Allicin and hesperidin protect sperm production from environmental toxins in mice

Abstract

This study investigates the protective effects of Allicin (A), a bioactive compound from garlic, and Hesperidin (HSD), a citrus flavonoid, against reproductive damage induced by para-nonylphenol (p-NP), an environmental pollutant, in mice. Fifty 8- to 10-week-old NMRI mice were divided into five groups: a control group, a group exposed to p-NP, and three groups treated with p-NP plus either Allicin, Hesperidin, or both. Treatments lasted for 35 days. Researchers analyzed sperm count, viability, motility, DNA damage, and morphology, alongside testicular parenchyma markers like antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels. Hormone levels (testosterone, LH, and FSH), testicular histopathology, apoptosis-related gene expression, and fertility indices were also evaluated. Results showed that mice treated with Allicin, Hesperidin, or both had reduced abnormal sperm morphology and DNA damage, with improvements in sperm motility, viability, and membrane integrity compared to the p-NP group. Antioxidant enzyme activities (TAC, SOD, GPx) and hormone levels increased, while MDA levels decreased, indicating reduced oxidative stress. Furthermore, expression of pro-apoptotic genes Bax and caspase-3 declined, while anti-apoptotic Bcl-2 expression rose. Treated mice demonstrated higher fertility indices and hormone levels. These findings suggest Allicin and Hesperidin mitigate p-NP-induced testicular damage by enhancing antioxidant defenses and regulating cell death pathways.

Introduction

Infertility among males is a rising concern, particularly in industrial areas where various environmental factors compromise reproductive health. Common issues include disruptions in sperm production, maturation, and fertilization capabilities, with environmental toxins, notably alkylphenols, playing a substantial role¹. Alkylphenol ethoxylates (APEs) serve as non-ionic surfactants, and their degradation product, para-nonylphenol (p-NP), is widely found in products like detergents, paints, phenolic resins, herbicides, and food packaging^2,3. Humans are commonly exposed to p-NP via skin contact, inhalation, and ingestion of contaminated food or water³. P-NP displays mild estrogenic effects^4,5, imitating estrogen by interacting with estrogen receptors at different life stages, from fetal to adult, thereby contributing to male reproductive issues⁶. Research shows that p-NP exposure during development can impact the male reproductive system in animal models, leading to reduced sperm production, undescended testicles, lower reproductive organ weight, and testicular anomalies⁷. Further studies have demonstrated that p-NP exposure at 250 mg/kg in adult rats causes hormonal disturbances, impacting testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels⁷. Like other environmental toxins, p-NP generates oxidative stress in the reproductive system. Research by Chitra et al.¹ has demonstrated that p-NP elevates oxidative stress levels within the epididymal sperm of adult rats. Studies also highlight that vitamin E (Vit. E), a strong free radical scavenger, is effective in preventing free-radical-induced membrane damage⁸. Moreover, vitamin E’s antioxidant properties are known to alleviate oxidative stress in testicle⁶.

Allicin (A; diallylthiosulphinate), a bioactive compound found in garlic (Allium sativum L.), has various biological properties, with a chemical structure of C6H10OS2. This compound is produced when garlic tissue is disrupted, converting the amino acid alliin (S-allylcysteine sulphoxide) through the enzyme alliinase⁹. Allicin can neutralize free radicals, thereby providing cellular protection against stress¹⁰. Studies have shown Allicin’s hypoglycemic and hypolipidemic effects in poultry¹¹, particularly in reducing liver enzymes such as ALT and AST, and enhancing immune responses by increasing antibody titers against pathogens like Newcastle disease virus in broiler chickens¹⁰. Recently, allicin’s status as a potent natural antioxidant has been highlighted, as it boosts the activity of internal antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), decreasing both inflammation and oxidative stress in male rabbits exposed to environmental toxins¹². In addition, El‐Ratel et al.¹³ showed that Allicin increased antioxidant enzyme activity, including SOD and glutathione peroxidase (GPx), which further confirmed its protective role in the male reproductive system of rats treated with doxorubicin.

Hesperidin (HSD; 3′,5,7-trihydroxy 4′-methoxy flavanone 7-rutinoside, also known as hesperetin 7-rutinoside) is a glycoside flavanone predominantly present in citrus fruits from the Rutaceae family¹⁴. Recognized as a natural antioxidant, HSD is known for its ability to reduce oxidative stress (OS)¹⁵. Research indicates that HSD has numerous pharmacological properties, including anti-inflammatory, anticancer, and antimicrobial effects^16,17. These applications are further supported by HSD’s high safety margin, non-accumulative nature, and minimal side effects^18–20. Studies have particularly underscored HSD’s protective effects against testicular toxicity in animals, attributing this to its antioxidant and free-radical-neutralizing capacities^21,22. Although various natural antioxidants have been studied for their roles in protecting against lead-induced testicular toxicity, which shares similar mechanisms of oxidative damage with p-NP, ^23,24, HSD’s specific effects have not been comprehensively examined in the context of p-NP exposure. This study evaluates the protective effects of the non-toxic antioxidants HSD and A on p-NP-induced testicular toxicity, focusing on sperm traits, biochemical markers, and histopathology. It addresses a critical gap in understanding natural antioxidants like Allicin and Hesperidin against reproductive toxicity caused by p-NP. Identifying safe therapeutic agents is crucial due to the widespread impact of p-NP on male fertility. By analyzing the effects of Allicin and Hesperidin, this research sheds light on strategies for mitigating environmental toxin-related reproductive harm, contributing to reproductive health and toxicology.

