of the Indian National Science Academy of New Delhi [5] for the use of experimental animals were followed. 2.2. Plant material Fresh seeds of C. cyminum were collected from the local market of Jaipur and were identified and authenticated in the
Department of Botany, University of Rajasthan, Jaipur. One hundred percent methanol extract was prepared by Soxhlet extraction for 72 h. Methanol was evaporated under reduced pressure to obtain the concentrated oily extract (15 g), which was then suspended in olive oil just before experimentation (C. cyminum methanol extract [CcMtE]).
Fig. 1. Effect of CcMtE on serum testosterone.
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Fig. 2. Effect of CcMtE on serum estradiol.
2.3. Acute toxicity studies The study of acute toxicity of the CcMtE was performed according to the method of Turner [6]. The LD50 value of the experimental drug was 1500 mg/kg body weight of male albino rats. 2.4. Treatment protocol and autopsy schedule Male albino rats of proven fertility were grouped as follows: Group I: control rats received 0.5 mL/day of the vehicle (i.e., olive oil).
Group IIA: rats treated with CcMtE at 100 mg/rat/day dose level. Group IIB: recovery after treatment of CcMtE at the 100 mg/rat/day dose level from Group IIA. Group IIIA: rats treated with CcMtE at 200 mg/rat/day dose level. Group IIIB: recovery after treatment of CcMtE at 200 mg/rat/day dose level from Group IIIA. An olive oil suspension of extract was prepared and administered daily by oral gavage for 60 days. Twenty-four hours after the last dose at Day 61, the animals were autopsied under light ether anesthesia.
Fig. 3. Effect of CcMtE on serum luteinizing hormone.
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Fig. 4. Effect of CcMtE on serum follicle-stimulating hormone.
2.5.2. Sperm parameters Sperm motility in the cauda epididymis and sperm density in the epididymis and testes were assessed by the method of Prasad et al. [7].
2.5.4. Serum hormonal assay The concentrations of serum testosterone, luteinizing hormone, follicle-stimulating hormone and estradiol levels were assayed from samples according to the standard protocol using radioimmunoassay. The serum concentration of testosterone was measured according to standard methods [8]; the sensitivity of the assay was 10 pg/mL. Luteinizing hormone and follicle-stimulating hormone were measured by the method of Moudgal and Madhwaraj [9], and serum levels of estradiol were analyzed using 125 I radioimmunoassay kit, which has been previously tested in rat sera against other radioimmunoassays [10].
2.5.3. Fertility test The mating test was performed from Days 55 to 60. The male rats were cohabited with proestrous female rats at a ratio of 1:3. Successful mating was confirmed by the presence of spermatozoa in the vaginal smear. The mated females were separated to note the implantation site on Day 16 of pregnancy through laparotomy.
2.5.5. Tissue biochemistry After autopsy, reproductive organs (i.e., testes, epididymis, seminal vesicles and ventral prostate) were removed, cleared of fat and connective tissue, weighed on electronic balance and kept at −4°C until assayed for various biochemical parameters. The estimation of protein [11] and sialic acid [12] were performed in the testis, epididymis, seminal vesicle and ventral prostate. Concentrations of
2.5. Parameters 2.5.1. Body weight and organ weights Initial and final body weights were recorded. The testes, epididymis, seminal vesicle and ventral prostate were excised, carefully removed from the surrounding fat and connective tissue and weighed on an electronic balance.
