Effect of L-Dopa on cypermethrin induced reproductive conditions in female Japanese quail, Coturnix coturnix japonica

Bindushree Baghel, S. K. Prasad*, Ranjan Lal

School of Studies in Life Science, Pt. Ravishankar Shukla University, Raipur-492010, Chhattisgarh, India.

Abstract

The aim of the present study was to test whether L-Dopa (L-Dihydroxyphenylalanin) treatment can protect cypermethrin induced toxic effect on female reproductive organs in Japanese quail, Coturnix coturnix japonica. The adult female Japanese quail of 6-7 weeks of age and 150-190 gm in weight were divided into four groups (6 birds each). Group-1 received normal saline and served as control.  Group-2 received cypermethrin (1mg/kg body weight) dissolved in corn oil. Group-3 received L-Dopa (5mg/100 mg body weight) and Group- 4 received both cypermethrin and L-Dopa. All treatments were given for 30 days. After completion of treatment body weight were recorded. The birds were anesthetized with ether at the end of the experiment. Oviduct and follicles were removed and fixed in bouin’s fluid for further study. Follicles weight, size and number of follicles CYP treated group showed significantly decreased value when compared to control and L-dopa group.  L-dopa treated group showed significantly increased value of oviduct weight, follicles weight and size whereas number of follicles were no significantly different when compared to control. CYP + L-dopa fed group showed significantly increased value of follicles weight and decreased number of follicles in comparison to control. Body weight did not show any significant change in the entire experimental group when compared to control. On the basis of the results, it may be concluded that L-Dopa might be a potent protective agent against CYP induced toxic effect on female reproductive conditions in Japanese quail.

Keywords:  L-Dopa, Pesticides, Cypermethrin, Reproduction, Female Japanese quail

Introduction

Cypermethrin (a pesticide) is widely used for the treatment of parasite to resolve the ectoparasitic infestation in poultry farm (Alves et al, 2016; Sivajothi et al, 2017). Parasitic infection in poultry farm causes concurrent infections which results in loss of productivity (White meat and egg products) of poultry industries which are a rich source of essential component of food and nutrition (Ghafoor et al, 2010). Excessive use of this pesticide is the major source of environmental hazards for living organisms including human beings, because it gets incorporated in the food chains (AM Abd-Alla et al, 2002). Many pesticides are known to cause degeneration of reproductive organs, inhibition of spermatogenesis, sterility and decrease in hormone and steroid levels. Among these, Cypermethrin is one of the widely used pesticides and it is considered as an endocrine disruptive chemical (Mnif et al, 2011). It influences the processes of gametogenesis and other sexual attributes (Crisp et al, 1998). It has been reported that when cypermethrin ingested with drinking water in different concentrations, a significant decrease on the fertility, epididymal and testicular sperm count was observed in male rats (Elbetieha et al, 2001). when cypermethrin were given in different doses, decrease in the body weight, testes and epididymal, seminiferous tubules diameter, degeneration of testicular structure and seminiferous tubules, reduced germ cell, sertoli cell, leydig cell and reduced serum level of testosterone have been observed in adult male rats (Fang et al, 2013).

Dopamine is a product of L-Dopa (L-Dihydroxyphenylalanine) metabolism, and was found to possess free radical scavenging activity and strong anti-oxidant capacity (Gow-Chin and Chiu-Luan, 1997; Kazuki and Hiroyuki, 2000). It has been hypothesized that L-Dopa stimulates the forebrain and hypothalamus, thus stimulating gonadotropin-releasing hormone (GnRH) secretion, which ultimately activates adenohypophyses to secrete luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Meites et al, 1977). Elevated levels of LH and FSH stimulate the process of spermatogenesis via testosterone in Japanese quail (Prasad et al, 2007; Prasad et al, 2009) and in rat (Ashidi et al, 2019) and mice (Concessao et al, 2020). On the basis of above considerations, the aim of the present study was designed to test whether L-Dopa (L-Dihydroxyphenylalanin) administration can protect cypermethrin induced toxic effect on female reproductive organs in Japanese quail, Coturnix coturnix japonica.

