|Year : 2020 | Volume
| Issue : 3 | Page : 142-147
Ascorbic acid and curcumin alleviate abnormal estrous cycle and morphological changes in cells induced by repeated ultraviolet B radiations in female Wistar rats
Gayatri Rai, Narendra Namdev, Payal Mahobiya
Department of Zoology, Dr Harisingh Gour (A Central University) Vishwavidhyalaya, Sagar (M.P.), India
|Date of Submission||07-Aug-2019|
|Date of Decision||29-Mar-2020|
|Date of Acceptance||11-Apr-2020|
|Date of Web Publication||20-May-2020|
Department of Zoology, Dr Harisingh Gour (A Central University) Vishwavidhyalaya, Sagar (M.P.)
Source of Support: None, Conflict of Interest: None
Objective: To study the protective effect of ascorbic acid and curcumin against the abnormal estrous cycle and morphological changes in cells induced by repeated ultraviolet B (UVB) radiation in female Wistar rats.
Methods: Sixteen female sexually mature Wistar rats weighing 130-150 g and aged 12-16 weeks were randomly divided into four groups. The control group received normal food and water ad libitum. The UVB group was exposed to a dose of 280 nm of UVB radiation for 2 h daily. The UVB+curcumin group received a dose of 280 nm of UVB radiation for 2 h daily and also an oral dose of curcumin (25 mg/kg body weight) daily. The UVB+ascorbic acid group received a dose of 280 nm of UVB radiation for 2 h daily and also an oral dose of ascorbic acid (250 mg/kg body weight) daily. All the treatments last for 15 consecutive days. Body and ovary weight and gonadosomatic index were measured. The stages (proestrus, estrus, metaestrus and diestrus) of the estrous cycle were determined by the cell types observed in the vaginal smear.
Results: UVB radiation caused irregular alterations on the estrous cycle and morphological changes of the female Wistar rat as compared with the control group. Ascorbic acid and curcumin protected UVB-induced estrous phases and their cells. But curcumin showed greater protection than ascorbic acid.
Conclusions: Ascorbic acid and curcumin at low doses can alleviate abnormal estrous cycle and morphological changes in cells induced by UVB radiations in female Wistar rats.
Keywords: Estrous cycle; Ultraviolet-B radiation; Vaginal smear; Ascorbic acid; Curcumin
|How to cite this article:|
Rai G, Namdev N, Mahobiya P. Ascorbic acid and curcumin alleviate abnormal estrous cycle and morphological changes in cells induced by repeated ultraviolet B radiations in female Wistar rats. Asian Pac J Reprod 2020;9:142-7
|How to cite this URL:|
Rai G, Namdev N, Mahobiya P. Ascorbic acid and curcumin alleviate abnormal estrous cycle and morphological changes in cells induced by repeated ultraviolet B radiations in female Wistar rats. Asian Pac J Reprod [serial online] 2020 [cited 2022 Nov 28];9:142-7. Available from: https://www.apjr.net/text.asp?2020/9/3/142/284276
| 1. Introduction|| |
Ultraviolet (UV) rays include a band of electromagnetic radiations with a wavelength from 200 nm to 400 nm. Being present in the sunlight, UV radiations are an important source of energy and have sufficient power to penetrate the body cells; consequently, the chemical and biological effects generated by these radiations are much greater than those by simple heating effects. Radiations emitted and transmitted through different sources are absorbed by the animal body, which tends to be very high levels of environmental toxin. UV radiations bear the ability to induce both positive and negative effects, thereby altering the well-being of both animals. UV radiations are non-ionizing and are classified into three types UVC, UVB, and UVA. UVC (200-280 nm, shortwave length) is more lethal than UVB (280-320 nm, medium wavelength) and UVA (320-400 nm high wavelength),,.
