Research Article

 

Histomorphological and Biochemical Studies in Ovaries of Female Tatera indica and Bandicota bengalensis Inhabiting South-West Region of Punjab in North India

 

Parkash Singh1*, Gurinder Kaur Sangha2

1Department of Medical and Biotechnology, Baba Farid College, Bathinda-151001, India; 2Department of Zoology, Punjab Agricultural University, Ludhiana-141004, India.

 

Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.

Received | June 28, 2018; Accepted | August 31, 2018; Published | September 26, 2018

*Correspondence | Parkash Singh. Department of Medical and Biotechnology, Baba Farid College, Bathinda-151001, India; Email: parkashsingh23@gmail.com

Citation | Singh P, Sangha GK (2018). Histomorphological and biochemical studies in ovaries of female tatera indica and bandicota bengalensis inhabiting south-west region of punjab in north india. Adv. Anim. Vet. Sci. 6(11): 480-485.

DOI | http://dx.doi.org/10.17582/journal.aavs/2018/6.11.480.485

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2018 Singh and Sangha. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Introduction

 

The development of modern technology and the rapid industrialization are among the foremost factor for environmental pollution. The environmental pollutants are spread through different channels many of which finally enter in to the food chain of livestock and man (Kaplan et al., 2010). Pesticides having metals and other agrochemicals are some of the major causes of environmental toxicity in farm animals (Rajaganapathy et al., 2011).The wide spread use of these chemicals in agriculture has caused environmental pollution and potential health hazards (Agarwal and Sharma, 2010). Several pesticides and environmental contaminants are potential endocrine disrupters and female reproductive system may be regarded as sensitive target for theses disrupters (Bretveld et al., 2006; Agarwal and Sharma, 2010). They can alter development of reproductive system as well as ovulation and implantation (Stamati et al., 2007, Gopinath, 2013).

 

The Malwa region of Punjab India is facing an unprecedented crisis of environmental health linked to indiscriminate, excessive and unsafe use of pesticides, fertilizers and poor ground water quality. Studies of this region have also highlighted a sharp increase in many other pesticide-related diseases, such as mental retardation and reproductive disorders (Mittal et al., 2014). The increase in genetic damage in animals indicates the potential genetic hazards posed by excessive use of pesticides in Bathinda district and the long-time over-use of pesticides appears to be a major cause for prevalence of various diseases in cotton cultivated districts of the Malwa region of Punjab (Kalra and Sangha, 2018). The high use of pesticides along with environmental and social factors is responsible for the high concentration of pesticide residues in the food chain of this region (Mittal et al., 2014). Earlier studies give some indication of increased reproductive risks of exposure to pesticides/heavy metals but the epidemiological evidences do not allow any clear inference to be drawn (Thakur et al., 2008, Singh et al., 2012, Mittal et al., 2014). The present study was designed to examine the possible effect of environmental contaminants on the female fertility indices and ovarian functions in Tatera indica and Bandicota bengalensis inhabiting South-West region of Punjab.

 

Material and Methods

 

During the study, the field rats i.e. Tatera indica and Bandicota bengalensis were collected from Bathinda district of South-West region of Punjab using multi catch rat traps. The same species of rats were collected from Punjab Agricultural University, Ludhiana and adjoining areas as they served as control rats (as in these areas the spray of pesticides is in the permissible limit). Approval of Institutional Animal Ethical Committee, Guru Angad Dev Veterinary and Animal Science University (GADVASU) Ludhiana was obtained for the usage of animals vide letter No. 3901-35 dated 06.08.2012. Animals were brought to laboratory and separated according to age and sex.

 

Organ Weight and Organosomatic Index

Following humane sacrifice, the reproductive organs viz. ovary, oviduct, vagina and uterus were excised cleaned of the adhering tissue and weighed separately. The organo-somatic index (OSI) was calculated by using the following formula as per Chattopadhya et al. (2011).

 

 

 

Biochemical Studies

For biochemical studies, one whole ovary was homogenized in 2 ml of phosphate buffer saline (PBS 0.1M, pH 7.4) and ovary homogenate was centrifuged at 3000 for r.p.m. for 10 min. The Supernatant was used for estimation of total soluble proteins and various enzymes. Protein was estimated by the method of Lowry et al. (1951). Acid phosphates (ACP) and alkaline phosphates (ALP) activity was measured in citrate buffer (0.05M pH 10.5) and glycine buffer (0.05M pH 10.5), respectively using p-nitrophenol phosphate as substrate following the method of Bessay et al. (1946). Total lipids from the ovaries tissue were extracted by the method of Folch et al. (1957). Total phospholipids were estimated by Ames, (1966) method and cholesterol was estimated by Chiamori and Henry, (1959) method from the above extracted lipids.