Materials and methods

Animals

The experiment was conducted in adult female poultry bird Japanese quail Coturnix coturnix japonica. Japanese quail belongs to the family Galliformes. This species is different from the common quail Coturnix coturnix. Japanese quails are widely used for the egg production, meat production and as a laboratory animal. Japanese quail maturation time period is 6-7 week but sex difference appeared about 3 weeks of age.  It is hardy and weighs in the range of male is 100-160gm and female bird is 150-190 gm. Males are characterized by rusty brown throat, breast feather and presence of cloacal gland. Female have pale breast feathers that speckled with dark coloured spots. Japanese quails were procured from a local Poultry farm, Vyavsay Kendra located at Gauravpath, Ravigram, Telibandha, Raipur (C.G).  The quail birds were acclimatized in the animal house of the school of studies in Life Science, Pt. Ravishankar Shukla University, Raipur, for one week before the experiment. Birds were provided with food and water ad libitum throughout the experiment.

Cypermethrin and L-DOPA

Cypermethrin [(RS)-α-Cyano-3- phenoxybenzyl-(1 RS, 3 RS, l RS, 3

RS)-3-2, 2-dichlorovinyl)-2, 2-dimethylcyclopropane carboxylate] (CMN920T8216B) was purchased from Sigma Aldrich and had a purity of about 95 %. L-Dopa (L-Dihydroxypheylalanine) (T-8382821) was purchased from Sisco Research Laboratory (SRL) and purity about 99%.

Experimental design

The adult female Japanese quail of 6-7 weeks of age and 150-190 gm in weight were divided into four groups (6 birds each). Group-1 received normal saline and served as control.  Group-2 received cypermethrin (1mg/kg bw) dissolved in corn oil. Group-3 received L-dopa (5mg/100mg bw) and Group- 4 received both cypermethrin and L-dopa. All treatments were given for 30 days. After completion of treatment body weight were recorded. The birds were anesthetized with ether at the end of the experiment. Oviduct and follicles were removed and fixed in bouin’s fluid for further study (Fig. 1). Birds were handled and maintained under standard management conditions as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India for the use of laboratory animals.

Fig. 1: Experimental Design

Statistical analysis

Values were expressed in terms of mean ± standard deviation (Mean ± SD) to differentiate between treatment groups. The data were analyzed in the MS Excel worksheet by Student’s‘t’ test and Analysis of variance (ANOVA) using SPSS (ver. 16.0 for Windows, SPSS Inc.). p (probability value) < 0.05 level was considered as statistically significant.

Result and discussion

Follicles weight, size and number of follicles CYP treated group showed significantly decreased value when compared to control and L-dopa group.  L-dopa treated group showed significantly increased value of oviduct weight, follicles weight and size whereas number of follicles were no significantly different when compared to control. CYP + L-dopa fed group showed significantly increased value of follicles weight and decreased number of follicles in comparison to control. Body weight did not show any significant change in the entire experimental group when compared to control (Fig. 2-6; Table-1-5; Plate -1).

Fig. 2: Effect of Cypermethrin and L-DOPA on body weight in female Japanese quail.  Values are mean ± S.E.        

Table- 1: One way analysis of variance (ANOVA) showing significant changes in the body weight of female Japanese quail

      Analysis of variance of data reveals that experimental groups differ significantly (F=1.075, i.e. p>0.05)

Fig. 3:  Effect of Cypermethrin and L-DOPA on oviduct weight in Japanese quail. Value are mean ± S.E.

Table-2: One way analysis of variance (ANOVA) showing significant changes in the oviduct weight of Japanese quail.

Analysis of variance of data reveals that experimental groups differ significantly (F=6.720, i.e. p<0.05)

Fig. 4: Effect of Cypermethrin and L-Dopon follicle weight in Japanese quail. Values are mean ± S.E.       

Table-3: One way analysis of variance (ANOVA) showing significant changes in the follicle weight of Japanese quail.