Mammalian germ cells are very sensitive to radiation, which can change the structure of the cell cytoplasm and nucleus  and affect the sensitivity of cells and tissues. The sensitivity of cells and tissues to the effects of radiation vary, with actively dividing cells (blood cells, embryonic cells and cells of gonads) being more sensitive. Cells exposed to radiation are more sensitive than the normal viable cells i.e. cells of the gonads (ovaries and testes), embryonic cells and blood cells. Many factors including age, stress, noise, light, temperature, nutrition, and social relationships impact the estrous cycle length ,,,,. Radiations also affect the reproductive behaviour by inducing imbalance of the production of the hormones by mammalian gonads .
In order to maintain healthy reproductive performance, breeding conditions (light cycle) and timing need to be properly controlled and regulated. The artificial light-dark cycle of an animal facility is critical to the synchronous development of eggs. Female rats should be allowed to stand around one week so as to acclimatize to the artificial conditions of light-dark cycle. The release of luteinizing hormone, a pituitary hormone that induces ovulation, is regulated by the light-dark cycle. The animal rooms are usually of 14 h light and 10 h dark cycles. The reproductive cycle of female rats called estrous cycle is categorized into four main stages i.e. proestrus, estrus, metestrus, and diestrus. The onset of sexual maturity is up to the age of 12 months and the mean cycle length is 4 days . Due to the short cycle length, rats are ideal models to investigate various changes of the reproductive cycle ,.
Previous studies concerned the reproductive system as well as the influence of the estrous cycle on the non-reproductive functions ,,, and vaginal smear cytology was used for the determination of the estrous cycle phases ,. The characterization of each phase is based on the proportion among three types of cells observed in the vaginal smear: epithelial cells, cornified cells, and leukocytes. The collection of vaginal secretions and the use of stained material generally take 1-2 hours or more. Although having much lesser penetrating power, UVB radiations are potent enough to cause DNA damage . UVB radiation is the main stimulator of the constitutive xanthine oxidase and nitric oxide synthase in human endothelial cells , as well as in human keratinocytes .
To the best of our knowledge, the present study is pioneering in its field, which for the first time demonstrates the effect of repeated UVB radiation exposure on the estrous cycle of female Wistar rats. In the present study, we also investigated the protective effect of antioxidants (ascorbic acid and curcumin) against the morphological changes in cells induced by UVB radiation.
| 2. Methods and Materials|| |
All the chemicals and reagents used were of analytical grade. Ascorbic acid was purchased from Sigma-Aldrich Co., USA. Curcumin was obtained from Himedia, India and crystal violet and rest of the chemicals used were obtained from Central Drug House (P) Ltd, New Delhi, India.
2.2. UV irradiation
UVB light (TL 20W/01 UVB Narrowband made in Germany), which emitted UVB in the range of 280nm (UVB), was used as the source to irradiate the Wistar rats. The irradiance was two hours daily for a period of 15 days .
2.3. Experimental animals and treatment
Female adult Wistar rats weighing 130-150 g and aged 12-16 weeks were purchased from the College of Veterinary Sciences and Animal Husbandry Mhow (22.55° N, 75.75° E, M.P), India. All animals (n=16) were housed in plastic cages and were fed on standard laboratory diet daily food and water ad libitum. Rats were kept on laboratory conditions, with standard temperature (20±2) °C, relative humidity of (40%–60%) and 12h/12h light and dark cycle.
The experimental rats were randomly divided into four groups, with 4 rats in each group. The control group received normal food and water ad libitum. The UVB group received a dose of 280 nm of UVB radiation for 2 h daily for a period of 15 days. The UVB+curcumin group received a dose of 280 nm of UVB radiation for 2 h daily and also an oral dose of curcumin (25 mg/kg body weight) daily for a period of 15 days. The UVB+ascorbic acid group received a dose of 280 nm of UVB radiation for 2 h daily and also an oral dose of ascorbic acid (250 mg/kg body weight) daily for a period of 15 days ,,.