Results

The analysis revealed that mice in the p-NP group displayed a notable reduction in sperm concentration and motility, along with decreased values for curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF) compared to other groups (p ≤ 0.05; Table 1). Treatment with Allicin (A) and Hesperidin (HSD) resulted in improvements in both total and progressive sperm motility, with increases in VCL, VSL, VAP, ALH, and BCF compared to the p-NP group (p ≤ 0.05; Table 1). Additionally, the p-NP group exhibited diminished plasma membrane functionality (PMF) and sperm viability, which were significantly avoided with A and HSD administration (p ≤ 0.05; Table 2). DNA damage in sperm and the frequency of abnormal sperm morphology were significantly elevated in the p-NP group but were notably reduced by A and HSD treatments (p ≤ 0.05; Table 2). Notably, the combined administration of A and HSD led to enhancements in these parameters, closely matching those observed in the control group.

Table 1.

Epididymal sperm concentration, total and progressive motilities, and motility characteristics in different experimental groups. Values are expressed as mean ± SD.

Analysis	Groups
	Control	p-NP	A + p-NP	HSD + p-NP	A + HSD + p-NP
Epididymal sperm concentration (106/mL)	32.17 ± 23.60a	16.38 ± 21.09e	24.29 ± 15.45d	26.35 ± 27.88c	29.45 ± 26.30b
Total motility (%)	79.96 ± 2.15a	45.88 ± 1.25e	63.01 ± 2.58d	68.96 ± 2.89c	75.19 ± 2.13b
Progressive motility (%)	40.85 ± 1.91a	8.47 ± 1.28d	29.24 ± 1.44c	30.81 ± 1.85c	34.67 ± 1.63b
VAP (μm/s)	29.39 ± 1.47a	14.99 ± 1.89d	18.46 ± 1.38c	19.43 ± 1.12c	25.26 ± 1.56b
VCL (μm/s)	83.80 ± 2.53a	43.15 ± 2.34e	68.30 ± 2.89d	72.81 ± 1.50c	78.48 ± 2.13b
VSL (μm/s)	23.47 ± 1.43a	11.32 ± 1.81d	15.82 ± 1.39c	17.26 ± 1.56bc	20.82 ± 1.47ab
LIN (%)	0.23 ± 0.04a	0.21 ± 0.03a	0.22 ± 0.03a	0.22 ± 0.02a	0.23 ± 0.03a
ALH (μm/s)	2.70 ± 0.03a	1.11 ± 0.02d	1.84 ± 0.03c	2.18 ± 0.02c	2.51 ± 0.03b
STR (%)	0.85 ± 0.03a	0.83 ± 0.04a	0.84 ± 0.03a	0.84 ± 0.04a	0.85 ± 0.02a
BCF (Hz)	12.56 ± 1.26a	6.40 ± 1.28c	8.21 ± 1.13b	8.98 ± 0.39b	11.61 ± 1.30a

p-NP, para-nonylphenol; A, allicin; HSD, hesperidin; VAP, Average path velocity; VCL, Curvilinear velocity; VSL, Straight line velocity; LIN, Linearity; ALH, Amplitude of lateral head displacement; STR, Straightness; BCF, Beat-cross frequency.

^a-eDifferent superscripts within the same row demonstrate significant differences (p ≤ 0.05).

Table 2.

Epididymal sperm plasma membrane functionality, DNA damage, viability and abnormal morphology in different experimental groups. Values are expressed as mean ± SD.

Analysis	Groups
	Control	p-NP	A + p-NP	HSD + p-NP	A + HSD + p-NP
Sperm plasma membrane functionality (%)	82.96 ± 3.81a	48.46 ± 3.69e	65.51 ± 2.53d	70.45 ± 1.27c	77.82 ± 2.18b
Sperm viability (%)	86.85 ± 3.24a	55.45 ± 2.42e	71.37 ± 2.37d	75.09 ± 2.41c	82.30 ± 2.53b
Sperm DNA damage (%)	5.12 ± 0.20d	28.95 ± 1.48a	15.30 ± 1.09b	11.42 ± 0.50c	9.25 ± 1.09cd
Sperm abnormal morphology (%)	7.27 ± 0.43 d	34.77 ± 1.31 a	18.43 ± 1.34b	16.15 ± 0.39b	11.92 ± 0.73c

p-NP, para-nonylphenol; A, allicin; HSD, hesperidin.

^a-eDifferent superscripts within the same row demonstrate significant differences (p ≤ 0.05).

In the p-NP group, serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were significantly lower than in the control group (p ≤ 0.05). In contrast, groups treated with A, HSD, or both showed significant increases in these hormone levels compared to the p-NP group (p ≤ 0.05; Fig. 1).

