Toxic Impact of Chlorpyrifos Insecticide on the Ovary of

Modern agriculture and vector-borne diseases are responsible for the rapid increase in pesticide usage, which is harmful to animals, particularly birds. The declining in the diversity of bird's species is a major concern Chlorpyrifos (CPF) is the commonly used insecticide in agriculture sector. Unfortunately, CPF is still being applied across the world. Objective: To evaluate the toxic impact of chlorpyrifos on the ovaries of the pigeon in relation to the reproductive toxicity. Methods: For this purpose, N=80 healthy (Columba Livia domestica) pigeons were used. Pigeons had been acclimated for �fteen (15) days and divided into four equal groups (A-D) Oral dosages 1.3 mg, 1.6 mg, and 2.1 mg per kg body weight per day of chlorpyrifos (CPF) were administered respectively to group B, C and D for 84 consecutive days and pigeons from group (A) assigned as control. Daily observations were made on stress-related signs and behavioral changes. (H & E) staining method was applied to examine histological change in the ovary of pigeons after exposure to CPF, likewise for the control. Results: Birds from groups B-D displayed notable clinical signs such as: tremors, salivation, open-mouth breathing, ru�ed feathers, lethargy and watery diarrhea and reduced food intake. The Histological examinations revealed that the ovary of pigeons from the B, C, and D showed notable variations such as follicular degeneration, vacuolation, hypertrophy, and �brosis after exposure to CPF. The intensity was on high doses as compared to control which were with normal ovarian articulation small containing big follicles of all diameters. Oocytes, thecal layers, granulosa layers (g), stromal Tissue, and nucleus contained yolk-like secondary oocytes, mature follicles. Conclusions: In current study it was concluded that, the CPF produced drastic impact on the ovary of pigeon that may be associated with alterations in reproduction.

The global usage of pesticide is increasing in agriculture sector to satisfy consumers' cravings for food, almost four million tons of pesticides were used worldwide, with Asian n at i o n s a cco u n t i n g fo r t h e m a j o r i t y ( 5 2 .8 % ) of consumption and Pakistan is on second number in consumption in this region [1][2][3].Chlorpyrifos (CPF) was originally developed to eradicate mosquitoes but CPF is now a versatile insecticide that is widely used in agriculture sector such as on cereals, cotton, fruits, nuts, vegetables, lawns, and ornamental plants [4].CPF is commonly used inside home to e ciently control pests including cockroaches, eas, spiders, and ies, even though it is predominantly used in agriculture [5].In addition, it is used locally to get rid of eas and lice on domesticated animals and in dog kennels.Indiscriminate use of chlorpyrifos cause serious problems endangering the ecosystem since their residue lingers in the environment and continuously puts non-target living beings like birds at risk for health glitches [6,7].CPF's primarily toxic mechanism is Acetylcholinesterase (AChE) inhibition, resulting in m u s c a r i n i c, b r o n c h o s p a s m , we a k n e ss , m u s c l e fasciculation, and CNS symptoms such tremors, convulsions, anxiety, and coma are among the symptoms [8].Synthetic insecticides like CPF, which are widely used, have the possible to harm development and reproduction.CPF affects the production of testosterone in animals and Copyright © 2024.Markhor, Published by Crosslinks International Publishers decreases the release of pituitary hormones such FSH and LH [9].According to reports, long term exposure of CPF have impact on reproductive system, which may result in sperm destruction, reduced semen quality, and changes in reproductive hormones [10] and at higher doses; these changes were also seen in total protein, liver enzymes, serum creatinine, as well as hematological and biochemical abnormalities [11].The toxic effects of CPF on birds' liver, plasma and brain demonstrates inhibition of cholinesterase activities within two hours of exposure with symptoms, including gasping, salivation, feces, trembling, drooping wings, convulsions, recumbence, and death.[12].The toxicological effects of OPs, such as chlorpyrifos, on hematology and serum biochemistry shows a signi cant drop in the values of erythrocytes, hemoglobin, and hematocrit in birds.Chicken treated with CPF results alterations in from serum biochemistry and histology of different organs including necrosis, hepatic hemorrhages and vacuolar disintegration of tubular epithelial cells' nephropathy.CPF also cause changes in hepatic sinusoids, alter fatty tissue, congest hepatic cells, and multiply bile duct cells.[13] Organophosphate (OP) insecticides, such as chlorpyrifos, demonstrates their toxicity by preventing the ner vous system's production of AChE, a crucial neurotransmitter in the central and peripheral nervous systems [14,15].This study was aimed to evaluate the harmful effects of chlorpyrifos on avian female reproductive organs and to gather data on the indiscriminate use of pesticides that result in the extinction of important bird species and a decline in avifaunal biodiversity.

