RETRACTED ARTICLE: Chlorantraniliprole (Coragen® 20% SC) exposure induced reproductive toxicity mediated by oxidative stress, apoptosis, and sperm quality deficient in male Wistar rats

Abstract

Pesticides can adversely affect reproduction by causing congenital abnormalities, fetal demise, and infertility. The reproductive toxicity of coragen, a modified ryanodine receptor-targeting insecticide with chlorantraniliprole concentrations of 20%, was examined in male rats. Twenty-one healthy male rats were randomly assigned to one of three groups: the control group, two orally administered with low (500 mg/kg) and high (1000 mg/kg) doses of coragen for 8 weeks. Exposure to coragen resulted in significant, dose-dependent changes in male reproductive hormones, steroidogenic enzymes, and an imbalance in the oxidant-antioxidant system. The treated groups revealed significantly higher lipid peroxidation levels than the control group. The effects were accompanied by damage to testicular tissue, modified testicular lactate dehydrogenase, reduced sperm motility and viability, and heightened sperm abnormalities. Elevated levels of pro-apoptotic proteins (caspase-3 and Bax) and decreased levels of anti-apoptotic protein (Bcl-2) provided evidence of apoptosis in both treatment groups. Moreover, coragen induced substantial DNA damage in the testicular tissue. The results indicate that the reproductive impairment caused by coragen may be ascribed to oxidative stress, hormonal disturbance, apoptosis, and damage to testicular DNA and finally might result in infertility and compromised reproductive function.

Introduction

Infertility is defined as the inability to achieve a pregnancy after 1 year of regular, unprotected sexual intercourse. Approximately 20 to 35% of couples experience infertility. Female factors account for 35% of infertility cases, while male factors contribute to 30%. In 20% of cases, both men- and women-induced factors contribute to infertility. In 15% of cases, the underlying reason remains unexplained, a condition known as idiopathic infertility (Moreira et al. 2021).

Male infertility is significantly more challenging to assess due to cultural factors, yet it still accounts for 30–50% of infertility cases. It occurs when sperm are unable to fertilize a regular egg successfully. Causes of male infertility include varicocele, congenital abnormalities, urogenital infections, genetic disorders, hormonal imbalances, and immunological issues. Environmental factors include radiation, obesity, and exposure to toxicants like pesticides (Ilgın et al. 2018).

Pesticides are synthetic chemicals commonly used to prevent crop losses caused by pests. Over the past half-century, their increasing use has become a primary pest control method. As endocrine-disrupting chemicals (EDCs), pesticides disrupt the body’s hormone production, release, and function, which are vital for growth, development, and reproduction. Exposure to pesticides can lead to reduced fertility, congenital disabilities, childhood cancers, and other developmental issues (Afify et al. 2018).

Approximately 50% of male infertility cases can be attributed to testicular toxicity (Abd-Allah and Abd El-Rahman 2023), with hormonal imbalances, environmental factors, lifestyle, and diet also playing significant roles (El-Naggar et al. 2022). Previous research has shown that pesticide exposure like iprodione and chlorpyrifos can negatively affect reproductive development and function (Hassan et al. 2021a, b). These pesticides are associated with reduced testicular size, impaired sperm quality, hormonal disruptions, oxidative stress, and activation of apoptosis-related enzymes. They can also alter gene expression related to steroidogenesis and spermatogenesis.

Imidacloprid insecticide has been associated with inducing reproductive damage in male rats by increasing serum FSH and LH levels and altering the testicular redox state. This leads to higher MDA levels, reduced GSH content, and decreased CAT activity in the testes. In addition to aberrant sperm counts, viability, motility, and morphology, histopathological abnormalities of the testes include significant degeneration and necrosis in germ cells and Leydig cells, seminiferous tubules atrophy, and suppressed expression of StAR and aromatase genes, along with lower blood testosterone levels (Mehanna et al. 2022). Similarly, hexachlorobenzene pesticide induces reproductive toxicity through oxidative stress, altered sperm parameters, apoptosis, and histological changes. It reduces the activity of gamma-glutamyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase while increasing malondialdehyde levels. This significantly reduces epididymal sperm count, motility, viability, hypo-osmotic tail swelling, and sperm morphological defects. It also causes testicular histological and histomorphometric abnormalities and elevates pro-apoptotic markers Bax, caspases-3, and −9 (Tijani et al. 2024).

Coragen is the brand name for chlorantraniliprole, an insecticide with an active component concentration of 18.5% or 20% SC. It is a novel Ryanodine receptor-targeting insecticide (Ryanoids) in the anthranilic diamides class, specifically targeting ryanodine receptors in lepidopteran species. They bind to cardiac and skeletal muscle calcium channels, blocking nervous transmission. Coragen is used in various crops to control all Lepidoptera species as well as some Coleoptera, Diptera, and Isoptera species such as codling moth (Cydia pomonella), apple fruit moth (Argyresthia conjugally), and free leaf living larvae. Ryanodine receptors in vertebrates are 400 to 3000 times less sensitive to chlorantraniliprole than in target insects, making it less hazardous to fish, birds, and mammals. However, animal studies have linked coragen exposure to weight loss, blood disorders, liver damage, and lung and spleen abnormalities (Dutta et al. 2014; Abdel-Mobdy et al. 2017; Meligi et al. 2019). Additionally, rats exposed to coragen have exhibited elevated uric acid levels, abnormal thyroid and sex hormone levels, increased white blood cell count, anemia, kidney failure, and low platelet count (Hassan et al. 2021a, b). While classified as non-toxic and non-carcinogenic (Abdel-Mobdy et al. 2021), human exposure to coragen has resulted in cardiac manifestation in a 26-year-old woman cardiac issues (Mishra et al. 2016) and intrauterine and genetic toxicity in female albino rats (Omar et al. 2022).

Studies have also shown that coragen can lower blood calcium levels and induce severe toxicity in rats. Sub-acute administration caused vomiting, nausea, eye swelling, nosebleeds, tongue discoloration, impaired coordination, and weakness (Kumar et al. 2013). Similarly, sub-lethal doses of coragen (2000 mg/kg) for 4 weeks resulted in oxidative damage, histopathological alterations in the spleen, lung, liver, and severe symptoms including nosebleeds, blindness, paralysis, and weakness (Meligi et al. 2019).

Limited information exists concerning the reproductive toxicity of coragen in males. This study seeks to explore the possible testicular toxicity of Coragen 20% SC in male rats and examine its effects on reproductive health by evaluating DNA integrity, apoptotic proteins, semen quality, testicular enzyme activity, and hormone levels.

Material and methods

Chlorantraniliprole

This study used the insecticide chlorantraniliprole, marketed under the trade name Coragen 20% SC. It has the chemical formula 3-Bromo-N-[4-chloro-2-methyl-6-[(methylamino) carbonyl]phenyl]−1-(3-chloro-2-pyridinyl)−1H-pyrazole-5 carboxamide and was obtained from the Food Machinery and Chemical Corporation (FMC), Egypt. This compound is classified as an anthranilic diamide insecticide and has an LD₅₀ greater than 5000 mg/kg body weight in male albino rats (Shallan Magdy et al. 2016).

Study strategy

Experimental animals

The present study was conducted on adult male Wistar rats (Rattus norvegicus) weighing 160–180 g, obtained from the animal house of the Faculty of Veterinary Medicine, Cairo University, Egypt. Animals were provided with standard food and water ad libitum. Before experimentation, rats were acclimatized for one week in plastic cages under 25 ± 3 °C, controlled humidity, and a 12-h light/dark cycle. The experimental protocols and procedures employed in this work received authorization from the Institutional Animal Care and Use Committee (IACUC) in accordance with Guide for the Care and Use of Laboratory Animals 8th Edition 2011under approval number CU/I/F/40/21 Cairo University, Faculty of Science, Egypt.