2.4. Sample collection
At the end of the experiment, animals were anesthetized and sacrificed by cervical dislocation; ovaries were dissected out, washed in ice-cold phosphate buffer saline and stored at -20 °C for further analysis.
2.5. Measurement of body and ovary weight
Measurement of body and ovary weight was done with an electronic balance (Sartorius, BP210 S). Body weight measurement was performed before and after the experiment. Ovary weight measurement was performed after the experiment.
2.6. Calculationof gonadosomatic index
Calculation of gonadosomatic index between body weight and gonad weight individually was done by the following formula.
Gonadosomatic index = Gonad weight × 100/Body weight
2.7. Estrous cycle
Estrous cycle, the main reproductive cycle of non-primate vertebrates such as mice, rats, horses, etc represented the cyclic pattern of the ovarian activity that allowed females to go from a period of reproductive receptivity to non receptivity, eventually leading to pregnancy after successful mating. In rodents such as rat the estrous occurred after every 4-5 days, with sequential stages of proestrus, estrus, metaestrus and diestrus which lasted for 1, 1, 1 and 2 days, respectively. The stages of the estrous cycle were estrus, metaestrus, diestrus and proestrus . These stages occurred in each cycle and in a sequential manner. The day of estrus was usually designated as day 1 of the cycle. The stages of the estrous cycle were best determined by the cell types observed in the vaginal smear. Normally, vaginal smears should be examined in the morning (08:00 to 09:00 a.m.).
Sexually mature female animals were selected and vaginal smears were collected by using a sterilized micropipette. The micropipette was filled with a small amount of double-distilled water and was then inserted into the vagina of the female rat. The vagina was flushed two to three times with the double-distilled water and then the fluid was placed onto a glass slide. The fluid was equally distributed onto the glass slides and the smear was stained with crystal violet (1%) and observed under a light microscope with 10× and 40× magnification (Carl Zeiss, Germany).
2.8. Statistical analysis
All statistical analysis was performed by using one-way analysis of variance. The data were expressed as mean±standard deviation (mean±SD). Dunnett test was applied for the comparison between the control group and each treated group individually. P < 0.05 was considered statistically different.
2.9. Ethics statement
This study was approved by the Department of Pharmaceutical Sciences Dr. Harisingh Gour Vishwavidyalaya (A Central University) Sagar (M.P.), India (Ethical registration No. 379/CPCSEA/IAEC- 2018/2017). Also, international guidelines were followed for the care and use of laboratory animals.
| 3. Results|| |
3.1. Body weight, ovary weight, and gonadosomatic index
Female Wistar rats in the UVB group showed a significant reduction in body weight as compared with the control group (P < 0.05). It was observed that curcumin and ascorbic acid treatment increased the body weight significantly (P < 0.05) as compared with the UVB group and no significant difference was found when compared with the control group. A significant decrease in ovary weight was observed in the female Wistar rats of the UVB group as compared with the control group (P < 0.01), while a significant increase was noted in the UVB+curcumin and UVB+ascorbic acid groups as compared with the UVB group. No significant difference was observed in UVB+curcumin and UVB+ ascorbic acid groups as compared with the control group. Gonadosomatic index decreased in the UVB group while increased in UVB+curcumin and UVB+ascorbic acid groups and no significant difference was found when compared with the control group [Table 1].
|Table 1: Body weight, ovary weight and gonado-somatic index in ovaries of female Wistar rats in all groups.|
Click here to view
3.2. Estrous cycle
In our experiment, it was found that the UVB exposure irradiated on whole body with a dose of 280 nm wavelength for 2 h expressively influenced the phases of estrous cycles [Figure 1], [Figure 2], [Figure 3], [Figure 4]. Exposure of UVB radiation on the animal showed significant differences and irregular alterations in the estrous cycle and their cells.