Experiment Design and Birds
The ovarian toxicity of chlorpyrifos pesticide was tested in 80 healthy female adult pigeons (Columba livia domestica).Before the experiment, all pigeons were vaccinated and acclimatized in a lab for 15 days.They lived in tidy wooden wire cages.Pigeons ate grains and seeds and drank clean water.After acclimation, they were placed into four equal groups of twenty pigeons (A, B, C, and D) and housed in various cages by tagging method.Their pre-test weights were recorded.Pigeons from groups B, C, and D were treated with CPF insecticide, whereas group A was the control.

Preparations for Insecticide and the Study Protocol
Toxicological methods determined the pesticide concentration (Chlorpyrifos 40EC) via LD50.Groups A, B, C, and D received oral treatment at 1/25th, 1/20th, and 1/15th of the LD50 (1.3 mg, 1.6 mg, and 2.1 mg/kg body weight/day) with one milliliter of maize oil for 84 days, while pigeons from A group acted as controls.The current study fed all pigeons the same amount of grains and seeds, 22% crude proteins, and water.Observation of Body Weights, Food Intake, And Clinical Signs The Histological Techniques Daily observations were made on clinical and stressrelated signs, symptoms, and behavioral changes based on LD50 of CPF technical information during the trial with a continuous observations on daily basis for their physical activates after doses administrated.And frequency of main only main clinical signs is shown in table form.Histology, body and organ weights and food consumption were assessed.After the experiment, eight pigeons from each group were randomly dissected and weighed.Absolute and relative ovarian in table form weights were recorded.For 24-48 hours, ovaries were immersed in Bouin's solution to analyses histological changes.Small ovary fragments were incorporated in para n wax after drying in an alcohol-graded series.Thin tissue slices (5-6 μm) were cut using a Rotary microtome and stained with Harris's hematoxylin (Gurr, 1956) and Eosin (Putt, 1948).Microphotography and histology studies were done using the Best Scope LCD digital biological LCD microscope BLM 260.

Statistics Analysis
One-way analysis of variance (ANOVA) was used to evaluate the data.Signi cant values were determined as Mean ± SD at the (P<0.05),(P<0.01), and (P<0.001)levels.The least signi cant difference (LSD) test was used to assess the mean difference between the groups using the statistical program Statistics 8.1.

R E S U L T S
The following is the presentation of the study's ndings about the toxicity of chlorpyrifos pesticide to exposed birds at various doses.The widespread toxicity of chlorpyrifos, a commonly used organophosphate pesticide has shown toxic effects on the reproductive systems of birds.

Clinical Observation and Feed Intake
During the trial, pigeon from groups B, C, and D showed symptoms.Tremors, lethargic or not moving, depressed and remained isolated and diarrhea (Table 1), after exposure to CPF open mouth breathing, salivation, tremors, and motor activity, isolation, watery diarrhea and decreased mating frequency due to not coupling with male pigeons were present in same cages.In comparison, the control birds were healthy.Group D birds were dangerous.CPF-exposed birds showed behavioral changes from the rst week of treatment, including a substantial decrease in feed consumption compared to the control group.

Absolute and Relative weights of the Ovary
Total and relative ovary weights the female pigeons were dissected and their ovaries weighed after the procedure.Table 3 shows that chlorpyrifos therapy lowered ovarian size and weight compared to the control group.Compared to the control group, chlorpyrifos insecticides signi cantly affected ovarian weights (P < 0.01) in birds from groups B, C, and D. Post-treatment gonado-somatic index and relative ovarian weights for all treatment groups were considerably (P < 0.01) lower than the control group (Table 3).