 

Hormone Assay

Blood sample from each rat was collected directly from heart by heparinised syringe in heparinised vials. Blood was centrifuged at 2300 r.p.m. for 15 minutes. Supernatant was obtained as plasma which was used for the determination of estrogen and progesterone concentration using ELISA kits.

 

Histological Studies

For histomorphological studies, ovary of Tatera indica and Bandicota bengalensis rats collected from Bathinda region and Ludhiana rats were fixed in alcoholic bouin’s solution for 24 hours. After complete fixation, the tissue was dehydrated in graded series of ethanol, cleared in xylene and embedded in paraffin wax (Melting point between 58-60°C). The 3-5µm thick sections were cut serially with the help of microtome and after usual de-waxing and rehydration in descending series of ethanol to water, the sections were stained in haematoxylin,- eosin, cleared in xylene and mounted in DPX for microscopic examination. All serial sections of the ovary were studied for various stages of development of follicles, total number of normal, atretic follicles, diameter of follicles and oocyte as described by Kaur and Guraya, (1983).

 

Statistical Analysis

Results were expressed as Mean ± standard error of mean (SEM) and subjected to student t-test. Results were considered statistically significant with p<0.05.

 

ResultS

 

There was significant decrease in the weight of ovary in Tatera indica and weight of vagina in female Tatera indica and Bandicota bengalensis Bathinda rats as compared to the Ludhiana rats (Table 1).The amount of total proteins was less in the ovaries of Tatera indica and Bandicota bengalensis female rats inhabiting Bathinda region of Punjab as compared to Ludhiana rats (Table 2). The level of various ovarian enzymes like acid phosphates (ACP) and alkaline phosphates (ALP) increased significantly in Tatera indica and Bandicota bengalensis Bathinda rats as compared to Ludhiana rats (Table 2). Hormonal balance is important to preserve female reproduction and maintain fertility. Changing level of estrogen or progesterone can disturb this balance. In the present study estrogen and progesterone level decreased in all Tatera indica and Bandicota bengalensis rats collected from Bathinda rats as compared to Ludhiana rats (Table 3).

 

Table 1: Relative weight of reproductive organs (g/100 g bw) in Tatera indica and Bandicota bengalensis female rats.

 

	T. indica		B. bengalensis
Organs	Ludhiana (Control)	Bathinda	Ludhiana (Control)	Bathinda
Ovary	0.030±0.001	0.021±0.001*	0.025±0.001	0.021±0.002
Oviduct	0.009±0.000	0.008±0.000	0.009±0.000	0.010±0.000
Uterus	0.096±0.003	0.090±0.006	0.094±0.003	0.100±0.005
Vagina	0.104±0.002	0.082±0.003*	0.108±0.002	0.088±0.002*

 

Values are Mean ± SE,

*Significant difference at (p<0.05) as compared to control

 

Table 2: Levels of Proteins, Total lipids, Cholesterol and Phospholipids (mg/g wet wt. of ovary) and activity of acid and alkaline phosphatase (ICU) in ovaries of female Tatera indica and Bandicota bengalensis.

 

	T. indica		B. bengalensis
Biochemical constituents	Ludhiana (Control)	Bathinda	Ludhiana (Control)	Bathinda
Proteins	8.054±0.546	6.802±0.291	8.215±0.855	7.946±0.245
Total lipids	14.25±1.200	13.65±0.854	19.20±0.145	14.85±0.478*
Cholesterol	9.180±0.865	5.905±0.435*	9.451±1.354	6.888±0.421*
Phospholipids	10.206±1.081	7.763±0.409*	10.486±1.610	9.153±0.874
ACP	3.476±0.434	5.380±0.493*	3.939±0.481	4.708±0.707
ALP	14.124±1.172	16.402±1.056	15.551±2.348	17.069±1.270

 

Values are Mean ± SE,

*Significant difference at (p<0.05) as compared to control

 

Table 3: Progesterone and Estrogen concentration (pg/ml) in ovaries of female Tatera indica and Bandicota bengalensis.