|Figure 1: Vaginal smears of proestrus stain with crystals violet in UVBirradiated Wistar rats treated with curcumin and ascorbic acid (Images show 10× and 40× magnification). Nucleated epithelial cells are observed in the control group (A1 and A2); nucleated epithelial cells in cohesive clusters are seen in the UVB group (B1 and B2); separation of cohesive clusters of epithelial cells is shown in the UVB+curcumin group (C1 and C2); epithelial cells appear in the UVB+ascorbic acid group (D1 and D2).|
Click here to view
|Figure 2: Vaginal smears of estrus stain with crystals violet in UVBirradiated Wistar rats treated with curcumin and ascorbic acid (Images show 10× and 40× magnification). Normal cornified squamous anucleated cells are shown in the control group (A1 and A2). Degraded cornified squamous fluid and structure are present in the UVB group (B1 and B2). Curcumin prevents the cornified squamous anucleated cells in the UVB+curcumin group (C1 and C2). Ascorbic acid has a curative effect on the degrading anucleated cells in the UVB+ascorbic acid group (D1 and D2).|
Click here to view
|Figure 3: Vaginal smears of metestrus stain with crystals violet in UVBirradiated Wistar rats treated with curcumin and ascorbic acid (Images show 10× and 40× magnification). A1 and A2 show the control group. In B1 and B2 (the UVB group), UVB changes the cornified epithelial cell. In C1 and C2 (the UVB+curcumin group), curcumin repairs the cell cornified structure. In D1 and D2 (the UVB+ascorbic acid group), ascorbic acid prevents cell cornified structure.|
Click here to view
|Figure 4: Vaginal smears of diestrus stain with crystals violet in UVBirradiated Wistar rats treated with curcumin and ascorbic acid (Images show 10× and 40× magnification). A1 and A2 show the control group. B1 and B2 (the UBV group) show aggregated white blood cells due to UVB irradiation. In C1 and C2 (the UVB+curcumin group), curcumin prevents aggregation of the white blood cells and cures mucus. In D1 and D2 (the UVB+ascorbic acid group), ascorbic acid prevents aggregation of white blood cells and less mucus is seen.|
Click here to view
During the proestrus phase, the appearance of epithelial cells was shown in different groups [Figure 1]. Nucleated epithelial cells were observed in the control group [Figure 1]A1 and [Figure 1]A2. Nucleated epithelial cells in cohesive clusters were seen in the UVB group [Figure 1]B1 and [Figure 1]B2. Separation of cohesive clusters of epithelial cells was shown in the UVB+curcumin group [Figure 1]C1 and [Figure 1]C2. Epithelial cells appeared in the UVB+ascorbic acid group [Figure 1]D1 and [Figure 1]D2
During the estrus phase, the control group showed normal cornified squamous anucleated cells and the predominance of nucleated and cornified epithelium cells was shown [Figure 2]A1 and [Figure 2]A2. The UVB treated group presented the degraded cornified squamous fluid and structure and showed less nucleated and cornified epithelial cells [Figure 2]B1 and [Figure 2]B2. In the UVB+curcumin group, curcumin prevented cornified squamous anucleated cells [Figure 2]C1 and [Figure 2]C2. In the UVB+ascorbic acid group, ascorbic acid showed a curative effect on the degrading anucleated cells [Figure 2]D1 and [Figure 2]D2. Compared with ascorbic acid, curcumin prevented the nucleated and cornified epithelial cells.
During the metestrus phase, the UVB group presented the emergence of neutrophils interspersed or clumped among the nucleated epithelial cells as compared with the conrtol group. UVB changed the cornified epithelial cell. Rats had fewer numbers of nucleated epithelial cells [Figure 3]B1 and [Figure 3]B2. As metestrus progressed, an increase of neutrophil number resulted in smears of less cellularity. In the UVB+curcumin group, curcumin repaired the cell cornified structure [Figure 3]C1 and [Figure 3]C2. In the UVB+ascorbic acid group, ascorbic acid prevented cell cornified structure [Figure 3]D1 and [Figure 3]D2.