Table 3 Effect of CcMtE on tissue biochemistry Treatment
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
Testis Cholesterol (mg/g)
Protein (mg/g)
Glycogen (mg/g)
Sialic acid (mg/g)
9.43±0.31 12.74±0.17⁎⁎ 8.60±0.07‡, ns 13.92±0.10⁎⁎,†† 10.18±0.09§, ns
213.75±5.62 167.09±2.51⁎⁎ 198.64±4.89‡, ns 153.30±1.33⁎⁎,†† 192.86±3.99§, ns
3.56±0.25 2.21±0.03⁎⁎ 2.85±0.12‡, ns 2.10±0.02⁎⁎,† 2.92±0.09§, ns
5.10±0.16 3.69±0.03⁎⁎ 4.86±0.08‡, ns 3.57±0.02⁎⁎,† 4.81±0.05§, ns
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01; ⁎⁎ p≤.001 compared with Group I (control); †p≤.01 and comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
††
p≤.001 compared with Group IIA; ‡p≤.001
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Table 4 Effect of CcMtE on tissue biochemistry Treatment
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
Protein (mg/g)
Sialic acid (mg/g)
Fructose (mg/g)
Cauda epididymides
Seminal vesicle
Ventral prostate
Cauda epididymides
Seminal vesicle
Ventral prostate
Seminal vesicle
238.41±2.19
208.64±4.08
189.86±1.52
5.64±0.25
5.22±0.21
5.31±0.07
4.90±0.18
200.42±2.00⁎⁎
183.31±2.22⁎⁎
162.20±1.33⁎⁎
3.75±0.04⁎⁎
4.05±0.08⁎⁎
4.26±0.06⁎⁎
3.72±0.07⁎⁎
229.97±5.11‡,ns
201.75±3.10‡,ns
180.20±2.94‡,ns
5.46±0.04‡,ns
5.07±0.06‡,ns
5.19±0.30‡,ns
4.83±0.20‡,ns
189.31±1.78⁎⁎,††
174.64±0.44⁎⁎,†
150.87±1.25⁎⁎,††
3.28±0.05⁎⁎,††
3.72±0.05†
3.96±0.06⁎⁎,†
3.18±0.09⁎⁎,††
226.86±6.59§,ns
199.75±2.31§,ns
179.19±2.62§,ns
5.28±0.12§,ns
4.98±0.12§,ns
5.10±0.02§,ns
4.80±0.14§,ns
Values are mean±SEM (n=10). Levels of significance: ns, nonsignificant; ⁎p≤.01 and ⁎⁎ p≤.001 compared with Group I (control); †p≤.01 and ††p≤.001 compared with Group IIA; ‡p≤.001 comparison between Group IIA and Group IIB; §p≤.001 comparison between Group IIIA and Group IIIB.
glycogen [13] and cholesterol [14] in the testes and fructose [15] in the seminal vesicle were also estimated. 2.5.6. Blood and serum biochemistry Blood from each animal was collected by cardiac puncture. Total red blood cell (RBC) and white blood cell (WBC) count [16], hemoglobin [17] and hematocrit [18] values were recorded. Blood urea, sugar [19], serum protein, cholesterol, phospholipid, bilirubin, glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were also recorded simultaneously using reagent kits (SPAN Diagnostics, Surat, India). 2.5.7. Histopathology and testicular cell dynamics Tissues were fixed in Bouin's fluid, sectioned at thickness of 5 μm and stained with hematoxylin and eosin for histopathological studies and discrimination of the stages of spermatogenesis by the method of Leblond and Clermont [20]. The Sertoli cells, spermatogonia, primary spermatocytes (preleptotene and pachytene), secondary spermatocytes and round spermatids were counted under magnification of ×800 [21]. Interstitial cell types such as mature, degenerating and fibroblast were also estimated using a differential cell count.
2.5.8. Statistical analysis Data are expressed as mean±SEM and analyzed for statistical significance using one-way ANOVA. Results were significant at p≤.001. 3. Results 3.1. Body and organ weights No significant changes were observed in the body weight of CcMtE-treated rats, whereas the weights of the testes, epididymides, seminal vesicles and ventral prostate of the CcMtE-treated group decreased significantly (p≤.001) but were restored after another 60 days of recovery period (Table 1). 3.2. Sperm dynamics and fertility index The sperm density in the testis and cauda epididymides and sperm motility in the cauda epididymis were found to be significantly reduced (p≤.001) after CcMtE administration (Table 2). Decline in fertility was 69.0% and 76.0% at dose levels of 100 and 200 mg/rat/day, respectively, in the extracttreated groups. The number of implantation sites and viable fetuses were markedly decreased (p≤.001) in Group II,
Table 5 Effect of CcMtE on blood analysis Treatment
RBC (million/mm3)
WBC (per mm3)
Hematocrit (%)
Hemoglobin (g/100mL)
Blood sugar (mg/dL)
Blood urea (mg/dL)
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
5.26±0.25 5.05±0.13, 5.32±0.18, 4.95±0.15, 5.21±0.14,
8548.75±50.00 8440.00±60.00, ns 8525.00±65.00, ns 8400.00±80.00, ns 8515.00±50.00, ns
45.60±1.92 44.05±1.51, ns 45.20±1.40 43.07±1.75, ns 45.00±0.99, ns
14.25±0.75 13.80±0.15, ns 14.15±0.08, ns 13.70±0.20, ns 14.05±0.10, ns
90.54±2.98 84.04±1.69, ns 90.05±1.12, ns 85.67±1.86, ns 88.27±0.98, ns
44.21±3.90 43.78±0.92, ns 44.11±0.85, ns 45.30±0.76, ns 42.20±1.21, ns
Values are mean±SEM (n=10). ns, nonsignificant.
ns ns ns ns
37.10±0.66, ns 35.10±1.85, ns 0.74±0.05, ns 45.26±1.02, ns
108.56±2.85, ns
104.21±2.06, ns
15,788.87±122.22, ns
16,133.31±177.82, ns
Values are mean±SEM (n=10). HDL, high-density lipoprotein; LDL; low-density lipoprotein; VLDL, very low density lipoprotein; ns, nonsignificant.