         Analysis of variance of data reveals that experimental groups differ significantly (F=23.321, i.e. p<0.001)

Fig. 5: Effect of Cypermethrin and L-DOPA on number of follicles in Japanese quail Values are mean ± S.E.

Table-4: One way analysis of variance (ANOVA) showing significant changes in the number of follicles of Japanese quail.

Analysis of variance of data reveals that experimental groups differ significantly (F=18.027, i.e. p<0.01)

Fig. 6:  Effect of Cypermethrin and L-DOPA on follicular size in Japanese quail.  Values are mean ± S.E.

Table-5: One way analysis of variance (ANOVA) showing significant changes in the follicular size of Japanese quail.

       Analysis of variance of data reveals that experimental groups differ significantly (F=52.097, i.e. p<0.001)

                    Plate-1:  Effect of Cypermethrin and L-DOPA on the size of follicles in Japanese quail.

A:  Photograph Showing the follicle in control group of Japanese quail

B:  Photograph Showing the follicle in CYP group of Japanese quail

C:  Photograph Showing the follicle in L-DOPA group of Japanese quail

D:  Photograph Showing the follicle in CYP+L-DOPA group of Japanese quail

Pesticides are widely used to control pests and vectors in agriculture and household. Residue from these pesticides has persisted in the environment and has been found in soil, fruit and vegetables. Cypermethrin causes suppressive effect on the production of reproductive hormones like luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol etc. and interferes with the structure and function of reproductive organs (Keenan et al, 2009; Zhou et al, 2018; Baghel and Prasad, 2021 a b).  Cypermethrin is a neurotoxic agent which might act on hypothalamus and anterior pituitary and may directly affect testis and can consequently affect sexual activity through the hypothalamo-hypophyseal function (Elbetieha et al, 2001; Fang et al, 2013). Some researchers have shown that cypermethrin induces dopaminergic neurodegeneration in rats (Singh et al, 2012 a; Khatab et al, 2016), however, others report revealed its response only when induced by any other chemical (Wolansky and Harrill, 2008). During the last two decades in maneb& paraquat reported to induce dopaminergic neurodegeneration after prolonged exposure in rats and mice (Chugh et al, 1992; Nasuti et al, 2007). Cypermethrin exposure decreased dopamine level. This could be due to either decrease in TH or inhibition of biosynthesis of dopamine or decrease in aromatic-L-amino-acid decarboxylase synthesis, as observed with deltamethrin (an analogue of cypermethrin) exposure (Mun et al, 2005). It is well known that Cypermethrin is a well-established modulator of dopamine and gamma-aminobutyric acid levels in brain (Patel et al, 2006). Exposure of pesticides determines progressive damage of the dopaminergic neurons in the substantia nigra. Cypermethrin induces the nigrostriatal dopaminergic neurodegeneration either alone or in blended with other neurotoxicants pesticide (Thiruchelvam, 2002; Logroscino, 2005; Liu and Shi, 2006; Singh et al, 2011; 2012 b).  It has also been reported that the regulation of reproductive conditions in Japanese quail might be associated with dopaminergic system of the brain (Prasad et al, 2007; Prasad et al, 2009; Yadav and Chaturvedi, 2018).

On the basis of the results of the present study it has been apparent that Cypermethrin induces impairment in reproductive indices and causes reproductive disruption. On other hand L-Dopa induces and maintains reproductive conditions. L-Dopa in combination with cypermethrin reduces disruptive effects of cypermethrin on reproductive indices in female Japanese quail. Therefore, it may be concluded that L-Dopa might be a potent protective agent against cypermethrin induced toxic effect on reproduction in female Japanese quail. In view of the importance of poultry industry and nutritional value of poultry products the finding of the present study may aid in the rational development of new strategies of poultry industry management aimed at improving the resources of food and nutrition (human health) and benefiting the economy.

Acknowledgement

The authors are thankful to UGC, New Delhi, Goveronment of India for financial support in the form of  Basic Science Research (BSR) Fellowship to Bindushree Baghel and CSIR-UGC, NET-JRF to Ranjan Lal.

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