During diestrus phase, the predominance of nucleated epithelium cells was shown in the control group [Figure 4]A1 and [Figure 4]A2. However, in the UVB group, UVB changed the cornified epithelial cell; mucous entrapped and distorted the epithelial cells, neutrophils and leukocytes were observed [Figure 4]B1 and [Figure 4]B2. In the UVB+curcumin group, curcumin repaired the cell cornified structure [Figure 4]C1 and [Figure 4]C2. In the UVB+ascorbic acid group, ascorbic acid prevented cell ornified structure [Figure 4]D1 and [Figure 4]D2.
| 4. Discussion|| |
The present study reveals that UVB exposure does influence the oestrus cycle in female rats. A whole estrous cycle in rats takes seven to fifteen days among UVB exposure days. This cycle involves four stages: proestrus, estrus, metestrus, and diestrus.
UVB radiation was non-ionizing radiation, which meant that this radiation was able to non-ionise the material in its path. In this case, the cells of the ovaries were actively divided into blood cells and ovarian gametes. These cells were very sensitive to the effect of radiation and as a result, damaged ovarian cells. The results of different phases of the estrous cycle were irradiated with UVB radiation . The rodent estrous cycle was very sensitive to alterations in the radiation. Our result showed the alteration of nucleated epithelium cells, cornified epithelium cells, leukocyte cells.
Morphological changes in vaginal smear were detected with conventional stains and crystals violet also showed significant changes. The striking change in the nucleated epithelial cells of proestrus-estrus is especially evident by this method . UV radiation affected the blood hormonal level significantly . Repeated UVB exposure hasan effect on the estrous cycle, presents a cluster of cells in the estrus cycle phases and reduces the ovary weight. In contrast, the estrous cycle becomes longer when mice are exposed to a repetition of gamma radiation . Previous studies showed thatwhole- body exposure to radiation could result in a slight decrease in weight gain of rats ,,. In our experiment, UVB exposure showed less significant alterations in the estrous cycle. Moreover, the UVB group showed tight clusters of cells and increased length of estrous phases. It was also evident from the results that the repeated UVB exposure significantly decreased body weight, ovary weight, and sexual maturity.
Both antioxidants (curcumin and ascorbic acid) showed the protective effect of the reproductive system, increased body and ovary weight as well as brought an increase in gonadosomatic index. Lower doses of antioxidants (curcumin and ascorbic acid) used in the present study (25 mg/kg and 250 mg/kg respectively) showed a preventive effect on different phases of the estrous cycle in female Wistar rats. However, higher dose concentration of these antioxidants (curcumin and ascorbic acid) induced the blockage of estrous phases and showed significant antifertility activity and elevated estrogenic activity, with inhibition of ovulation and impairment of fertility . Additionally, a higher dose of another medicinal plant such as Calotropis procera (25, 50 and 100 mg/kg of dry roots), Rivea hypocrateriformis (200 and 400 mg/kg body weight), Momordica chrantia (25, 100 mg/g body weight), Momordica cymbalaria (250 and 500 mg/kg body weight), Gacinia kola (200 mg/kg body weight) were also reported to affect the estrous cycle phases and showed reduced fertility in mammals . Moreover, the estrus cycle in the rats treated with a high dose of Momordica cymbalaria extracts also showed a decrease in the duration of estrus and metestrus phases and prolongation of the proestrus phase . A high dose of alcoholic extract of Azadirachts indica flower disrupts the estrous cycle in Sprague-Dawley rats and causes a partial block in ovulation and hence has the potential to develop female contraception.
In conclusion, UVB exposure causes significant disturbance in the estrous cycle and leads to irregularity in the estrous cycle and their cells. UV radiations have effects on the reproductive cycles and result in abnormalities. Both curcumin and ascorbic acid protect estrous cycle and increase body and ovary weight besides maintaining the gonadosomatic index.
Conflict of interest statement
The authors declare that there is no conflict of interest.