Fig. 1.

The hormonal assays of the (a) Follicle-stimulating hormone (FSH), (b) Luteinizing hormone (LH), and (c) testosterone for the treated groups and control. p-NP: para-nonylphenol; A: Allicin; HSD: Hesperidin. Different superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

Histopathological analysis of the testes in the p-NP group revealed significant structural damage, including seminiferous tubule atrophy, intraepithelial vacuolization, and pronounced hypocellularity (Fig. 2). Tubular connective tissue exhibited lesions such as tears, empty spaces, blocked blood vessels, inflammatory infiltrations, fluid accumulation, and expanded interstitial spaces. Degeneration of Leydig cells with pyknotic nuclei and detachment of Sertoli cells from germ cells, alongside irregular, amorphous, and smaller nuclei, were also observed. Johnsen’s scoring confirmed germ cell degeneration, disorganization, and desquamation in the p-NP group (Table 3). These changes were significantly more severe compared to other groups (p ≤ 0.05; Table 3). Degeneration of germ cells during spermatogenesis in the p-NP group led to reduced SCI, RI, and STsD, adversely affecting the MI (p ≤ 0.05; Table 3). Additionally, a decrease in the SI and TDI was observed in the p-NP group, indicating impaired spermatogenic progression. Testicular histology parameters, including biopsy scores, spermiogenesis index (SPI), Leydig cell nuclear density (LCND), and TDI, were lower in the p-NP group but showed significant improvement with A, HSD, or combined A + HSD treatments (p ≤ 0.05; Table 3).

Fig. 2.

Testicular histo-architecture in different experimental groups. (A) control; (B) para-nonylphenol; (C) para-nonylphenol + Allicin; (D) para-nonylphenol + Hesperidin; (E) para-nonylphenol + Allicin + Hesperidin (hematoxylin and eosin staining, 400 ×). The black arrows represent the sperm density in the seminiferous tubule lumen, which was significantly lower in group B compared to the control. Groups C, D, and E showed improvements in sperm density compared to group B. The asterisks indicate disorganization and disturbance in the seminiferous tubules, with group B showing the most severe effects, while groups C, D, and E exhibited lesser degrees of disruption.

Table 3.

Histological parameters and reproductive organ weights in different experimental groups. Values are expressed as mean ± SD.

Analysis	Groups
	Control	p-NP	A + p-NP	HSD + p-NP	A + HSD + p-NP
length of the right testis (cm)	0.79 ± 0.03a	0.65 ± 0.04d	0.70 ± 0.03c	0.74 ± 0.04b	0.77 ± 0.03ab
length of the left testis (cm)	0.80 ± 0.02a	0.68 ± 0.03d	0.72 ± 0.04c	0.76 ± 0.03b	0.78 ± 0.04ab
Diameter of the right testis (cm)	0.50 ± 0.02a	0.39 ± 0.03d	0.43 ± 0.02c	0.45 ± 0.03bc	0.48 ± 0.03ab
Diameter of the left testis (cm)	0.50 ± 0.03a	0.38 ± 0.05d	0.43 ± 0.04c	0.46 ± 0.03bc	0.49 ± 0.04ab
1st-day body weight (g)	25.20 ± 0.05a	25.16 ± 0.04a	25.09 ± 0.04a	25.27 ± 0.05a	25.22 ± 0.04a
35-day body weight (g)	26.54 ± 0.05a	18.68 ± 0.05c	21.96 ± 0.06bc	22.43 ± 0.04b	24.71 ± 0.04ab
Right testis weight (g)	0.113 ± 0.001a	0.070 ± 0.002b	0.096 ± 0.003a	0.105 ± 0.003a	0.110 ± 0.004a
Left Testis weight (g)	0.112 ± 0.003a	0.069 ± 0.002b	0.094 ± 0.001a	0.103 ± 0.004a	0.110 ± 0.002a
Epididymis weight (g)	0.045 ± 0.003a	0.031 ± 0.002d	0.035 ± 0.003c	0.037 ± 0.003c	0.041 ± 0.004b
Testis/body weight (%)	0.44 ± 0.003a	0.28 ± 0.002c	0.37 ± 0.004b	0.38 ± 0.003b	0.40 ± 0.004b
Johnsen score	9.46 ± 0.23a	5.63 ± 0.25d	7.21 ± 0.24c	7.45 ± 0.20c	8.51 ± 0.23b
Cosentino score	1.0 ± 0.02c	3.41 ± 0.02a	2.39 ± 0.01b	2.21 ± 0.03b	1.25 ± 0.02c
Seminiferous tubule diameter (µm)	53.91 ± 1.31a	33.52 ± 1.20d	39.43 ± 1.31c	42.65 ± 1.29c	48.24 ± 1.41b
Sertoli cell index	86.78 ± 2.87a	70.89 ± 2.35d	74.15 ± 2.93c	77.34 ± 2.71c	82.11 ± 2.82b
Repopulation index	72.62 ± 2.81a	45.31 ± 1.53e	57.25 ± 2.85d	61.47 ± 2.77c	67.45 ± 2.48b
Miotic index	2.74 ± 0.04a	1.13 ± 0.02c	1.84 ± 0.03b	2.02 ± 0.02b	2.46 ± 0.03a
Leydig cell nuclear diameter (µm)	6.21 ± 0.26d	8.19 ± 0.21a	7.23 ± 0.34b	7.14 ± 0.24b	6.51 ± 0.27c
Tubular differentiation index (%)	86.09 ± 2.70a	70.47 ± 2.41d	76.04 ± 2.64c	78.19 ± 2.84c	82.24 ± 2.49b
Spermiogenesis index (%)	83.26 ± 2.26a	58.89 ± 2.95d	72.64 ± 2.12c	76.68 ± 2.43b	79.41 ± 3.15b