Effects on Absolute Body and Organ Weight Body Weights
The body weight of treated birds in the B, C, and D groups were signi cantly in uenced by CPF exposure.Birds in groups B, C, and D had signi cantly lower body weights (P < 0.05) compared to the control group, with a considerable decline (P < 0.01) in birds of groups C and D. Each group's birds' body weight was recorded every fortnightly.All treatment groups' weight losses were recorded throughout the study.The pigeons on insecticide chlorpyrifos exposure reduced birds' body when compared their body weights from on the end of trail and with control group.At the end of experimental study, the control group's body weight signi cantly higher than groups B, C and D's (Table 2).

Histology of Chlorpyrifos Insecticide-Exposure and Control Ovaries
The control group ovaries had healthy, large and small follicles with intact granulosa membranes.Microscopic images of control birds' ovaries revealed all diameters of healthy follicles, follicular epithelium thickness (including granulosa and theca cells), and maturation differed (Figure 2).

D I S C U S S I O N
Our study showed that, the birds without treatment were healthy and active, with no clinical problems while birds treated with higher chlorpyrifos doses displayed mild to severe indications as tremors, diarrhea, dullness, stumbling gait, and reduced crowing.On other hand chickens fed chlorpyrifos shows these same results in accordance with our results [16][17][18].In our investigation, chlorpyrifos caused cholinergic toxicity.All subjected birds had signs of behavioral changes not interested to feed intake similarly to previous evidences [19][20][21].In our study the pigeon's treated with chlorpyrifos exhibited signi cant declines in body weight and feed de ciency in pigeons from group B-D, which was contrary to the results of the previous investigation [22].Furthermore, the treated birds with high dose CPF exposure displayed clinical signs as dullness, and decreased appetite for food in pigeons from group C and D. The earlier studies found no statistically signi cant body weight or feed e ciency decreases in chlorpyrifos-treated birds.In birds, sub-lethal pesticide exposure caused lethargy, depression, and decreased food consumption [23,24].Whereas same with CPF exposure reduced hatch rates and increased infant deformities.No changes were made to newborn chick body or brain weights by CPF.Oral CPF at 55 mg/kg body weight may improve layers [25][26][27][28].The adverse effects of chlorpyrifos rst were mild but worsened.The metabolites of chlorpyrifos cause neurotoxicity and tissue damage [29,30].Therefore, CPF inhibits acetyl cholinesterase (AChE), which stops neurotransmission at cholinergic synapses [31,32].Pathos-morphological studies assessed organ weights and shapes, while histopathological studies showed pesticide damage to vital organs and tissues [33].Critical evaluations of organophosphate pesticide impact on avian systems reveal population turnover may affect pesticide-induced adult mortality, reduced fecundity, and partial sterility [34].Our results were also in accordance with reported results wherein mortality was recorded due to the toxicity of CPF in pigeons of group B, C and D. Since wild birds only reach sexual maturity once a year, these two factors may dramatically impact their population [35].OP such as CPF pesticides in uence almost every bodily function, therefore long-term exposures may affect demographics and ecology [36].Female quail with 100 ppm parathion on day 10 have considerably lower plasma progesterone, corticosterone, and luteinizing hormone than those with 0 or 25 ppm [37].Short-term parathion use disrupts steroid hormones, producing reproductive issues in quail [38].In present study compared to birds in the control group, the toxic effects of chlorpyrifos insecticides on ovarian weights were very signi cant, birds belonging to groups B, C, and D. CPF-treated birds had less number of large follicles, damaged and degraded follicles in their ovaries.Ovarian architecture, follicle size, and maturity were normal in control birds.The dose-dependent damage to stomal cells induced hypertrophy, vacuolation, and connective tissue regressing follicles and eggs has been studied in ecological theory and practice in birds [39].In current study CPF produced follicular deterioration where just one follicle (secondary oocytes) and a few tiny follicles were visible in the ovary of the pigeon belonging to group B. Group C, pigeon's ovaries showed severe alterations in the stroma cells, including brosis and hypertrophy, vacuolation in stomas, stromal cell hypertrophy and connective tissue regressing follicles.In the ovary of group D, stromal vacuolation is clearly visible connective tissue, and stromal cells damage.These observations revealed that birds of groups B, C, and D had signi cantly altered ovaries and female reproductive organ system as a result of exposure to chlorpyrifos insecticides.Same results reported that complex network of physiological reactions is being discovered by studying chlorpyrifos' effects on pigeon ovaries, demonstrating bird reproductive systems' vulnerability to human toxins [40].Current study highlighted ecological stewardship to conser ve ecosystems and the need for tougher pesticide controls to protect animals.So, this new study will demonstrate the need for coordinated bird population and biodiversity