Payal Mahobiya designed the experiment plan and supported Gayatri Rai. Gayatri Rai managed the experimental animals, performed the treatment and completed data analysis and wrote the manuscript. Narendra Namdev assisted in collecting the veginal smear and the treatment. Payal Mahobiya and Gayatri Rai contributed to the editing and completion of the manuscript. All authors read and approved the final manuscript.
| References|| |
Rai G, Kumar A, Mahobiya P. Effect of radiation on thyroid gland of Wistar rat. Int J Biol Res
De Laat A. Van Der Leun JC. De Gruijl FR. Carcinogenesis induced by UVA (365-nm) radiation: The dose-time dependence of tumor formation in hairless mice. Carcinogenesis
Ferguson GW, Gehrmann WH, Chen TC, Dierenfeld ES, Holick MF. Effects of artificial ultraviolet light exposure on reproductive success of the female panther chameleon (Furcifer pardalis)
in captivity. Zoo Biol
Sudatri NW, Yulihastuti DA, Suaskara IBM, Suartini NM. Estrous cycle of mice (Musmusculus
L.) exposed by repeated gamma rays radiation. Int J Pure App Biosci
Li S. Davis B. Evaluating rodent vaginal and uterine histology in toxicity studies. Birth Defects Res B
Westwood FR. The female rat reproductive cycle: A practical histological guide to staging. Toxicol Pathol
Campbell CS, Ryan KD, Schwartz NB. Estrous cycle in the mouse: Relative influence of continuous light and the presence of a male. Biol Reprod
Paccola CC, Resende CG, Stumpp T, Miraglia SM, Cipriano I. The rat estrous cycle revisited: A quantitative and qualitative analysis. Anim Reprod
Cora MC, Kooistra L, Travlos G. Vaginal cytology of the laboratory rat and mouse: Review and criteria for the staging of the estrous cycle using stained vaginal smears. Toxicol Pathol
Singh R, Nath R, Mathur AK, Sharma RS. Effect of radiofrequency radiation on reproductive health. Indian J Med Res
Ivell R. Research in reproduction: Challenges, needs, and opportunities. Front Physiol
-Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: Some helpful considerations. Brazil J Biol
Marcondes FK, Miguel K, Melo IL, Spadari-Bratfisch RC. Estrous cycle influences the response of female rats in the elevated plus-maze. Physiol Behav
Marques DA, de Carvalho D, da Silva GS, Szawka RE, Anselmo-Franci JA, Bicego KC, et al.
Influence of estrous cycle hormonal fluctuations and gonadal hormones on the ventilatory response to hypoxia in female rats. Pflμgers Arch-Eur J Physiol
Vanderlei LCM, Marcondes FK, Lanza LLB, Spadari-bratfisch RC. Influence of the estrous cycle on the sensitivity to catechol amines in right atria from rats submitted to foot shock stress. Can J Physiol Pharmacol
Spadari-bratfisch RC, Nunes IS, Vanderlei LCM, Marcondes FK. Evidence for b2-adrenoceptors in right atria from female rats submitted to foot shock stress. Can J Physiol Pharmacol
Chateau D, Geiger JM, Samama B, Boehm N. Vaginal keratinization during the estrous cycle in rats: A model for evaluating retinoid activity. Skin Pharmacol
Hoar W, Hickman CP. Ovariectomy and the estrous cycle of the rat. In: Hoar W, Hickman CP (eds.). General and comparative physiology
. 2nd ed. New Jersey: Prentice Hall; 1975, p. 260
Rinnerthaler M, Bischof J, Streubel MK. Oxidative stress in aging human skin. Biomolecules
Halliday GM, Damian DL, Rana S. The suppressive effects of ultraviolet radiation on immunity in the skin and internal organs: Implications for autoimmunity. J Dermatol Sci