Experimental design

Coragen 20% SC was administered orally through gastric gavage three times a week for 8 weeks. Three groups of seven male rats each were established as follows:

• Group I: The control group received distilled water orally.

• Group II (low dose group, LD): Intoxicated rats received 500 mg/kg of Coragen 20% SC.

• Group III (high dose group, HD): Intoxicated rats received 1000 mg/kg of Coragen 20% SC, according to (Kumar et al. 2013; Dutta et al. 2014).

Sample preparation

At the beginning of the experiment, all animals were weighed using an automatic balance. Upon termination of the study, rats were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Subsequently, the reproductive organs, including the testes, epididymis, and seminal vesicles, were carefully dissected, examined, and weighed. Blood samples were collected via cardiac puncture. Serum was obtained by centrifuging the blood at 3000 rpm for 15 min.

The oxidative status biomarkers

Testicular tissues were homogenized in a cold phosphate-buffered saline (PBS) solution (pH 7.4) at a ratio of 5–10 ml per gram of tissue. After homogenization, the mixture was centrifuged at 4000 rpm for 15 min. Moreover, the supernatant was collected to analyze oxidative stress and antioxidant parameters. Malondialdehyde (MDA) levels were measured using Bio Diagnostic commercial kits (Cat. No. MD 25 29) following the methods of Satoh (1978) and Ohkawa et al. (1979). Catalase activity was assessed using a colorimetric technique (Cat. No. CA 25 17) based on Aebi (1984) and Fossati et al. (1980). Reduced glutathione (GSH) levels were determined with a colorimetric method (Cat. No. GR 25 11) as described by Beutler et al. (1963). Superoxide dismutase (SOD) activity was measured using the method of Nishkimi et al. (1972) (Cat. No. SD 25 21).

Estimation of testicular enzymes

Testicular lactate dehydrogenase activity was measured using an ELISA kit from Elabscience (USA, CAT No. E-EL-R0338). This kit follows the Sandwich-ELISA method. The micro-ELISA plate is pre-coated with an antibody specific to Rat D-LDH. Samples are added to the wells and combined with the specific antibody. A biotinylated detection antibody is specific to rat D-LDH, and Avidin-HRP conjugate is added and incubated. Unbound components are washed away. A substrate solution is added, and wells containing rat D-LDH, biotinylated antibody, and Avidin-HRP conjugate turn blue. The reaction is stopped with a stop solution, causing the color to turn yellow. The optical density (OD) is measured spectrophotometrically at 450 ± 2 nm.

Apoptotic markers assay

A 20% testicular tissue homogenate was prepared by homogenizing 1 g of tissue in 5 mL of cold phosphate buffer saline (PBS, 0.5 g Na₂HPO₄ and 0.7 g NaH₂PO₄ per 500 mL deionized water, pH 7.4). The homogenate was centrifuged at 4000 rpm for 15 min at 4 °C. The supernatant was subjected to an extraction process to quantify caspase-3, Bax, and Bcl-2 using ELISA kits purchased from CUSABIO (USA, CAT No. CSB-E08857r and CSB-E08854r) and Elabscience (USA, CAT No. E-EL-R0098, respectively), following the manufacturer’s protocols.

Estimation of DNA damage using comet assay

Testicular DNA damage was assessed using the single-cell gel electrophoresis (SCGE)/comet assay, a method developed by Singh et al. (1988). Testicular tissue was minced into fine particles in 1 mL of cold (HBSS) containing 20 mM EDTA and 10% DMSO. After allowing the tissue to settle, a 5–10 µL aliquot was mixed with 75 µL of low melting point agarose for DNA damage analysis. DNA damage was evaluated using a fluorescent microscope equipped with a 40 × objective and Komet 5 image analysis software (Kinetic Imaging, Ltd., Liverpool, UK) connected to a charge-coupled device (CCD) camera. Ethidium bromide (EtBr)-stained DNA was visualized to assess DNA damage qualitatively and quantitatively. DNA migration length and the percentage of migrated DNA were measured, and the program computes the tail and olive moments.

Reproductive hormone profile

Serum samples from all groups were tested for testosterone (CUSABIO, USA, CAT No. CSB-E05100r), follicle-stimulating hormone (FSH) (Kamiya Biomedical Company, USA, CAT No. KT-15332), and luteinizing hormone (LH) (CUSABIO, USA, CAT No. CSB-E12654r) using a competitive inhibition enzyme immunoassay. The microtiter plate was pre-coated with hormone-specific antibodies. Standards or samples were added to the wells with a specific antibody, and Horseradish Peroxidase (HRP) was conjugated specifically for hormones. After incubation, the unbound conjugate was washed off. A substrate solution was added, and color development, inversely proportional to hormone concentration, was measured. The reaction was stopped, and the color intensity was recorded.

Evaluation of steroidogenic enzyme

The ELISA kit utilizes the Sandwich-ELISA method to measure steroidogenesis enzymes, including steroidogenic acute regulatory protein (LS Bio, USA, Catalog No. LS-F49581) and 17β-hydroxysteroid dehydrogenase (17βHSD) (AFG scientific, USA, Catalog No. EK7210951). The microtiter strip plate is pre-coated with an antibody specific to the target enzyme. Standards or samples are added to the wells, allowing the antigen to bind to the antibody. A Horseradish Peroxidase (HRP)-conjugated antibody specific to the target enzyme is then added and incubated. Unbound components are washed away, and a TMB substrate solution is added, resulting in a color change. After the stop solution is added, the optical density (OD) is measured at 450 nm. The OD value is proportional to enzyme concentration, and enzyme levels in the samples are calculated by comparing the OD to a standard curve.

Testicular Histopathology

Testicular tissues were fixed in 10% buffered neutral formalin for 24 h, followed by dehydration in a graded series of alcohols (methyl, ethyl, and absolute ethyl), and cleared with xylene. The tissues were then embedded in paraffin wax. Four-micrometer-thick sections were cut, mounted on glass slides, deparaffinized, and stained with hematoxylin and eosin. The slides were examined under a light microscope, and images were captured. Histological procedures were performed according to standard protocols (Drury & Wallington 1980; Khidr et al. 2017).

Morphometric analysis

Approximately 30 circular or nearly circular seminiferous tubules were randomly selected from each group. Tubular diameter, lumen diameter, and germinal epithelium height were measured at 100 × magnification using Image J software (Version 1.53i).

Johnsen score

Twenty randomly selected inter-tubular regions in testicular tissue sections were examined under a light microscope. Mean Johnson’s testicular biopsy scores (MJTBS) were calculated according to Johnsen’s previously established method (Johnsen 1970).

Sperm analysis

The cauda epididymis was carefully separated from the testis and minced in phosphate-buffered saline to acquire a suspension, which was then dispersed and filtrated to remove tissue fragments.

Determination of sperm viability

A 5 μl sample was evenly distributed on a clean glass slide and allowed to air-dry. The dried samples were stained with 1% eosin and 10% nigrosine. Following the method of Morakinyo et al. (2009), sperm viability was assessed by bright-field microscopy at a magnification of 1000 × , examining a minimum of 100 sperm cells.

Determination of sperm abnormalities

A single drop of Eosin Y stain was added to the sperm suspension and incubated for 30 min. Subsequently, thin smears were prepared on clean glass slides and allowed to air-dry. Sperm viability was assessed by examining a minimum of 100 spermatozoa per animal under a microscope. Sperm were categorized as normal or abnormal based on the criteria outlined by Naryana et al. (2002).