Figure 1 :
Figure 1: Comparison of Daily Feed Intake (grams per day) in Pigeons: Control (Group A) vs. Chlorpyrifos Exposed Groups (Groups B, C, and D)Effects on Absolute Body and Organ Weight Body WeightsThe body weight of treated birds in the B, C, and D groups were signi cantly in uenced by CPF exposure.Birds in groups B, C, and D had signi cantly lower body weights (P < 0.05) compared to the control group, with a considerable decline (P < 0.01) in birds of groups C and D. Each group's birds' body weight was recorded every fortnightly.All treatment groups' weight losses were recorded throughout the study.The pigeons on insecticide chlorpyrifos exposure reduced birds' body when compared their body weights from on the end of trail and with control group.At the end of experimental study, the control group's body weight signi cantly higher than groups B, C and D's (Table2).
exhibited highly signi cant (P** < 0.01) (P*** < 0.01) reduction in feed intake in each pigeon from group B, C, D, in comparison with daily feed intake of each pigeon from control group (A) The Mean values are in Figure 1.Six birds in Group B, eight in Group C, and ten in Group D died from CPF toxicity in the second to sixth week of treatment.No birds in the control group died.Table 3: Ovarian weight (Absolute) and relative gonadsomatic index in Chlorpyrifos-treated birds of treated groups B to D with a signi cant increase (P < 0.01) ** and *** 1 1 as compared to group A 1 Mean Values not sharing the same letters are signi cantly different from each other The Impact of Chlorpyrifos Insecticides on the Ovarian Health of Treated Avian Species Ovaries General and Morphological Observations Birds' single functional left ovary contains a columnar epithelium with numerous cortical follicles of various sizes and shapes.Blood vessels were visible in the control group birds' yolky ovum.All treated birds' ovaries had tiny, translucent, white bubbles that resembled follicles in groups B, C, and D. The control birds' ovaries had healthy, yolky follicles of various sizes.
. Markhor, Published by Crosslinks International Publishers Microphotography of the Control group pigeon's ovary shows small and big follicles of all diameters.Oocytes (o), thecal layers (t), granulosa layers (g), stromal Tissue (s), and the nucleus (n) contain yolk-like secondary oocytes, mature follicles, and little yellow follicles (Primary oocytes) H & E. X Histological analysis of group B's ovary showed (Figure3) that the follicles had degenerated and were beginning to burst the columi.

Figure 2 :
Figure 2: Microphotography Section of the Ovary of the Control Group

Figure 3 :
Figure 3: Microphotography Shows One Secondary Oocyte-Containing Follicle in Group B Pigeon's Ovary

Figure 4 :
Figure 4: In ovary group C, microphotography showed a simple epithelial membrane of follicles rupturing and destroying chromatin.Degenerate follicles, bloody hypertrophy or brosis, non-growing primary follicles, and stromal vacuolation are apparent in the photograph.forty from H&E. 40 X.

Figure 5 :
Figure 5: The Ovary of D group pigeons contains single primary follicle oocyte and mature follicles were absent in this image and stoma with full vacuolation without connective, nervous, and vascular tissue.

Table 1 :
The Frequency of Clinical Signs Observed in Pigeons from Group B-D Exposed to Chlorpyrifos Insecticide

Table 2 :
The Fortnightly Body Weights of the Birds Exposed to Chlorpyrifos Insecticide as Compared to the Control Group