 

Hormones	T. indica		B. bengalensis
	Ludhiana (Control)	Bathinda	Ludhiana (Control)	Bathinda
Estrogen	370.25±15.07	411.00±12.32	359.10±18.00	268.01±8.24
Progesterone	327.53±11.24	294.22±9.02	261.24±21.64	189.33±14.15

 

Values are Mean ± SE,

*Significant difference at (p<0.05) as compared to control

 

Table 4: Percentage of atresia in ovaries of female Tatera indica and Bandicota bengalensis.

 

Species	Area	Condition of Ovary	Number of primary follicles	Number of secondary follicles	Number of tertiary follicles	Number of pre antral follicles	Number of antral follicles	Number of Corpus Luteum
Tatera indica	Ludhiana (Control)	Normal	26.22±1.34	11.00±1.02	3.00±0.02	11.05±3.22	6.64±0.22	4.00±1.00
		Atretic	9.25±0.028	3.00±0.24	1.02±0.03	1.33±0.88	2.22±0.88	1.22±0.02
		% Atresia	26	21.4	25.5	10.7	25	23.3
	Bathinda	Normal	18.22±2.10	9.00±2.22	3.24±0.07	7.02±1.00	4.02±0.25	2.00±0.25
		Atretic	5.00±0.12	3.22±0.42	1.44±0.33	4.22±0.24	2.00±0.15	-----
		% Atresia	21.53	26.3	30.7	37.5	33.2	-----
Bandicota bengalensis	Ludhiana (Control)	Normal	23.25±2.34	9.00±1.24	4.24±0.425	12.24±2.25	6.24±0.20	5.25±1.02
		Atretic	11.00±1.24	4.02±0.22	2.00±0.01	5.24±1.02	2.45±0.25	1.00±.025
		% Atresia	32.1	30.17	32.05	29.9	28.19	16.00
	Bathinda	Normal	18.00±2.45	11.84±2.63	3.55±0.34	10.00±2.04	4.25±1.25	4.25±1.00
		Atretic	7.45±1.22	5.26±1.00	1.96±0.22	6.25±0.86	2.96±.45	2.00±0.425
		% Atresia	29.27	30.76	35.57	38.46	41.05	32.00

 

Values are Mean ± SE,

*Significant difference at (p<0.05) as compared to control

 

Table 5: Diameter of follicles (µm) in ovaries of female Tatera indica and Bandicota bengalensis.

 

	Tatera indica		Bandicota bengalensis
Category of follicles	Ludhiana (Control)	Bathinda	Ludhiana (Control)	Bathinda
Primary	111.40±21.15 (32.5-147.5)	105.00±21.86 (32.5-150)	109.44±19.06 (45-123.55)	99.44±18.52 (35.2-147)
Secondary	116.90±21.70 (75-180)	114.50±14.92 (80-150)	116.2±9.27 (90.25-140.25)	106.20±13.41 (78.5-150.5)
Teritary	133.40±5.56 (111.5-140.25)	139.50±6.79 (127.5-162.5)	139.2±15.63 (111-180.25)	139.25±11.54 (111-175)
Preantral	174.62±9.91 (158.25-205.25)	178.50±9.162 (172.5-202.5)	187.64±12.88 (150-180.5)	187.64±4.59 (175.2-198.5)
Antral	167.05±14.97 (140-223.25)	190.00±11.70 (152.5-222.5)	200.50±9.78 (150.25-230)	200.51±12.94 (160.5-229)

 

Values are Mean ± SE,

*Significant difference at (p<0.05) as compared to control; Figures in parenthesis indicates the range of diameter

 

 

The present study revealed that the number of normal follicles in all the stages of follicles was higher in Ludhiana Tatera indica and Bandicota bengalensis and their number decreased in the ovaries of rats collected from Bathinda region (Table 4; Plate 1 Fig a-h). The average diameter of all growing follicles was also less in the ovaries of Bathinda rats than PAU control rats (Table 5).

 

Discussion

 

The loss of organ weight in ovaries was probably due to alteration in reproductive hormones (Liu et al., 2006). The decrease in weight and size of ovaries can be due to extensive fibrosis and atretic follicles (Baligar and Kaliwal, 2002; Gopinath, 2013). Organophosphates like chlorpyrifos, diazinon, methyl parathion, dimethoate and monocrotophos given to female albino rats have also resulted in significant decrease in the ovarian weights (Kaur and Dhanju, 2005; Johari et al., 2010; Nishi and Hundal, 2013).