Determination of sperm counting

A 10 μl aliquot of diluted sperm was loaded onto a Neubauer hemocytometer and allowed to settle for 5 min before microscopic examination. Sperm concentration, expressed as millions per milliliter, was determined using a light microscope at 400 × magnification, following the method described by Lowe and Jeffrey (1990).

Determination of sperm motility

Sperm were observed under a light microscope after being placed on a clean slide and on a heated stage maintained at 37 °C. Sperm motility was determined by counting the number of motile and non-motile sperm and calculating the percentage of motile sperm.

Statistical analysis

All values are presented as mean ± standard deviation (SD). Data were analyzed using IBM-SPSS statistical software (version 25). One-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparison test for pairwise comparisons. Statistical significance was set at P < 0.05. Cohen’s d effect size was calculated using GPower software (version 3.1.9.4), with effect sizes interpreted as follows: < 0.20 (weak), 0.21–0.50 (modest effect), 0.50–0.8 (moderate), and > 0.8 (strong). Pearson correlation analysis was used to assess relationships between parameters, with correlation coefficients (r) and P-values calculated to evaluate the strength and significance of associations.

Results

Impact of coragen on the body weight change and weights of the reproductive organ

Compared to the control group, the low-dose group (500 mg/kg) showed a moderate effect size (d = 0.63, d = 0.63) for body weight change, while the high-dose group (1000 mg/kg) showed a small effect size (d = 0.32).

In either treated group, there were no significant changes in the absolute reproductive organ weights of the right testis, left testis, and left epididymis. Effect sizes for these organs were small (right testis, d = 0.30 and d = 0.10; left testis, d = 0.38 and d = 0.30; left epididymis, d = 0.20 and d = 0.30), indicating minimal changes.

In the high-dose group (1000 mg/kg), more pronounced effects were observed in the right epididymis and seminal vesicle, with large effect sizes (d = 1.0 and d = 1.2, respectively). In contrast, the low-dose group (500 mg/kg) showed small effect sizes for both organs (right epididymis, d = 0.40; seminal vesicle, d = 0.40).

Regarding the relative weights of the reproductive organs, both treated groups showed a slight reduction in the left and right testis compared to the control group. In the low-dose group, the right testis exhibited a small effect size (d = 0.30), while the high-dose group showed a moderate effect size (d = 0.60). The left testis showed small effect sizes in both treated groups (d = 0.14 and d = 0.30). The relative weight of right epididymis and left epididymis showed no effect (d = 0) in rats treated with coragen (500 mg/kg) of right epididymis and both treated groups of left epididymis. However, in the high-dose group (1000 mg/kg), a reduction in the relative weight of the right epididymis was observed, with a large effect size (d = 1.30), and a reduction in the relative weight of the seminal vesicle was noted in both treated groups. The low-dose group (500 mg/kg) showed a small effect size (d = 0.40), while the high-dose group (1000 mg/kg) exhibited a large effect size (d = 1.5) Table 1.

Table 1.

Body weight change and the absolute and relative weights of reproductive organs for all experimental groups

Items		Control	Coragen-1/10 LD50 (500 mg/kg)	Coragen-1/5 LD50 (1000 mg/kg)	P-value	f-value
Body weight change (g)		85.57 ± 21	73.57 ± 16.83	79.14 ± 18.95	0.51	0.69
Effect size (d)		-	0.63	0.32
Absolute wight (g)	Right testis	1.44 ± 0.10	1.40 ± 0.16	1.45 ± 0.1	0.78	0.25
	Effect size (d)		0.3	0.1
	Left testis	1.41 ± 0.1	1.36 ± 0.16	1.44 ± 0.09	0.48	0.77
	Effect size (d)		0.38	0.3
	Right epididymis	0.2 ± 0.03	0.19 ± 0.02	0.17 ± 0.03	0.19	1.83
	Effect size (d)		0.4	1
	Left epididymis	0.18 ± 0.03	0.17 ± 0.06	0.19 ± 0.04	0.76	0.27
	Effect size (d)		0.2	0.3
	Seminal vesicle	1.17 ± 0.38	1.03 ± 0.29	0.82 ± 0.18	0.11	2.55
	Effect size (d)		0.4	1.2
Relative weight (%)	Right testis	0.58 ± 0.06	0.56 ± 0.08	0.54 ± 0.07	0.7	0.36
	Effect size (d)		0.3	0.6
	Left testis	0.56 ± 0.06	0.55 ± 0.08	0.54 ± 0.07	0.88	0.13
	Effect size (d)		0.14	0.3
	Right epididymis	0.08 ± 0.01	0.08 ± 0.01	0.06 ± 0.02	0.15	2.09
	Effect size (d)		0	1.3
	Left epididymis	0.07 ± 0.01	0.07 ± 0.02	0.07 ± 0.02	0.93	0.07
	Effect size (d)		0	0
	Seminal vesicle	0.46 ± 0.12	0.41 ± 0.12	0.30 ± 0.08a	0.04	3.74
	Effect size (d)		0.4	1.5

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

The oxidative status

Coragen administration negatively impacted antioxidant balance, as displayed in Table 2. Exposure to 1/10 LD₅₀ coragen caused a notable reduction in SOD levels with (d = 0.5) indicating a small effect, a marked increase in CAT levels with a large effect (d = 1.8), and a modest increase in GSH levels with a small effect size (d = 0.3). In contrast, rats administered 1/5 LD₅₀ coragen showed a marked reduction in SOD and CAT levels along with an elevation in GSH levels with a large effect size of (d = 2.9, d = 8.1 and 2.1), respectively.

Table 2.

Impact of coragen exposure on testicular oxidative stress parameters

Groups	Control	Coragen-1/10 LD50 (500 mg/kg)	Coragen-1/5 LD50 (1000 mg/kg)	P-value	f-value
MDA (nmol/g)	94 ± 10.2	133 ± 14.1a	157 ± 8.83ab	0	1039.89
Effect size (d)		3.2	6.6
SOD (U/gm)	1053 ± 81.5	1017.25 ± 60.9a	830 ± 73.6ab	0	1620.13
Effect size (d)		0.5	2.9
Catalase (U/g)	98.75 ± 2.63	108 ± 6.68a	79.50 ± 2.08ab	0.00002	45.37
Effect size (d)		1.8	8.1
GSH (mmol/g)	3.71 ± 0.58	3.90 ± 0.66	4.76 ± 0.43ab	0.0004	20.76
Effect size (d)		0.3	2.1

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. “b” indicates significance compared to coragen 1/10 LD₅₀. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

Compared to the control group, both the low-dose and high-dose treatment groups had markedly elevated lipid peroxidation biomarker (MDA) levels with a large effect size (d = 3.2 and 6.6), respectively. Notably, the high-dose group showed a more pronounced increase in MDA and GSH levels while demonstrating a greater decrease in SOD and CAT activity compared to the low-dose group.

The impact of coragen on testicular enzymes

Rats administered a low dose (500 mg/kg) and a high dose (1000 mg/kg) revealed a significant increase in testicular lactate dehydrogenase levels compared to the control group, with a large effect size (d = 18.3 and 14.4). The increase was more pronounced in the low-dose group, as shown in Table 5.

Table 5.