17.00±0.33, ns 41.80±0.56, ns 85.01±1.65, ns
105.55±3.33, ns 15,988.86±166.66, ns
93.41±2.60, ns
35.77±0.90, ns 32.00±2.10, ns 0.69±0.085, ns 53.95±0.58, ns 16.43±0.45, ns 38.18±1.82, ns 82.16±2.25, ns
107.27±1.82, ns 15,888.87±88.89, ns
91.44±1.97, ns
36.33±0.26, ns 36.70±2.10, ns 0.76±0.02, ns 46.38±1.16, ns 17.50±0.50, ns 41.66±1.66, ns 87.50±2.50, ns
34.44±0.26, ns 33.50±1.45, ns 0.68±0.06, ns 49.03±1.57, ns 17.33±0.50, ns 40.90±2.72, ns
38.77±1.17 0.78±0.05 44.19±3.25 104.44±4.44 16,188.86±144.44
Control rat testes show different stages of spermatogenesis in the seminiferous tubule. Spermatids are located peripherally near the lumen of the seminiferous tubule prior
Group I: control or vehicle-treated Group IIA: CcMtE 100 mg/rat/day Group IIB: CcMtE 100 mg/rat/day (recovery) Group IIIA: CcMtE 200 mg/rat/day Group IIIB: CcMtE 200 mg/rat/day (recovery)
3.7. Histological studies
Total protein (mg/dL)
C. cyminum extract brought about a significant reduction in most of the germinal cell types. Spermatogonia decreased significantly at both dose levels of 100 and 200 mg/rat/day. The preleptotene and pachytene spermatocytes were also reduced significantly (pb.01) (Fig. 5). The number of mature Leydig cells decreased significantly (pb.01), whereas significant elevation was seen in degenerating Leydig cells at the 100 and 200 mg/rat/day dose levels (Fig. 6).
Table 6 Effect of CcMtE on serum analysis
3.6. Testicular cell dynamics
Treatment
Total cholesterol (mg/dL)
Blood estimation (i.e., RBC, WBC, hemoglobin, hematocrit, blood sugar and blood urea) was found to be within the normal range as with control animals (Table 5). Serum protein, cholesterol, phospholipid, bilirubin, GOT and GPT contents of serum did not change significantly (Table 6).
90.47±1.97, ns
Phospholipids (mg/dL)
3.5. Hematological and serological parameters
86.66±3.30, ns
Triglyceride (mg/dL)
3.4.2. Epididymis, seminal vesicle and ventral prostate In response to CcMtE treatment, a significant reduction in the sialic acid and total protein contents of the epididymis, seminal vesicle and ventral prostate was observed in all the animals in Groups II and III (Tables 3 and 4). A marked reduction (p≤.01) in the fructose of seminal vesicle was also observed (Table 4). All the parameters were found to be within the normal range after the 60-day recovery period.
103
89.29±2.81, ns
HDL-cholesterol (mg/dL)
VLDL-cholesterol (mg/dL)
3.4.1. Testis Following CcMtE administration to rats, testicular cholesterol content increased, whereas protein content decreased significantly (p≤.001). The levels of sialic acid and glycogen of CcMtE-treated rats decreased significantly (p≤.001) (Table 3).
17.75±0.25
3.4. Tissue biochemistry of reproductive organs
42.49±0.83
LDL-cholesterol (mg/dL)
Bilirubin (%)
Serum testosterone level of CcMtE-treated rats decreased highly significantly (p≤.001) with respect to the control group. The decrease in serum testosterone concentration recovered to normal values following the withdrawal period of 60 days. Contrary to this, no significant differences were found in the level of serum estradiol, follicle-stimulating hormone and luteinizing hormone between the controls and treated rats in all the treatment as well as in the recovery groups (Figs. 1–4).
88.75±1.25
3.3. Serum hormonal concentration
95.82±1.78
GOT (U/L)
GPT (U/L)
whereas no implantation sites or viable fetuses were recorded in the female rats that cohabited with Group III-treated male rats; the placebo group was found to be normal. After the recovery period, fertility was 91.3% and 86.7% in rats treated with CcMtE 100 and 200 mg/rat/day, respectively.