p-NP, para-nonylphenol; A, allicin; HSD, hesperidin.

^a–fDifferent superscripts within the same row demonstrate significant differences (p ≤ 0.05).

In the p-NP group, significant reductions in tissue total antioxidant capacity (TAC), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were noted (p ≤ 0.05). These levels were significantly higher in the A and HSD groups, nearing those in the control group (p ≤ 0.05; Fig. 3). Malondialdehyde (MDA) levels, a marker of oxidative stress, were significantly elevated in the p-NP group compared to the other groups (p ≤ 0.05; Fig. 3). However, administration of A and HSD had a protective effect, significantly reducing MDA levels relative to the p-NP group (p ≤ 0.05). The combination of A and HSD avoided TAC, GPx, SOD, and MDA levels to values comparable to those in the control group (p ≤ 0.05; Fig. 3).

Fig. 3.

Biochemical findings in different experimental groups. p-NP: para-nonylphenol; A: Allicin; HSD: Hesperidin. (A) Total antioxidant capacity (TAC); (B) glutathione peroxidase (GPx); (C) superoxide dismutase (SOD); (D) Malondialdehyde (MDA). Different superscripts demonstrate significant differences (p ≤ 0.05; Mean ± SD).

No initial differences in body weight were observed between the groups. However, after 35 days, testicular weights in the p-NP group were significantly reduced compared to other groups (p ≤ 0.05). Epididymal weights in the p-NP group were also lower but showed notable improvement following A and HSD treatments (p ≤ 0.05). Additionally, the ratio of testis weight to body weight was significantly improved in the A and HSD groups relative to the control (p ≤ 0.05). Although testis diameter showed no significant differences across groups, the p-NP group had shorter testis lengths than the other groups (p ≤ 0.05; Table 3). The testis weight reduction correlated with a decline in spermatogenesis efficiency, as reflected in Johnsen’s and Cosentino’s scores (Table 3).

Quantitative RT-PCR analysis of mRNA expression for Bax, caspase-3, and Bcl-2 genes revealed elevated levels of caspase-3 mRNA and Bax in the p-NP group, in contrast to other groups (p ≤ 0.05; Fig. 4). In the p-NP group, expression of pro-apoptotic genes Bax and caspase-3 was significantly elevated, while anti-apoptotic Bcl-2 expression was reduced compared to the control group (p ≤ 0.05). Consequently, the Bax/Bcl-2 ratio was significantly higher in the p-NP group (p ≤ 0.05). Treatment with Allicin, Hesperidin, or both led to a significant decrease in Bax and caspase-3 expression and an increase in Bcl-2 expression, resulting in a lower Bax/Bcl-2 ratio compared to the p-NP group (p ≤ 0.05; Fig. 4).

Fig. 4.

Reverse transcription-polymerase chain reaction findings in different experimental groups. p-NP: para-nonylphenol; A: Allicin; HSD: Hesperidin. The densities of Bcl-2 (a), Bax (b), and Caspase-3 (c) mRNA levels in testicular parenchyma were measured by densitometry and normalized to the 18SrRNA mRNA expression level. Significant differences between groups are indicated by different superscripts (p ≤ 0.05; Mean ± SD).

All animals displayed mating ability, as indicated by positive mating indices with untreated female partners. However, a lower number of males in the p-NP group succeeded in impregnating females (p ≤ 0.05; Table 4). Administration of A, HSD, and A + HSD significantly increased the number of males achieving successful fertilization compared to the p-NP group (p ≤ 0.05; Table 4). Both male fertility and pregnancy indices were significantly lower in the p-NP group (p ≤ 0.05; Table 4), but these parameters were substantially improved in groups receiving A, HSD, and the combined A + HSD treatment relative to the p-NP group (p ≤ 0.05; Table 4). Additionally, the number of pregnant females per group closely matched improvements in male fertility indices, suggesting a direct relationship between sperm quality and reproductive success.

Table 4.

Fertility indexes of adult male rats exposed to heat stress after natural mating with non-exposed females.