Impact of coragen exposure on reproductive hormone levels, testicular enzymes, and the steroidogenic enzymes

Groups	Control	Coragen-1/10 LD50 (500 mg/kg)	Coragen-1/5 LD50 (1000 mg/kg)	P-value	f-value
Testosterone (ng/ml)	3.93 ± 0.16	1.37 ± 0.05a	2.88 ± 0.19ab	0.000	14.89
Effect size (d)	-	21.6	6
FSH (IU/L)	1.46 ± 0.3	2.34 ± 0.3a	2.19 ± 0.4a	0.00002	48.02
Effect size (d)	-	3	2
LH (IU/L)	2.75 ± 0.38	4.15 ± 0.3a	3.55 ± 0.37ab	0	118.4
Effect size (d)	-	4.1	2.1
LDH (p mol/ml	0.87 ± 0.08	2.095 ± 0.05a	1.832 ± 0.05ab	0.000	519.383
Effect size (d)	-	18.3	14.4
STAR (ng/ml)	3.94 ± 0.48	2.75 ± 0.15a	3.63 ± 0.17b	0.000	19.974
Effect size (d)	-	3.3	0.86
17- β HSD (ng/ml)	2.72 ± 0.08	1.16 ± 0.12a	2.05 ± 0.07ab	0.000	335.906
Effect size (d)	-	15.3	8.9

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. “b” indicates significance compared to coragen 1/10 LD₅₀. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

Estimation of apoptotic markers

Coragen administration induced apoptosis in testicular tissues, as presented in Table 3. Both treated groups exhibited a marked increase in pro-apoptotic markers, including caspase-3 and Bax, with a large effect size (d = 2.2 and 3 for caspase-3, d = 5.4 and 4.9 for Bax). In contrast, the anti-apoptotic marker Bcl-2 was significantly reduced in both treated groups, with a large effect size (d = 6.2 and 3.1) for the 500 mg/kg and 1000 mg/kg doses. However, the lower dose of coragen caused a more pronounced increase in caspase-3 and Bax levels and a more substantial decrease in Bcl-2 levels compared to the higher dose.

Table 3.

Impact of coragen exposure on testicular apoptosis markers

Groups	Caspase-3 (ng/mg)	Bcl-2 (ng/mg)	Bax (ng/mg)
Control	0.79 ± 0.03	11.98 ± 0.68	2.77 ± 0.28
Coragen-1/10 LD50 (500 mg/kg)	1.08 ± 0.18a	8.53 ± 0.39a	4.89 ± 0.48a
Effect size (d)	2.2	6.2	5.4
Coragen-1/5 LD50 (1000 mg/kg)	0.88 ± 0.03	9.88 ± 0.68ab	4.10 ± 0.26ab
Effect size (d)	3	3.1	4.9
P-value	0.009	0.00007	0.00005
f-value	8.32	33.9	36.98

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. “b” indicates significance compared to coragen 1/10 LD₅₀. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

Testicular DNA damage

The comet assay results, presented in Table 4, show significant DNA damage following coragen exposure. The groups exposed to coragen (500 mg/kg) showed a notable increase in comet percentage, tail length, DNA in the tail (%), tail moment, and olive moment compared to the control group with large effect sizes (d = 19.6, 2.9, 7, 2.7, and 3.8) respectively. The group exposed to coragen (1000 mg/kg) exhibited higher marked elevation in comet percentage, tail length, DNA in the tail (%), tail moment, and olive moment with effect sizes 41.6, 5.2, 4.3, 5.8, and 3.6 respectively, indicating a large effect. The greatest impact was observed in the high-dose group (Fig. 1). Significant differences were also found between the two treated groups for comet percentage, tail length, tail moment, and olive moment. In contrast, changes in DNA in the tail percentage were insignificant.

Table 4.

The comet assay parameters for all experimental groups

Groups	Control	Coragen-1/10 LD50 (500 mg/kg)	Coragen-1/5 LD50 (1000 mg/kg)	P-value	f-value
Comet %	11.32 ± 0.36	21.52 ± 0.64a	26.29 ± 0.36ab	0	2355.87
Effect size (d)		19.6	41.6
Tail length (px)	8.91 ± 0.54	10.27 ± 0.38a	11.61 ± 0.5ab	0	72.26
Effect size (d)		2.9	5.2
% DNA in tail	4.49 ± 0.28	6.48 ± 0.29a	6.48 ± 0.6a	0	67.62
Effect size (d)		7	4.3
Tail moment	0.38 ± 0.05	0.59 ± 0.1a	0.70 ± 0.06ab	0	42.07
Effect size (d)		2.7	5.8
Olive moment	0.68 ± 0.05	0.85 ± 0.04a	1.01 ± 0.13ab	0	34.03
Effect size (d)		3.8	3.6

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. “b” indicates significance compared to coragen 1/10 LD₅₀. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

Fig. 1.

Illustration representing testicular DNA as per comet assay. a, b Control group, the cells had spherical, tail-free cells and undamaged nuclei. c, d Low dose group exposed to 500 mg/kg of coragen and e, f high dose group exposed to 1000 mg/kg of coragen showed a high degree of DNA damage in both treated groups

Reproductive hormone profile

Coragen administration significantly altered reproductive hormone levels, as presented in Table 5. Serum testosterone levels were markedly lower in the treatment groups compared to the control, with large effect sizes (d = 21 for the low dose and d = 6 for the high dose). Additionally, both treatment groups exhibited significant increases in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, with large effect sizes (d = 3 and 2 for FSH, d = 4.1 and 2.1 for LH). Notably, the low-dose group showed a more pronounced decrease in testosterone and greater increases in FSH and LH compared to the high-dose group. Significant differences between the treated groups were observed in testosterone and LH levels, while changes in FSH levels were not statistically significant.

The steroidogenic enzymes

Exposure to coragen pesticides resulted in a marked decline in steroidogenic acute regulatory protein (STAR) expression in the low-dose group (d = 3.3) and a less pronounced decrease in the high-dose group (d = 0.86), both showing a strong effect size relative to the control. Both treatment groups exhibited a notable reduction in 17β-hydroxysteroid dehydrogenase (17β-HSD) expression with a large effect size (d = 15.3 and d = 8.9), as indicated in Table 5. Significant differences in STAR and 17β-HSD expression were observed between the two treated groups.

Testicular histopathology

The testicular sections of the control group exhibited the typical structure of seminiferous tubules, including spermatogenic cells, germ cells, and organized interstitial cells. The germinal epithelium lined the oval seminiferous tubules, which contained two types of cells: germ cells and Sertoli cells. Sertoli cells, interspersed among the spermatogenic cells, had visible nucleoli. The spermatogenic cells were arranged in rows, representing different stages of spermatogenesis, including spermatogonia at the basement membrane, primary spermatocytes, spermatids, and mature spermatozoa within the lumen. Leydig interstitial cells were observed in the interstitium (Fig. 2a, b).

Fig. 2.