Bald T, Quast T, Landsberg J. Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma. Nature
Akindele AJ, Adeneye AA, Salau OS, Sofidiya MO, Benebo AS. Dose and time-dependent sub-chronic toxicity study of hydroethanolic leaf extract of Flabellaria paniculata
Cav. (Malpighiaceae) in rodents. Front Pharmacol
Peepre K, Deshpandey U, Choudhary PS. Role of antioxidants on thyroid hormones in Wister rats. Int J Sci Res
Sharaf HA, Morsy FA, Shaffie NM, El-Shennawy AT. Histological and histochemical study on the protective effect of curcumin on ultraviolet irradiation induced testicular damage in albino rats. J Cytol Histol
McLean AC, Valenzuela N, Fai S, Bennett SAL. Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification. J Visual Exp
Yener T, Turkkani TA, Aslan H, Aytan H, Cantug CA. Determination of oestrous cycle of the rats by direct examination: How reliable? Anat Histol Embryol
Araujo MC, Antunes LM, Takahashi CS. Protective effect of thiourea, a hydroxyl-radical scavenger, on curcumin-induced chromosomal aberrations in an in vitro
mammalian cell system. Teratog Carcinog Mutagen
Gava N, Clarke CL, Byth K, Arnett-Mansfield RL, deFazio A. Expression of progesterone receptors A and B in the mouse ovary during the estrous cycle. Endocrinology
Turker H. Effect of ultraviolet radiation on total plasma T3, total plasma T4 and TSH hormones in molerat (Spalax leucodon). Gazi Univ J Sci
Kumlin T, Iivonen H, Miettinen P, Juvonen A, van Groen T, Puranen L, et al.
Mobile phone radiation and the developing brain behavioral and morphological effects in juvenile rats. Radiat Res
Adaramoye OA, Adedara I, Farombi EO. Possible ameliorative effects of kolaviron against reproductive toxicity in sub-lethally whole body gamma-irradiated rats. Exp Toxicol Pathol
Usikalu MR, Rotimi SO, Oguegbu AE. Effect of exposure of 900 MHz radiofrequency radiation on rat brain. Eur J Exp Biol
Thakur S, Bawara B, Dubey A, Nandini D, Chauhan NS, Saraf DK. Effect of Carum carvi
and Curcuma longa
on hormonal and reproductive parameter of female rats. Int J Phytomed
Circosta C, Sanogo R, Occhiuto F. Effects of Calotropis procera
on oestrous cycle and on oestrogenic functionality in rats. Farmaco
Shivalingappa H, Satyanarayan ND, Purohit MG, Sharanabasappa A, Patil SB. Effect of ethanol extract of Rivea hypocrateriformis
on the estrous cycle of the rat. J Ethnopharmacol
Sharanabasappa A, Vijayakumar B, Patil SB. Effect of Momordica charantia
seed extracts on ovarian and uterine activities in albino rats. Pharmaceut Biol
Koneri R, Balaraman R, Saraswati CD. Antiovulatory and abortifacient potential of the ethanolic extract of roots of Momordica cymbalaria
Fenzl in rats. Indian J Pharmacol
Akpantah AO, Oremosu AA, Noronha CC, Ekanem TB, Okanlawon AO. Effects of Garcinia kola
seed extract on ovulation, oestrous cycle and foetal development in cyclic female Sprague-Dawley rats. Niger J Physiol Sci
Raj A, Singh A, Sharma A, Kumar P, Bhatia V. Antifertility activity of medicinal plants on reproductive system of female rat. Int J Bio-Engineer Sci Tech
Koneri R, Saraswati CD, Balaraman R, Ajeesha EA. Anti-implantation activity of the ethalonic root extract of Momordica cymbalaria
Fenzl in rats. Indian J Pharmacol
Gbotolorum SC, Osinubi AA, Noronha CC, Okanlawon AO. Antifertility potential of neem flower extract on adult female Sprague-Dawley rats. Afr Health Sci
[Figure 1], [Figure 2], [Figure 3], [Figure 4]