Analysis	Groups
	Control	p-NP	A + p-NP	HSD + p-NP	A + HSD + p-NP
Number of females	20	20	20	20	20
Number of females mated	20	20	20	20	20
Number of males mated	10	10	10	10	10
Number of males impregnating females	10	1	4	5	8
Number of females pregnant	20	1	5	7	15
Female mating index (%)	100a	100a	100a	100a	100a
Male mating index (%)	100a	100a	100a	100a	100a
Pregnancy index (%)	100a	5d	25c	35c	75b
Male fertility index (%)	100a	5d	40c	50c	80b

p-NP, para-nonylphenol; A, allicin; HSD, hesperidin. Female mating index = number of females mated/number of females × 100; Male mating index = number of males mated/number of males × 100; Pregnancy index = number of females pregnant/number of females mated × 100; Male fertility index = number of males impregnating females/number of males mated × 100.

Discussion

Nonylphenol is an environmental toxin known for its harmful effects on various organs, including the testes. Its toxicity primarily arises from its ability to generate reactive oxygen species (ROS), which disrupt the endocrine and reproductive systems²⁵. By interfering with hormonal balance, nonylphenol contributes to structural damage in the testes, negatively affecting sperm production and inducing cell death in the germinal layers²⁶. One of the main mechanisms underlying its detrimental effects is the induction of oxidative stress. Previous research has shown that nonylphenol exposure lowers the activity of critical antioxidant enzymes such as glutathione reductase and superoxide dismutase, leading to oxidative damage in testicular parenchyma²⁷. The resulting oxidative stress promotes free radical formation, which attacks cellular membranes and damages testicular cells. The estrogen-like properties of para-nonylphenol, combined with its oxidative stress-inducing potential, significantly contribute to the observed damage in testicular parenchyma²⁵. Numerous studies have demonstrated the effectiveness of antioxidants in countering the harmful effects of this pollutant^28–30. This research assesses how Allicin (A) and Hesperidin (HSD) impact fertility indices, testicular structure, and spermatogenesis in mice exposed to para-nonylphenol. Results show that A and HSD enhance sperm quality, reduce testicular damage and apoptosis, restore oxidant-antioxidant balance, and improve fertility after exposure. Additionally, the Bax/Bcl-2 ratio, linked to apoptosis, decreased significantly with A and HSD, offering protection to testicular cells. These findings align with Azizi and Mehranjani²⁸, which found that antioxidants like green tea extract mitigate p-NP-induced damage, extending this knowledge to Allicin and Hesperidin. Although our study primarily investigates male reproductive parameters, future studies need to ascertain the effect on female reproductive health and offspring outcomes to facilitate clearer appreciation of the benefits afforded by such antioxidants.

Sperm, being rich in polyunsaturated fatty acids, are particularly prone to damage caused by ROS, which can impair membrane integrity and DNA stability. These factors contribute to the toxicity associated with para-nonylphenol exposure³¹. Previous findings have reported a significant decline in sperm count in animals subjected to para-nonylphenol³². Similar to other environmental toxins, para-nonylphenol disrupts the delicate equilibrium between antioxidants and free radicals, resulting in elevated oxidative stress and altered sperm function¹. Furthermore, lipid peroxidation, triggered by oxidative stress, can adversely affect sperm motility³². The decline in motility is linked to a reduction in the mitochondrial density within the sperm midpiece, which is essential for generating movement³³. Research also indicates that low doses of para-nonylphenol inhibit ATP synthesis in the mitochondria, which may further impair motility³⁴. In this study, it was observed that exposure to para-nonylphenol reduced sperm motility and led to mitochondrial dysfunction, while A, HSD, or their combination improved motility and prevented damage. The decline in sperm motility and viability observed in the p-NP group can be attributed to oxidative stress-induced damage to sperm membranes and mitochondrial dysfunction. Allicin and Hesperidin, through their antioxidant properties, mitigated these effects by reducing lipid peroxidation and enhancing mitochondrial function, as evidenced by improved sperm motility and viability. The doses of Allicin (30 mg/kg) and Hesperidin (100 mg/kg) were selected based on their proven efficacy in previous studies, offering physiologically relevant antioxidant protection against oxidative stress in rodent models. These results are consistent with the findings of Abdel-Daim et al.³⁵, who reported that Allicin improves mitochondrial function in doxorubicin-treated rats, and Olayinka & Adewole²¹, who observed that Hesperidin restores sperm motility in finasteride toxicity.

When oxidative damage occurs due to an imbalance favoring free radicals over antioxidants, various biological mechanisms and molecules can be affected. Sperm rely on both enzymatic and non-enzymatic antioxidant defenses to mitigate this damage³⁶. We observed reduced redox enzyme activity in mice exposed to para-nonylphenol. Treatment with A and HSD was found to counteract the para-nonylphenol-induced decrease in enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) within testicular parenchyma. Para-nonylphenol also significantly elevated levels of malondialdehyde (MDA), a marker of lipid peroxidation, in testicular parenchyma, which indicates oxidative damage affecting cellular membranes and leading to cell death^37,38. Previous studies also linked toxic exposure to increased lipid peroxidation^39–41. In fact, our observations are consistent with previous ones, while treatment with A and HSD reduced MDA levels previously increased by exposure to para-nonylphenol, which showed a protective effect against lipid peroxidation and supported antioxidant defense. Taken together, these findings support the findings of Bisht et al.⁴², who have attributed oxidative stress to infertility in rodent models and are expanding the use of antioxidants such as A and HSD in clinics.