Photomicrographs of hematoxylin and eosin-stained testicular tissue, comparing histological changes across three experimental groups: a, b Control testicular tissue displaying interstitial and seminiferous tubules (ST). Germinal epithelium (G) was stratified and lined each tubule. The spermatid (hollow arrow), spermatogonia (bifid arrow), spermatocytes (notch arrow), and spermatozoa (S) are signs of healthy spermatogenic cells. Leydig's interstitial cells can be seen (arrow). c–f The low-dose coragen treatment group (500 mg/kg) revealing disorganized seminiferous tubules and a significant loss in the spermatogenic cell lineage (double head arrow). A set of seminiferous tubules with considerable damage to their architecture and basement membrane disruption and vacuolation (wavy arrow). Within the lumen of specific seminiferous tubules, injured spermatogenic cells (Sloughed germ cells) were found falling (bold arrow). In the inter-tubular connective tissue, there was a decrease in interstitial cells (arrow) and an increase in interstitial width(triangle). g–i The high-dose coragen treatment group (1000 mg/kg) showing disorganized seminiferous tubules and a significant loss in the spermatogenic cell lineage (double head arrow). A set of seminiferous tubules with considerable damage to their architecture and basement membrane disruption. Seminiferous tubules that are aberrant with uneven shape and irregular basement membrane (curved arrow) and vacuolation (wavy arrow). In the inter-tubular connective tissue, there was a decrease in interstitial cells (arrow) and an increase in interstitial width (triangle). There was a hemorrhagic area in the interstitial space (star). Absence of spermatozoa, spermatid, and spermatocyte

In contrast, the testicular tissue of the treated groups showed various degrees of damage; notable abnormalities included disruption of the basement membrane, degeneration of spermatogenic cell lineage, vacuolization, reduced interstitial cells, and increased interstitial width in the inter-tubular connective tissue and significant disorganization and damage to the seminiferous tubules. Meanwhile, the low-dose (500 mg/kg) group exhibited injured spermatogenic cells within the lumen of specific seminiferous tubules (Fig. 2c–f). The high-dose (1000 mg/kg) group displayed an irregular basement membrane, a hemorrhagic area in the interstitial space, and a substantial number of seminiferous tubules devoid of spermatozoa, spermatids, and spermatocytes (Fig. 2g–j).

The Johnson Score outcomes supported these findings, with a marked decline in scores in the rats treated with low dose (500 mg/kg) and high dose (1000 mg/kg) of coragen, compared to the control with effect size (d = 2.1 and d = 3), respectively, which indicates a large effect size, with the higher dose (1000 mg/kg) displaying a marked decreased in Johnson score than lower dose (500 mg/kg) group. There was a substantial difference between both treated coragen groups; 1/5 LD₅₀ significantly reduced the Johnson score compared to 1/10 LD₅₀.

Morphometric outcomes

The morphometric analysis of all groups is displayed in Table 6. The group administered with 1/10 LD₅₀ of coragen exhibited a slight decrease in tubule diameter, a minor increase in lumen diameter, and a notable reduction in germinal thickness with a small effect size for tubule diameter (d = 0.3) and lumen diameter (d = 0.5) and a large effect size for germinal thickness (d = 2.3).

Table 6.

Testicular morphometric analysis and Johnson score for all experimental groups

Groups	Control	Coragen-1/10 LD50 (500 mg/kg)	Coragen-1/5 LD50 (1000 mg/kg)	P-value	f-value
Tubule diameter (µm)	282.90 ± 28.6	272.50 ± 33.1	261.24 ± 38a	0.03	3.68
Effect size (d)	-	0.3	0.6
Lumen diameter (µm)	126.73 ± 16.7	134.46 ± 15.7	141.89 ± 21.7a	0.003	6.05
Effect size (d)	-	0.5	0.8
Germinal thickness (µm)	59.52 ± 5.6	46.73 ± 5.3a	38.45 ± 6.3ab	0	119.2
Effect size (d)	-	2.3	3.5
Johnson score	9.72 ± 0.35	6.91 ± 1.83a	5.10 ± 2.16ab	0	69.97
Effect size (d)	-	2.1	3

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. “b” indicates significance compared to coragen 1/10 LD₅₀. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

In contrast, the group administered 1/5 LD₅₀ of coragen revealed a marked decrease in tubule diameter and germinal thickness, along with a notable increase in lumen diameter. Effect sizes were moderate for tubule diameter (d = 0.6) and large for lumen diameter and germinal thickness (d = 0.8 and d = 3.5). The high-dose group showed a more significant reduction in tubule diameter and germinal thickness and a greater increase in lumen diameter than the low-dose group.

Sperm quality outcome

The sperm concentration and percentages of motility, viability, and abnormality are reported in Table 7. Coragen treatment markedly reduced sperm concentration, motility, and viability in both treated groups compared to the control group, with large effect sizes (d = 40, 19.7 for concentration; d = 1.5, 3.6 for motility; and d = 12.2, 7.3 for viability). Sperm abnormalities were remarkably higher in the low-dose (500 mg/kg) group compared to the high-dose (1000 mg/kg) group, with large effect sizes at both doses (d = 10 and d = 1.3). These abnormalities included various sperm defects such as lack of hook heads, banana-shaped heads, amorphous shape heads, small heads, and tail defects (Fig. 3). Specifically, the low dose (500 mg/kg) group showed a strong effect size for hookless heads (d = 2), amorphous heads (d = 10.9), banana-shaped heads (d = 2.4), and tail defects (d = 15.5). In contrast, the high dose (1000 mg/kg) group showed no effect size for hookless heads, small heads, and large heads (d = 0), a small effect size for banana-shaped heads (d = 0.4), and a large effect size for amorphous heads (d = 5.5) and of tail defects (d = 1.9). However, the low-dose group exhibited more significant negative impacts on sperm quality, with greater reductions in motility, viability, and increased abnormalities compared to the high-dose group.

Table 7.

Impact of coragen exposure on sperm quality parameters

Groups	Control	Coragen-1/10 LD50 (500 mg/kg)	Coragen-1/5 LD50 (1000 mg/kg)	P-value	f-value
Sperm motility %	45.00 ± 1.58	0.00a	19.00 ± 1.0ab	0.00	2187.14
Effect size (d)	-	40	19.7
Sperm Conc. (106/mol)	53.20 ± 4.97	44.20 ± 6.87a	40.40 ± 1.14a	0.007	7.71
Effect size (d)	-	1.5	3.6
Live sperms %	72.20 ± 1.92	48.80 ± 1.92a	60.60 ± 1.14ab	0.00	236.02
Effect size (d)	-	12.2	7.3
Sperms abnormalities %	17.80 ± 1.64	42.80 ± 3.1a	19.80 ± 1.3b	0.00	205.32
Effect size (d)	-	10	1.3
Without hook head %	1.80 ± 0.45	3.20 ± 0.84a	1.80 ± 0.45b	0.004	8.91
Effect size (d)	-	2	0
Amorphous shape head %	4.40 ± 0.55	10.40 ± 0.55a	7.40 ± 0.55ab	0.00	150
Effect size (d)	-	10.9	5.5
Banana shape head %	0.60 ± 0.55	1.80 ± 0.45a	0.80 ± 0.45b	0.004	8.86
Effect size (d)	-	2.4	0.4
Small head %	0.00	0.20 ± 0.45	0.00	0.39	1.00
Effect size (d)	-	0.6	-
Large head %	0.00	0.00	0.00	-	-
Effect size (d)	-	-	-
Tail defect %	11.00 ± 0.71	27.20 ± 1.30a	9.80 ± 0.55	0.00	574.1
Effect size (d)		15.5	1.9

The values (n = 7) are expressed as the mean ± SD. The letter “a” denotes significance (P < 0.05) against the control group. “b” indicates significance compared to coragen 1/10 LD₅₀. Effect size d was interpreted as follows: < 0.20 is a weak effect, 0.21–0.50 is a modest effect, 0.50–0.8 is a moderate, and > 0.8 is a strong effect

Fig. 3.