 

The decrease in the level of proteins in Bathinda rats as compared to the control rats may indicate the induced degenerative changes in the ovaries of rats or general disturbance of the proteins anabolism which may be due to androgen/estrogen deficiency leading to impaired game to genesis (Bretveld et al., 2006). Low levels of lipids, phospholipids and cholesterol in these rats indicated decreased steroidogenic activity in atretic follicles and degenerating corpora lutea and are due to loss of intracellular membranes in degenerating tissues (Wellington et al., 2004). Organophosphates like methyl parathion, dimethoate and monocrotophos when given to female albino rats at a dose level of 1/5th of LD50 had also resulted in significant decrease in concentration of proteins, lipids, phospholipids and cholesterol in ovaries of treated rats (Kaur and Dhanju, 2005). Shakoori et al. (1992) also reported a significant reduction in cholesterol levels in rats treated with cypermethrin. Phosphatases are involved in many different processes that require mobilization of phosphate ions or dephosphorylation as part of anabolic, catabolic or transfer processes (Kaur and Dhanju, 2004). A change in enzyme activity is generally related to intensity of cellular damage (Muthuviveganandavel et al., 2008). The peroxidation of membrane phospholipids not only alters the lipid milieu and structural and functional integrity of cell membrane, but also affects the activities of various membrane bound enzymes including ATPases (Rhodes et al., 1984).

 

It has been reported that the insecticides may destroy endocrinologic homeostasis by suppressing Gn-RH release, may act directly on the gonadotropins to alter the gonadotropin synthesis and secretion or indirectly by altering the pituitary cell responsiveness to GnRH or gonadal steroids which result in the alterations in the levels of FSH and LH affecting the feed-back mechanisms (Stoker et al., 1993). Earlier studies have demonstrated that various pesticides cause a decrease in circulating estradiol and progesterone levels in rats, monkeys and human beings and result in the endocrinal dysfunction (Bretveld et al., 2006; Johari et al., 2010). Evidences have also suggested that organochlorine pesticides, even at low concentrations, may disrupt the endocrine system which was responsible for proper hormone balance (Mantovani, 2002; Figa-Talamanca et al., 2001).

 

The quantitative assessment of follicle number is an indicator of the normal function as well as toxic responses in the ovary (Plowchalk et al., 1993). Earlier studies have also reported reduction of different types of healthy follicular stages with concomitant increase in atresia in rats and mice treated with different pesticides (Baligar and Kaliwal, 2002; Koc et al., 2009). Ataya et al. (1988) have reported that the cyclophosphamide is found to inhibit the development of the antral follicles in rats, thereby increasing atretic follicles in rats through interfering with hormonal ovarian follicular development and reduces estradiol. It has also been shown that the ovarian androgen and inhibin secretion by follicles may be an important part in the regulation of FSH secretion and follicular growth (Evans et al., 1997). A wide variety of environmental agents may be responsible for modifying the neurotransmitter levels that would act at the level of hypothalamus to adversely affect the reproductive functions. Organophosphates like chlorpyrifos, methyl parathion, dimethoate and monocrotophos given to female albino rats have also resulted in increased atresia of follicles in treated rats as compared to control rats (Kaur and Dhanju, 2005; Nishi and Hundal, 2013). The increase in antral follicles artesia and ovarian epithetlial proliferation may be either by directly targeting the tissues or indirectly via alteration of endogenous hormones (Borgest et al., 2002; Bretveld et al., 2006; Nishi and Hundal, 2013). The increase in genetic damage in rats indicates the potential genetic hazards posed by excessive use of pesticides in Bathinda district. The long-time over-use of pesticides appears to be a major cause for prevalence of various diseases in Malwa region of Punjab (Kalra and Sangha, 2018).

 

Conclusion

 

The alteration in biochemical constituents of ovary and decrease in ovarian weight, number of healthy follicles may be due to the reduced synthesis of steroids in the Tatera indica and Bandicota bengalensis female rats inhabiting Bathinda district of Punjab that can be attributed to pesticides evidenced by their presence in pesticide residue analysis.

 

Acknowledgements

 

The author is very thankful to the UGC that awarded Rajiv Gandhi National Fellowship (RGNF) for the financial support, head department of Zoology, Punjab Agricultural University Ludhiana and Punjab for providing necessary facilities to carry out the research work.