Spermatogenesis generates significant free radicals, requiring a balance with antioxidants to maintain the testes’ oxidative stability, as they are susceptible to oxidative stress⁴³, and can suffer considerable damage if the antioxidant defense system is compromised. Nonylphenol (NP) has been shown to impair cell connectivity by lowering connexin protein expression, which can lead to cell death and defects in both spermatogenic and Sertoli cells, potentially reducing the epithelial cell layer²⁹. This effect can also impair the blood testis barrier, increasing the risk of tissue swelling⁴⁴. Additionally, NP exposure can interrupt B-type spermatogonia in the G1 phase of cell division by inhibiting cyclin 1 protein expression, which is essential for mitosis²⁹. In a study by Kim et al.⁴⁵, exposure to NP at 50 μg/L reduced the seminiferous tubules’ size, thickened the basement membrane, and caused testicular degeneration in mice. Azizi and Mehranjani²⁸ found that 200 mg/kg of para-nonylphenol reduced key testicular parameters for 56 days. However, green tea extract improved these histological parameters. Our findings support these findings and show that A and HSD mitigated the damage caused by para-nonylphenol exposure. It is important to note that the paraffin embedding technique used in this study may have caused tissue retraction, potentially affecting the interpretation of histopathological findings. Future studies should consider using alternative embedding methods, such as HistoResin, to preserve testicular morphology better.

Excess free radicals in the testes can damage germ cells, spermatozoa, somatic cells, and the testicular microenvironment by disrupting DNA, RNA, and protein integrity³⁶. Such damage interferes with the testis’s endocrine functionality, ultimately stalling spermatogenesis and spermiogenesis⁴⁶. Testosterone plays a pivotal role in spermatogenesis, acting indirectly by fostering interactions between Sertoli cells⁴⁷. Evidence indicates that exposure to toxic chemicals in rats results in a decline in testosterone synthesis, accompanied by a substantial reduction in circulating sex hormone levels³⁷. The estrogen-like effect of p-NP may inhibit FSH because estrogen negatively controls FSH secretion⁴⁸. Estrogen regulates inhibin expression⁴⁹, an important factor that regulates FSH secretion by Sertoli cells⁵⁰. Therefore, one possibility is that p-NP may interfere with FSH levels by influencing inhibin expression, which is responsible for its role in low sperm production³². p-NP also blocks testosterone production by inhibiting 17α-HSD and disrupting the cAMP pathway in Leydig cells⁵¹. Our results in this study show that A and HSD eliminate this inhibition. We suggest that these compounds protect testicular cells from p-NP-induced oxidative stress, thus enhancing interactions between testosterone, LH, FSH, Sertoli cells, and spermatogenesis. Future studies are needed to find out whether A and HSD can reverse damage in chronic exposure scenarios.

Excessive free radicals contribute to various pathologies, inducing cell death and linking oxidative stress to spermatogenic cell apoptosis⁵². Apoptosis, a form of programmed cell death, occurs under both physiological and pathological conditions and is triggered by specific intracellular or extracellular signals that activate genetic pathways⁵³. Certain chemicals have been identified as inducers of apoptosis via the activation of caspase-3⁵⁴. Nonylphenol promotes oxidative stress by generating reactive oxygen species such as hydrogen peroxide²⁶. This leads to increased Bax activity and the release of cytochrome c from mitochondria, which activates the Apaf1/Caspase-9 complex^33,55. Nonylphenol exposure induces apoptosis in germ and Leydig cells, reducing sperm count and production^29,32. In this study, p-NP treatment increased caspase-3 and Bax while reducing Bcl-2 levels, suggesting increased apoptosis in testicular parenchyma. Antioxidants A and HSD counteracted these effects by inhibiting P-NP-triggered genes associated with apoptosis. The lower Bax/Bcl-2 ratio underscores the anti-apoptotic effects of A and HSD and thus supports previous findings on citrus flavonoids¹⁴.

Infertility is associated with oxidative damage, reduced antioxidant defenses, and increased ROS production⁵⁶. Reduced testosterone and sperm quality, often with lower testicular weight, was associated with infertility⁵⁷. P-NP exposure significantly reduced fertility rates in rats⁵⁸ and mice⁵⁹, and certain antioxidants were unable to mitigate these effects. Exogenous antioxidants such as vitamins C, A, E, and trace minerals improved oxidative stres⁶⁰ and embryonic survival rates⁶¹. In this study, fertility indices were significantly reduced in the p-NP group, where only 5% of males were able to successfully impregnate females. This drastic decline in fertility can be attributed to observing sperm DNA damage, abnormal morphology, and reduced motility, all essential for successful fertilization. Our results show that daily A and HSD supplementation increased fertility indices in mice, which is due to better membrane stability, better mitochondrial function, and higher levels of antioxidants, which had a positive effect on sperm mobility and viability⁶². This study confirms that A and HSD improve sperm quality, integrity maintain testicular parenchyma, and improve fertility rates. Treatments with Allicin and Hesperidin improved fertility indices, likely due to their protective effects on sperm quality and testicular health. Additionally, post-testicular events, such as impaired sperm maturation in the epididymis, may have further contributed to the reduced fertility in the p-NP group, which warrants further investigation.