Photomicrographs of sperm morphology following coragen treatment. a Sperm with normal morphology. b–g Sperm with morphological abnormalities; banana head without hook (notch arrow), fold tail (bold arrow), amorphous hook (dotted arrow)

Correlations between different variables

Pearson correlation analysis of coragen-induced lipid peroxidation in intoxicated rats revealed significant relationships with various biological markers. Lipid peroxidation showed a strong positive correlation with DNA damage, as measured by the Olive Tail Moment (OTM) and apoptotic marker protein Bax, with correlation values of 0.889 and 0.682, respectively. Conversely, lipid peroxidation exhibited a significant negative correlation with sperm parameters, including sperm motility and the percentage of live sperm, with correlation values of − 0.764 and − 0.685, respectively. Additionally, a significant negative correlation was found between OTM and the percentage of live sperm and sperm motility, with correlation values of − 0.586 and − 0.636, respectively. Bax showed a significant positive correlation with testosterone and sperm abnormalities, with values of 0.691 and 0.656, respectively. At the same time, it was negatively correlated with the percentage of live sperm and sperm motility, with values of − 0.795 and − 0.790, respectively. Caspase-3 demonstrated a significant positive correlation with Bax and testosterone, with correlation values of 0.621 and 0.681, respectively. Sperm abnormalities were significantly negatively correlated with the percentage of live sperm and sperm motility, with correlation values of − 0.864 and − 0.842, respectively. Furthermore, Johnson’s score was significantly correlated with testicular tubule diameter and germinal thickness, with correlation values of 0.202 and 0.574, respectively, as shown in Table 8.

Table 8.

Pearson correlations between different variables

Correlations
		Olive tail moment	Bax	Live sperm %	Motility
MDA	Pearson correlation	.889**	.682*	− .685*	− .764**
	Sig. (2-tailed)	0.000	0.015	0.014	0.004
		Testosterone	Live sperm %	Sperm abnormality	Motility
Bax	Pearson correlation	.691*	− .795**	.656*	− .790**
	Sig. (2-tailed)	0.013	0.002	0.021	0.002
		Bax	Testosterone
Caspase-3	Pearson correlation	.621*	.681*
	Sig. (2-tailed)	0.031	0.015
		Live sperm %	Motility
OTM	Pearson correlation	− .586*	− .636*
	Sig. (2-tailed)	0.045	0.026
		Motility	Live sperm %
Sperm abnormality	Pearson correlation	− .842**	− .864**
	Sig. (2-tailed)	0.000	0.000
		Tubule diameter	Tubule thickness
Johnson’s score	Pearson correlation	.202*	.574**
	Sig. (2-tailed)	0.039	0.000

^**Correlation is significant at the 0.01 level (2-tailed)

^*Correlation is significant at the 0.05 level (2-tailed)

Discussion

Pesticide use is a global concern due to its adverse environmental and human health impacts. These chemicals can enter the body through dermal contact, ingestion, or inhalation, reaching cellular and nuclear levels. Pesticides can induce cytotoxicity directly or indirectly by generating free radicals. Consequently, multiple biochemical markers should be assessed to evaluate the physiological status of damaged organs and tissues.

Pesticides contribute significantly to male infertility, primarily by disrupting spermatogenesis (Kumar and Singh 2022). These effects include reduced sperm count, decreased sperm motility, and hormonal imbalances. Moreover, pesticide exposure contributes to abnormal sperm morphology, increased sperm DNA damage, and adverse changes in testicular histology.

With its limited vascularization, the testis is typically exposed to low oxygen levels, which are crucial for spermatogenesis. However, its high unsaturated fat content makes it particularly vulnerable to oxidative and peroxidative damage (Mohamed et al. 2019).

A disruption in the balance between reactive oxygen species and antioxidant defense mechanisms can compromise the integrity of the hypothalamic-pituitary–gonadal axis, impairing spermatogenesis and contributing to male infertility (Mahmoud et al. 2024). Coragen treatment further disrupts the antioxidant defense system and provokes oxidative damage (Abdel-Mobdy et al. 2021), impairing male fertility by damaging sperm phospholipids, DNA, and proteins. This oxidative damage triggers apoptotic cell death, ultimately reducing sperm quality and function (Albarakati et al. 2020). The antioxidant defense system and free radical formation are balanced by the antioxidant enzymes that scavenge reactive oxygen species. In this study, coragen administration induced oxidative stress by decreasing SOD levels across the treatment groups. CAT levels showed a dose-dependent response, with a significant decrease in the high-dose group and a significant increase in the low-dose group. GSH levels were elevated, indicating a potential compensatory response to counteract oxidative stress. This increase in GSH likely reflects an adaptive mechanism to mitigate oxidative damage caused by coragen exposure, compensating for the dysregulation of SOD and CAT. The dose-dependent changes in CAT levels suggest that lower doses of coragen may trigger an adaptive antioxidant response to minimize oxidative damage.

In contrast, higher doses may overwhelm this protective mechanism, reducing both SOD and CAT activities despite the increase in GSH. The reduction in CAT activity indicates that coragen diminishes the antioxidant capacity of testicular cells, causing oxidative stress, while the reduction in SOD activity likely led to an accumulation of superoxide anions, intensifying lipid peroxidation. Malondialdehyde (MDA), a hallmark of lipid peroxidation, was significantly elevated in both treatment groups, with the high-dose group showing the most pronounced increase in MDA levels. Lower pesticide doses may cause gradual and cumulative damage, while higher concentrations lead to rapid and severe toxicity.

Additionally, pesticides can persist in the body with chronic exposure. Previous studies have shown similar effects. Abdel-Mobdy et al. (2021) demonstrated that coragen exposure at doses of 1/20 and 1/40 LD₅₀ induced oxidative stress in rats by decreasing SOD, GPX, and GST activities, while increasing lipid peroxidation (LPx) levels. Meligi et al. (2019) reported that coragen administration altered the activities of glutathione peroxidase (Gpx) and superoxide dismutase (SOD) in plasma, leading to oxidative stress. Dutta et al. (2014) found that coragen exposure increased thiobarbituric acid-reactive substances (TBARS), a marker of lipid peroxidation, leading to oxidative stress. Bantu and Vakita (2013) showed that coragen enhanced the activities of Gpx and SOD in treated fish tissues. Further studies of diamide insecticides also support these findings. Meng et al. (2022a, b) revealed that chlorantraniliprole exposure caused significant oxidative stress in the liver of zebrafish, marked by increased malondialdehyde (MDA) levels and subsequent apoptosis, as indicated by changes in apoptosis-related gene expression. Similarly, cyantraniliprole elevated lipid peroxidation levels in the reproductive organs of female Wistar rats and increased SOD activity in the uterus and GPx activity in the ovary (da Silva Scarton et al. 2022). Cyantraniliprole also induced oxidative stress and DNA damage in earthworms (Eisenia fetida) (Qiao et al. 2019; Xue et al. 2023).

Rats exposed to flubendiamide exhibited increased MDA levels and decreased cellular glutathione levels, along with reduced activities of SOD, CAT, GPx, and glutathione-S transferase (Mandil et al. 2016). In addition, Cui et al. (2017) demonstrated that three diamides—flubendiamide, chlorantraniliprole, and cyantraniliprole—induced acute and long-term toxicity in Daphnia magna, leading to a significant increase in reactive oxygen species (ROS), elevated catalase (CAT) activity, and a significant reduction in the activities of antioxidant enzymes, including SOD and GPx.

LDH, a key enzyme involved in energy metabolism, is essential for normal testicular function. It supports energy production, cellular processes, and hydrogen transport within sertoli and spermatogenic cells. Changes in LDH levels are linked to impaired testicular function and damage to Sertoli and germ cells (Mohamed et al. 2019). Coragen administration in rats, even at low doses, increased lactate dehydrogenase (LDH) levels in the testes, suggesting potential testicular dysfunction. This increased LDH can be attributed to several potential mechanisms, primarily linked to oxidative stress and disruption of cellular energy metabolism in testicular cells. Oxidative stress from coragen exposure can cause membrane and mitochondrial damage, causing cell dysfunction or death, and subsequently releasing LDH.