 

Conflict of interest

 

The authors declare that there is no conflict of interests regarding the publication of this paper.

 

Author’s contribution

 

Both the authors made substantial contributions to the design, acquisition of data, analysis and interpretation of data; Authors participated in drafting the article or revising it critically for important intellectual content; and gave final approval of the version to be submitted and any revised version.

 

References

 

• Agarwal A, Sharma B (2010) Pesticide induced oxidative stress in mammalian system. Int. J. Biol. Med. Res. 1: 90-104.

• Ames BN (1966). Estimation of phospholipids. In methods in enzymology: (Eds. E. F. Newfeld and V. Griessburg) Academic Press, New York, 196, p 11.

• Ataya K, Tadros M, Mohammed S (1988). Cyclophosphamide inhibits antral follicular development in the rat ovary. Biol. Reprod. 38:157.

• Baligar PN, Kaliwal BB (2002). Reproductive toxicity of carbofuran to the female mice: effects on estrous cycle and follicles. Ind. Hlth. 40:345-352 . https://doi.org/10.2486/indhealth.40.345

• Bessey OA, Lowry OH, Bruck MJ (1946). A method for the determination of alkaline phosphatase with five cubic millimeter of serum. J. Biol. Chem. 164: 321-329.

• Borgest C, Symonds D, Mayer LP, Hoyer PB, Flaws JA (2002). Methoxychlor may cause ovarian follicular atresia and proliferation of the ovarian epithelium in the mouse. Toxicol. Sci. 68: 473-478. https://doi.org/10.1093/toxsci/68.2.473

• Bretveld RW, Thomas CMJ, Scheepers PTJ, Zielhuis GA, Roeleveld N (2006). Pesticide exposure: The hormonal function of the female reproductive system disrupted. Reprod. Biol. Endocrinol. 4:30-36 https://doi.org/10.1186/1477-7827-4-30

• Chattopadhyay A, Podder S, Agarwal S, Bhattacharya S (2011) (Fluoride-induced histipathology and synthesis of stress protein in liver and kidney of mice. Ach. Toxicol. 85: 327-335. https://doi.org/10.1007/s00204-010-0588-7

• Chiamori NBS, Henry RJ (2011). Study of ferric chloride method of determination of total cholesterol and cholesterol esters. Am. J. Clin. Pathol. 31: 305-309. https://doi.org/10.1093/ajcp/31.4.305

• Evans ACO, Fortune JE (1997). Selection of the dominant follicle in cattle occurs in the absence of differences in the expression of messenger ribonucleic acid for gonadotropin receptors. Endocrinol. 138: 2963–2971. https://doi.org/10.1210/endo.138.7.5245

• Figa-Talamanca I (2006). Occupational risk factors and reproductive health of women. Occ. Med. 56(8): 521-531. https://doi.org/10.1093/occmed/kql114

• Folch I, Leas M, Sloanestansley GH (1957). A simplified method for isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226: 197-209.

• Gopinath C (2013). Toxicity and pathology of female reproductive tract. Cell. Biol. Toxicol. 131-114. https://doi.org/10.1007/s10565-013-9244-3

• Johari H, Shariati M, Abbasi S, Sharifi, Askari HR (2010). The effects (2of diazinon on pituitary-gonad axis and ovarian histological changes in rats. Iran. J. Reprod. Med. 8(3): 125-130.

• Kalra S, Sangha GK (2018). Assessment of genotoxic potential in rats collected from Bathinda district of south-west Punjab. Adv. Anim. Vet. Sci. 6(3): 108-112. https://doi.org/10.17582/journal.aavs/2018/6.3.108.112

• Kaplan GG, Hubbard H, Korzenik J, Sands EB, Panaccione R, Ghosh S, Wheeler AJJ, Villeneuve PJ (2010). The Inflammatory Bowel Diseases and Ambient Air Pollution: Novel Assoc. 105(11): 2412–2419.

• Kaur P, Guraya SS (1983). Follicular development in the Indian mole rat, Bandicota bangelensis during Non breeding season. Proc. Indian. Natn. Sci. Acad. 54(4): 239- 244.

• Kaur S, Dhanju CK (2004). Enzymatic changes induced by some organophosphorous pesticides in female rats. Ind. J. Exp. Biol. 42: 1017-19.