Conclusion

In summary, this study represents the first investigation into the preventive effects of A and HSD against testicular damage caused by p-NP exposure. Both A and HSD effectively prevent the morphological and histological damage induced by p-NP, thereby preserving testicular functionality. Their combined administration improves endocrine balance, boosts antioxidant defenses, enhances fertility parameters, and promotes sperm quality. The reduction in the Bax/Bcl-2 ratio further underscores their anti-apoptotic effects. These findings contribute to the understanding of testicular protection mechanisms and suggest antioxidant-based approaches as potential strategies to mitigate the harmful impact of p-NP on reproductive health. Future studies should explore the long-term effects of these compounds and their possible applications in human reproductive health.

Methods

Ethics statements

This study followed the ethical guidelines provided by the Animal Ethics Committee of the Islamic Azad University, Iran, under approval number IR-IAU-2/24/40.

Chemicals

Essential chemical reagents were also purchased from long-standing and renowned suppliers such as Sigma-Aldrich (St. Louis, Missouri, USA) and Merck (Darmstadt, Germany) to achieve high quality and reliability. Allicin (CAS number: 539-86-6, PubChem CID: 65036), Hesperidin (CAS number: 520-26-3, PubChem CID: 10621), p-nonylphenol (p-NP) (CAS number: 104-40-5, PubChem CID: 1752), and p-nonylphenol (CAS number: 104-40-5, PubChem CID: 1752) were purchased from Sigma-Aldria. The remaining chemicals were of analytical quality and were purchased from Merck.

Animals

Fifty adult male mice, with an average weight of 27.0 ± 2.0 g and aged between 8 and 10 weeks, were obtained from the Animal Resource Center of Urmia University of Medical Sciences in Urmia, Iran. The mice were kept in standard plastic cages within a regulated environment with a 12-h light/dark cycle, temperature maintained at 20–22 °C, and relative humidity of 50 ± 10%. For one week before the experiment, the mice had unrestricted access to standard laboratory chow and tap water.

Experimental protocol

Following one week of acclimatization, mice were distributed randomly into five groups containing n = 10 animals each. Sample size was calculated using G*Power analysis (v3.1.9.7) with parameters of an effect size = 0.8, α = 0.05, and power = 0.95. The doses of p-NP (200 mg/kg)⁶³, Allicin (30 mg/kg)⁶⁴, and Hesperidin (100 mg/kg)⁶⁵ were selected based on previous studies demonstrating their efficacy in inducing reproductive toxicity and providing antioxidant protection, respectively. The p-NP dose reflects environmentally relevant exposure levels known to cause testicular damage, while Allicin and Hesperidin doses were chosen based on their established antioxidant effects in rodent models. The duration of 35 days was chosen to cover a significant portion of the spermatogenic cycle in mice, which is approximately 35–40 days, allowing for assessing effects on spermatogenesis⁵². Groups are specified as follows:

• Group 1 (control): Received a daily oral dose of corn oil (0.1 mL/kg) administered by intragastric gavage⁶³.

• Group 2 (c group): Administered p-NP orally at a daily dose of 200 mg/kg (dissolved in corn oil) through intragastric gavage⁶³.

• Group 3 (A + p-NP): Received p-NP as previously described and an additional daily oral dose of Allicin (A) at 30 mg/kg (dissolved in corn oil) via gavage⁶⁴.

• Group 4 (HSD + p-NP): Treated with p-NP as described, alongside a daily oral dose of Hesperidin (HSD) at 100 mg/kg (dissolved in corn oil) via gavage⁶⁵.

• Group 5 (A + HSD + p-NP): Received the p-NP treatment, along with daily oral doses of both A (30 mg/kg) and HSD (100 mg/kg), all administered via gavage.

After the 35-day experimental period, male mice were mated with untreated female mice to assess reproductive parameters. The male mice were sacrificed on the third day after mating to ascertain the existence of mature sperm in the epididymal cauda. Euthanasia was carried out using an intraperitoneal (IP) injection of a solution containing ketamine (80 mg/kg) and xylazine (10 mg/kg). The two anesthetics were obtained from Alfasan, the Netherlands^{66, 36}.

Fertility indexes

Natural mating tests were carried out with 10 treated male mice (n = 10 per treatment) and 20 untreated female mice BALB/c (n = 20 per treatment; average weight of 20.0–25.0 g and aged between 8 and 12 weeks) in a ratio of 1:2 males to females in separate cages for up to 72 h. Vaginal smears were collected 24, 48, and 72 h after pairing to detect the presence of sperm, which signaled successful mating and was marked as gestational day 0 (GD0). At the end of this period, males were separated from females. Pregnant females at GD17 were anesthetized with a combination of ketamine (10% at 80 mg/kg/IP) and xylazine (2% at 10 mg/kg/IP) from Alfasan, Netherlands, before euthanasia³⁶.