Additionally, low-dose coragen may interfere with mitochondrial function or glycolytic pathways in testicular cells. Since LDH plays a crucial role in anaerobic glycolysis by converting pyruvate to lactate, testicular cells may shift to anaerobic metabolism to maintain energy production, leading to a compensatory increase in LDH secretion. The decreased sperm motility in the low-dose group further supports this hypothesis. Increased LDH levels can shift sperm cell metabolism toward anaerobic glycolysis. Since sperm motility depends on energy production, particularly ATP generation via oxidative phosphorylation in mitochondria, it negatively impacts sperm quality and sperm quality. In line with these findings, studies on the herbicide carbofuran have shown that prolonged exposure increases testicular LDH activity and negatively affects epididymal sperm parameters, such as reduced motility and increased abnormalities (Sobhy et al. 2017).

Our findings suggest that coragen-induced reproductive failure primarily results from oxidative damage in testicular cells, which activates the intrinsic apoptotic pathway. This is supported by the upregulation of caspase-3 and Bax, along with the downregulation of bcl-2. Germ cell apoptosis may also result from low testosterone levels, as adequate testosterone is essential for normal spermatogenesis and germ cell survival. Thus, alterations in oxidative stress and testosterone biosynthesis in the testes may lead to germ cell death, ultimately inhibiting spermatogenesis (Verma and Singh 2017).

Consistent with the current findings, Meng et al. (2022a, b) reported that flubendiamide, a widely used diamide insecticide, induced oxidative stress and apoptosis in the liver of zebrafish. Alterations in catalase activity marked this, MDA and glutathione levels, and increased mRNA expression of p53, PUMA, caspase-3, caspase-9, and Apaf-1, while decreasing the Bcl-2/Bax ratio. Similarly, flubendiamide triggered oxidative stress and apoptosis in the leafworm Spodoptera litura, with elevated MDA levels, reduced antioxidant enzymes activities (SOD, CAT, and GST), and increased expression of apoptotic genes (caspase-1, −3, and −5) (Jameel et al. 2023).

Coragen exposure in rats led to reduced Bcl-2 and increased Bax levels. It is causing DNA fragmentation in sperm chromatin, negatively affecting sperm motility and morphology. Additionally, coragen elevated lipid peroxidation, disrupting cellular membranes and causing DNA damage (Hathout et al. 2021). This damage disrupts critical proteins and nucleic acids, alters gene expression, and regulates apoptotic genes. The high-dose treatment group exhibited more pronounced DNA damage, likely due to higher levels of MDA, which generate more free radicals and cause oxidative stress. This increased oxidative stress may further affect the expression and function of apoptotic genes, such as those encoding caspase-3, Bax, and Bcl-2. In contrast, the low-dose group experienced less oxidative stress, likely triggering apoptotic pathways earlier, leading to poorer sperm quality.

Interestingly, the low-dose group showed higher levels of apoptotic proteins than the high-dose group. This may be because the lower dose may induce earlier activation of apoptotic pathways, leading to elevated levels of apoptotic proteins. In comparison, the higher dose caused more extensive DNA damage, which may have altered or inactivated the apoptotic genes, resulting in less pronounced changes in apoptotic protein levels. These findings suggest a non-linear dose–response, where lower doses trigger earlier apoptotic effects, while higher doses cause more severe DNA damage and oxidative stress, disrupting gene expression and apoptotic protein regulation. This evidence supports the idea that the high-dose treatment significantly impacts DNA damage, which in turn affects apoptotic gene expression and protein levels.

Testicular DNA damage is a critical factor contributing to male infertility. The comet assay provided a sensitive measure of DNA strand breakage and revealed a significant increase in testicular DNA damage following coragen exposure, indicating substantial DNA fragmentation and cellular disruption. This can impair spermatogenesis, alter gene expression, and disrupt fertility-related processes. The failure of antioxidant enzymes to adequately remove excess reactive oxygen species (ROS) likely caused DNA strand breaks and increased malondialdehyde (MDA) levels. These findings align with those of Omar et al. (2022), who reported significant DNA damage in the liver and kidney tissues of both mothers and fetuses exposed to coragen. Additionally, Said et al. (2021) observed increased testicular DNA fragmentation following sulfx treatment, suggesting impaired spermatogenesis.

Endocrine-disrupting chemicals (EDCs), including pesticides, can interfere with hormone production or mimic their effects, often disrupting the hypothalamic-pituitary–gonadal (HPG) axis. This axis regulates gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gland to produce FSH and LH, signaling the leydig cells in the testes to secrete testosterone. Disruptions in this hormonal cascade can lead to impaired spermatogenesis and sperm quality. Several studies have shown that pesticides can negatively impact the production of sex hormones, contributing to male reproductive dysfunction (Ghafouri-Khosrowshahi et al. 2019). Our findings indicate that coragen disrupted male hormone levels in rats by significantly decreasing testosterone and increasing FSH and LH compared to the control group. These hormonal changes suggest dysfunction in the HPG axis (Ben Abdallah et al. 2012), likely due to oxidative stress from high ROS levels and antioxidant imbalance, leading to leydig cell damage. The reduced testosterone production could also be linked to the inhibition of testicular steroidogenesis, as evidenced by decreased steroidogenic enzymes. The elevated LH and FSH levels reflect the negative feedback regulation of the hypothalamic–pituitary‐testicular (HPT) axis in response to low testosterone, further disrupting spermatogenesis and steroidogenesis. In line with these findings, Hassan et al. (2021a, b) reported a decrease in total and free testosterone in rats treated with coragen, impacting their reproductive function. In male rats, thiamethoxam exposure led to increased levels of FSH and LH with decreased testosterone levels (Abd-Allah and Abd El-Rahman 2023). Elevated FSH levels suggest damage to the germinal epithelium, often linked to azoospermia or oligospermia in infertile males. Additionally, elevated levels of FSH and LH indicate hypergonadotropic hypogonadism, which can result from testicular failure, agenesis, seminiferous tubular disease, or androgen production abnormalities (Abd El-Rahman and Omar 2022).

Oxidative stress in the testis can impair both steroidogenesis and spermatogenesis. The steroidogenic acute regulatory (StAR) protein plays a crucial role in steroidogenesis by transporting cholesterol from the cytosol to the mitochondria. There, CYP11A1 converts cholesterol into pregnenolone. Pregnenolone is further converted into dehydroepiandrosterone and androstenedione by steroidogenic enzymes such as CYP17A1, 3β-HSD, and 17β-HSD, and ultimately into testosterone (Xia et al. 2024). Our study found that coragen downregulated testicular StAR and 17β-HSD enzymes, indicating a disruption in testosterone production and a potential disturbance in the HPG axis (Tijani et al. 2024). Notably, this effect was more pronounced in the low-dose treatment group, suggesting that even minimal environmental exposure to coragen could disrupt the endocrine system and impact male reproduction. These results raise concerns about the reproductive toxicity of coragen (Lahimer et al. 2023).

Similarly, imidacloprid insecticide has been shown to suppress testicular StAR expression and reduce testosterone levels, accompanied by increased FSH and LH levels and altered redox balance in the testes (Mehanna et al. 2022). Hexachlorobenzene (HCB) pesticide also disrupts male reproduction by increasing FSH and LH levels while decreasing testosterone and reducing steroidogenic enzyme activity, including 3β-HSD and 17β-HSD. HCB exposure is also linked to elevated pro-apoptotic markers (Bax, caspases-3, and −9) and histological testicular abnormalities (Stephanie et al. 2023).