• Kaur S, Dhanju K (2005). Biochemical effects of some organophosphorus pesticide on the ovaries of albino rats. Indian J. Physiol. Pharmaco. 49(2):148-152.

• Koc ND, Kayhan FE, Sesal C, Muslu MN (2009). Dose-dependent effect of endosulfan and malathion on adult Wister abinorats ovaries. Pak. J. Biol. Sci.12(6) 498-503. https://doi.org/10.3923/pjbs.2009.498.503

• Liu P, Song XX, Wen WH, Yuan WH, Chan XM (2006). Effects of mixed cypermethrin and methylparathion on endocrine hormone levels and immune function of rat: II. Interaction. Wei. Sheng. Yan. Jiu. 35: 531-533.

• Lowry OH, Rosebrough NJ, Farr AL, Randall AJ (1951) Protein measurement with folin phenol reagent. J. Biol. Chem.193:265-75.

• Mantovani A (2002). Hazard identification and risk assessment of endocrine disrupting chemicals with regard to developmental effects. Toxicol.181-182: 367-370. https://doi.org/10.1016/S0300-483X(02)00468-7

• Mittal S, Kaur G, Vishwakarma GS (2014). Effects of Environmental Pesticides on the Health of Rural Communities in the Malwa Region of Punjab, India: A Review Human. Ecolo. Risk. Assess. 20: 366–87. https://doi.org/10.1080/10807039.2013.788972

• Muthuviveganandavel V, Muthuraman P, Muthu S, Srikumar K (2008). A study on low dose cypermethrin induced histopa­thology, lipid peroxidation and marker enzyme changes in male rat. Pest. Bichem. Physiol. 91:12-16. https://doi.org/10.1016/j.pestbp.2007.11.010

• Nishi K, Hundal SS (2013). Chlorpyrifos induced toxicity in reproductive organs of female Wister rats. Food. Chem. Toxicol. 62: 732- 738. https://doi.org/10.1016/j.fct.2013.10.006

• Plowachalk DR, Smith BJ, Mattison DR (1993). Assessment of toxicity to the ovary using follicle quantitation and morphometrics. In: Methods in toxicology, eds. By Heindel JJ, RE Chapin. 3: 57-58.

• Rajaganapathy V, Xavier F, Sreekumar D, Mandal PK (2011). Heavy Metal Contamination in Soil, Water and Fodder and their Presence in Livestock and Products: A Review. 4(3): 234-249.

• Rhodes C, Jones BK, Croucher A, Huston DH, Logan CJ, Hopkins R, Hall BE, Vickers JA (1984). The bioaccumulation and biotransformation of cis-trans cypermethrin in the rat. Pestic. Sci. 15: 471-480 https://doi.org/10.1002/ps.2780150508

• Shakoori AR, Aslam F, Sabir MA (1992). Effect of prolonged administration of insecticide (cyhalothrin/karate) on the blood and liver of rabbits. Folia. Biol. 4: 91-99.

• Singh P, Sangha GK, Khera KS (2012). Histomorphological observations in male rats inhabiting South-West region of Punjab. Proceeding of international conference on sustainable agriculture for food and livelihood security, E-P83 Crop Improvement Special Issue Short Communication. 1569-1570.

• Singh VK, Saxena PN (2012). Effect of Singh, A. and M. I. Kaur: A health surveillance of pesticide sprayers in Talwandi Sabo area of Punjab, North-West India. J. Hum. Ecol. 37(2): 133-137. https://doi.org/10.1080/09709274.2012.11906457

• Stamati PN, Hans L, Howard CV (2007). Pesticides as endocrine disrupters: Identification of hazards for female reproductive functions. Reprod. Hlth. Environ. 2: 227-248.

• Stoker TE, Goldman JM, Cooper RL (1993). The dithiocarbamate fungicide thiram disrupts the hormonal control of ovulation in the female rat. Reprod. Toxicol. 7: 211-218. https://doi.org/10.1016/0890-6238(93)90226-W

• Thakur JS, Rao TB, Rajwanshi A, Parwana HK, Kumar R (208). Epidemilogical study of high cancer among rural agricultural community of Punjab in northern India. Int. J. Environ. Res. Pub. Health. 5:399-407.

• Wellington LB, Silvia MSC, Carnieri E, Maria M, Maria B (2004). Effects of deltamethrin on functions of rat liver mitochondria and on native and synthetic model membranes. Toxicol. 152: 191-202.