38.66±1.27
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Fig. 5. Effect of CcMtEs on testicular cell dynamics.
to being released, and the lumen contains sperm (Fig. 7). Administration of C. cyminum extract at 100 and 200 mg/ rat/day dose levels for 60 days produced histoarchitectural alterations in the seminiferous tubules and arrested spermatogenesis (Figs. 8 and 9). However, the alterations recovered after 60 days of recovery period (Figs. 10 and 11).
4. Discussion The body weight of CcMtE-treated rats remained unchanged throughout the experiment. Regular body weight
examination was done to obtain the necessary health information of the animals for interpretation of reproductive effects [22]. The weight of reproductive organs, specifically the testes, is important to confirm reproductive competence in male animals [23]. The results of the present study showed that CcMtE treatment did not cause alterations in the weight of the vital organs but caused marked decrease in testicular weight, which may be attributed to the alterations in kinetics of spermatogenesis due to the loss of germinal cell [24]. Decreased weight of the accessory sex gland indicates glandular tissue atrophy [25]. Reduced protein content may be another reason for the diminutive growth rate of any
Fig. 6. Effect of CcMtEs on Leydig cell counts.
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Fig. 7. Section of control rat testis. Different stages of spermatogenesis can be seen in the seminiferous tubule. Spermatids are located peripherally near the lumen of the seminiferous tubule prior to being released, and the lumen contains sperm.
organ, which is proportional to its protein content [26] and also due to the absence of the spermatogenic elements in the testes. The accessory sex organs (i.e., epididymis, seminal vesicle and ventral prostate) are androgen-dependent, relying on testosterone for their growth and function [27]. Oral CcMtE administration reduces the level of serum testosterone, testicular weight and other accessory sex organ weights of the experimental rats. Their weight loss reflects a decline in bioavailability and production of androgens [28]. Treatment with CcMtE was efficient in suppressing the male reproductive potency because it reduces sperm dynamics. Inadequate sperm concentrations with sluggish or immotile sperm tend not to penetrate the cervical mucosa. Thus, sterility results from the failed fertilization of the ova [2]. A significant decrease in serum testosterone concentration might be due to the adverse effect of CcMtE treatment on the testicular hormonal milieu [29]. C. cyminum extract directly affects the testicular hormonal system without changing the levels of other endocrine hormones [2], which is also supported by the decreased number of mature Leydig cells, the main source of testosterone. Significantly decreased glycogen content may be due to the inhibition of glycolysis during spermatogenesis [1]. The developing spermatozoa consume glucose as well as fructose
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Fig. 8. Rat testis treated with 100 mg/rat/day for 60 days. The reduced size of seminiferous tubule and inhibition of spermatogenesis can be seen. Degenerated Leydig cells are also seen, and the lumen contains cellular debris.
as energy source. Fructose is the main source of energy of spermatozoa. [30]. The results from this study indicate that CcMtE caused a decrease in the fructose level because the inhibition of fructose and the decrease in sperm motility are always correlated [31]. The reduced content of sialic acid might alter the acrosomal membrane structural integrity, which eventually affects the metabolism, motility and fertilizing capacity of spermatozoa [32]. Testicular cholesterol plays a pivotal role as a precursor molecule in steroid hormone synthesis [33]. Increased accumulation of the testicular cholesterol suggests that it is not used in the testosterone biosynthesis, which corroborates that CcMtE inhibits steroidogenesis [29]. The nontoxic behavior of the C. cyminum was confirmed by unaltered hematological indices and clinical parameters (i.e., serum protein, cholesterol, phospholipids, triglyceride, high-density lipoprotein cholesterol, very low density lipoprotein cholesterol, low-density lipoprotein cholesterol, bilirubin, GOT and GPT), which indicate normal functioning of vital organs. From the present study, it can be concluded that C. cyminum is capable of suppressing male fertility that can be reversed after the cessation of treatment without altering the general metabolism. Hence, the possible contraceptive efficacy of C. cyminum seed extract should be considered in the development of a potent herbal male contraceptive.
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Fig. 9. Rat testis treated with 200 mg/rat/day for 60 days. Increased interstitial space with reduced amount of connective tissue and decreased diameter of seminiferous tubule can be seen. Degenerative Leydig cells can be seen as well.
Acknowledgments The authors are thankful to the head of the Department of Zoology and the coordinator of the Center of Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur, for providing necessary facilities and to the University Grant Commission, Regional Office, Bhopal, Madhya Pradesh, India, for financial support.
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Fig. 11. Recovered rat testis after treatment with 200 mg/rat/day for 60 days. The seminiferous tubule shows active spermatogenesis, with all successive spermatogenic cells; the lumen is filled with spermatozoa.
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