Histopathology is a crucial tool for assessing male reproductive toxicity. Insecticides can directly damage the testes and suppress androgen production, impairing fertility (Zakzook et al. 2020). The biochemical abnormalities were consistent with the histopathological findings, showing testicular alterations across different dose groups. Coragen-treated rats exhibited dilated, congested blood vessels, irregular seminiferous tubules, and a wide interstitium in the testes. Spermatogenic cells were disorganized, lacking spermatozoa in the lumen, with detached basal layer cells and intensely pigmented spermatogonia nuclei (Hassan et al. 2021a, b). Similar damage was observed in rats exposed to flubendiamide, which showed testicular congestion, deteriorating germ cells with nuclear pyknosis, necrosis, vacuolation, and spermatid loss (Mandil et al. 2016).

Coragen likely binds to the lipid components of the mitochondrial fraction within germ cells, disrupting testicular tissue and contributing to infertility. This damage is reflected in reduced seminiferous tubule diameter and germinal epithelium thickness. These findings are consistent with those of Said et al. (2021), who reported that Sulfix induced testicular damage by decreasing seminiferous tubule size and width due to spermatogenic cell destruction. The Johnson scores results aligned with the histological observations, as coragen-treated groups exhibited significantly lower Johnson scores than the controls. The Johnson score indicates that the reduction in spermatogonia and spermatocyte counts may be attributed to testicular oxidative injury (Nna et al. 2019).

A study involving 2122 individuals linked altered sperm parameters to occupational pesticide exposure. The researchers observed that males exposed to pesticides had lower semen volume, motility, and vitality (Daoud et al. 2017). Similarly, the current study found significant decreases in sperm count, motility, and viability in both treatment groups, accompanied by impaired spermatogenesis and various sperm morphological abnormalities, which were more pronounced in the low-dose group. The decrease in sperm motility in the low-dose group was attributed to an increased incidence of tail defects. In contrast, reduced sperm concentration indicated testicular cell damage caused by coragen, which disrupts spermatogenesis and decreases sperm count and viability (Somade et al. 2021). Most abnormal sperm in the epididymis are hormone-dependent (Hassan et al. 2021a, b), and testicular apoptosis is influenced by hormonal imbalances (Asadi et al. 2021). Consequently, both elevated and reduced serum hormone levels can negatively impact sperm DNA integrity and semen quality, contributing to male infertility (Bahrami et al. 2023).

Since testosterone promotes spermatogenesis, insufficient levels of this hormone may lead to poor sperm quality (Saber et al. 2021). Hormonal changes, particularly elevated levels of FSH and LH, can impair sperm parameters such as concentration, motility, and morphology. In line with these findings, Ghasemian et al. (2017) reported that elevated FSH and LH levels were associated with reduced sperm motility, increased sperm count, and morphological abnormalities. These studies emphasize the negative impact of hormonal imbalances on sperm quality.

Another contributing factor to lower sperm quality is coragen’s ability to induce oxidative stress through the increased generation of ROS. Elevated ROS levels cause DNA damage, further decreasing sperm motility and count. This oxidative damage leads to malformed sperm heads damaged acrosomes, and abnormal midpieces. Spermatozoa are highly susceptible to lipid peroxidation (Gamal et al. 2022), which damages sperm membranes, causing motility loss and membrane disintegration (Adedara et al. 2018). Additionally, peroxidation of phospholipids, proteins, and nucleotides within the testes can alter cytoplasmic organelles, causing severe degeneration of seminiferous tubules. This testicular toxicity impairs sperm function, reducing motility, count, and vitality and increasing sperm abnormalities (El-Okle et al. 2016). Malathion administration for 60 days (three times per week) decreased sperm motility and count while increasing abnormal sperm morphology (Gaber et al. 2023). Similarly, abamectin demonstrated a dose-dependent decline in motile sperm and an increase in aberrant sperm forms, highlighting its adverse impact on male sperm quality (Kolianchuk et al. 2023).

Coragen pesticide exhibits a non-linear dose–response relationship regarding male reproductive health, with distinct effects at both low and high doses. At low doses, prolonged exposure and potential bioaccumulation may lead to a gradual, insidious impact on male fertility. This occurs primarily through hormonal imbalances that disrupt endocrine function, impairing sperm quality and morphology via specific signaling pathways. Also, prolonged exposure at low doses may increase apoptotic protein levels, compromising reproductive health. In contrast, high-dose exposure causes more immediate and severe effects. It induces significant oxidative stress and lipid peroxidation, damaging cellular and DNA. This oxidative damage is associated with the inactivation or alteration of apoptotic genes, further disrupting reproductive function. As a result, high doses cause more direct and acute damage to sperm DNA and reproductive structures.

Overall, the impact of coragen on male fertility is multifaceted. Low doses primarily affect hormonal balance, sperm quality, and apoptosis regulation, while high doses lead to severe oxidative damage and DNA alterations, compromising the structural integrity of the reproductive system. The severity and nature of these effects are linked to dosage and exposure duration.

Conclusion

The present investigation provides evidence that exposure to coragen has adverse effects on male reproductive health through the disruption of endocrine function and the induction of reproductive toxicity. Supporting evidence comprises heightened DNA damage, oxidative stress in the testicles, and stimulation of apoptosis. The alterations manifest in modified testicular histology, resulting in compromised sperm quality and finally affecting male fertility. The results emphasize the possible risks of coragen on the male reproductive system. Additional investigation is necessary to clarify the fundamental processes of coragen’s toxicity and its complete influence on male biological reproduction.

The study offers important insights into the reproductive toxicity of coragen; however, some limitations need to be acknowledged. The doses administered in the study (500 mg/kg and 1000 mg/kg) may not correspond to human exposure levels. Determining the possible hazards at lower, environmentally applicable doses is needed. The eight-week exposure duration may inadequately reflect the long-term effects of coragen. Studies on chronic exposure are essential to assess the cumulative effects.

Further research is needed to comprehensively understand the mechanisms underlying Coragen’s toxicity and its long-term effects on male reproductive health. This includes examining the precise molecular pathways associated with Coragen-induced toxicity, evaluating the cumulative impacts of extended exposure to Coragen, and assessing the ecological consequences associated with the application of Coragen.

Future research needs to investigate the impacts of lower, environmentally relevant doses of Coragen to evaluate the possible hazards to human wellness more accurately. Additionally, investigating the molecular mechanisms contributing to Coragen’s toxicity, including how it collaborates with particular cellular processes, can yield significant knowledge of its mechanism of action.

Author contribution

H.A.A.R. and A.E.W.G. contributed to the research's conception, design, and execution. S.A.M.S. and H.A.A.R. performed the experiments. S.A.M.S. conducted the data collection, analysis, interpretation, graphing, and manuscript writing. H.A.A.R. contributed to evaluating and interpreting the data, revising and editing the manuscript, and final approval. All authors read the final manuscript and approved the submission. The authors declare that all data were generated in-house and that no paper mill was used.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

All source data for this work (or generated in this study) are available upon reasonable request.

Declarations

Ethical approval

The experimental protocols and procedures utilized in this study were approved by the Institutional Animal Care and Use Committee (IACUC), (approval number CU/ I/ F/ 40/21) Cairo University- Faculty of Science- Egypt.

Consent to participate

Not available.

Consent for publication

All authors have approved the final manuscript for publication.

Competing interests

The authors declare no competing interests.