Levels of plasma reproductive hormone

Following euthanasia on day 35, blood was collected via cardiac puncture into plain sample bottles for hormone level analysis. Euthanasia was conducted using an intraperitoneal (IP) injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), sourced from Alfasan, Netherlands³⁶. Serum samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) using a radioimmunoassay technique provided by DIA source. For testosterone extraction, serum was mixed with diethyl ether, vortexed, and the organic phase was collected and evaporated. The dried extract was reconstituted in assay buffer. The antibody used for testosterone showed cross-reactivity of less than 0.01% with other steroids. The intra-assay and inter-assay coefficients of variation for FSH were 3.5% and 5.2%, respectively, and for LH were 4.1% and 6.3%, respectively⁵².

Sperm collection

At the end of the experiment, all mice were weighed before euthanasia. Euthanasia was performed via intraperitoneal (IP) injection of ketamine (80 mg/kg) and xylazine (10 mg/kg), sourced from Alfasan, Netherlands³⁶. Subsequently, the epididymis and testes were carefully excised by dissection, and their absolute and relative weights were recorded. The sperm was extracted from the epididymis of the cauda by dissecting it into a petri dish containing 1 ml of human tube fluid (HTF). This medium, which contained sperm-carrying tissue, was incubated at 37°C in a 5% CO₂-rich environment for 30 minutes³⁶.

Sperm analysis

Count of sperm

Sperm concentration was assessed with a Brand (Germany) Neubauer hemocytometer after diluting devitalized samples (1:5) with distilled water^67,36.

Motility of sperm

Sperm motility, including progressive and total motility and motility characteristics, was evaluated at room temperature using Test Sperm 3.2 (Videotest, Russia) (Table 5). A 10 μL sample was examined with an Olympus BX41 microscope (Japan)⁵².

Table 5.

Parameter settings for the computer-assisted semen analysis.

Parameter	Setting
Frame rate	60-Hz
Number of frames	30
Duration of capture	1 s
Stage temperature rate	37 °C
Minimum cell size	4 pixels
Cell size	13 pixels
Minimum contrast	30
Cell intensity	75 pixels
Chamber type	Slide-Coverslip (22*22 mm)
Volume per slide	7 μL
Chamber depth	≈ 20 μm
Minimum number of field analysis	500 cells
Image type	Phased contrast
Average path velocity (VAP)	50.0 μm/s
Straightness (	50%
VAP cutoff	10.0 μm/s
Straight line velocity cutoff	0.0 μm/s
Curvilinear velocity	> 180 μm/s
Amplitude of lateral head	> 9.5 μm
Mean linearity	< 38%

Sperm viability and morphology

The morphology and viability of sperm were examined using eosin nigrosin stain in accordance with World Health Organization (WHO) guidelines⁶⁸.

DNA damage of sperm

Concentrated sperm smears were exposed to a 1:3 methanol and acetic acid solution for two hours, air-dried and then subjected to an acridine orange solution. The stained sperm samples were examined under fluorescence microscopy⁵².

Plasma membrane functionality (PMF) of sperm

The hypoosmotic swelling test was conducted to evaluate sperm PMF. A 10 μL sperm sample was combined with 100 μL of a hypoosmotic solution containing fructose and sodium citrate, followed by incubation at 37 °C for one hour. PMF was assessed at 400 × with an Olympus BX41, identifying curled or swollen tails⁵².

Enzymatic antioxidant activity assessment

Left testicle from each mouse was homogenized in 1000 μL of lysis buffer. After centrifuging the homogenates at 9000 rpm for 15 min, the supernatant was collected and used for subsequent biochemical analyses, as per the methods described by Sadeghi Rad et al.³⁶. Total protein concentration was determined using the Bradford method, and all enzymatic activities were normalized to the total protein content. Testicular TAC, GPx, and SOD activities were measured using Naxifer, Nagpix™, and Nasdox kits (Navand Salamat, Iran). MDA levels were assessed using the Nalondi™ kit (Navand Salamat, Iran) at 535 nm and reported as nmol/g protein³⁶.

Testicular histopathology and histomorphometry

Testicular parenchyma was fixed in 10% formalin, dehydrated through graded ethanol, and embedded in paraffin. Sections 7 μm thick were prepared with a microtome and stained using hematoxylin and eosin (H&E). The spermiogenesis index (SPI) was calculated as the ratio of sperm-containing tubules to those without. Other indices, including Sertoli cell index (SCI), meiotic index (MI), tubular differentiation index (TDI), and repopulation index (RI), were analyzed following standard protocols⁵². Johnsen’s score was applied to assess the quality of seminiferous tubules (Table 6). Testicular injury was graded based on the Cosentino system: Grade 1 (normal), Grade 2 (mild disruption), Grade 3 (moderate disorganization), and Grade 4 (severe damage with necrosis)³⁶.

Table 6.

Johnsen scoring system used for testicular damage evaluation.

Johnsen score	Description of histological criteria
10	Full spermatogenesis
9	Slightly impaired spermatogenesis, many late spermatids, disorganized epithelium
8	Less than five spermatozoa per tubule, few late spermatids
7	No spermatozoa, no late spermatids, many early spermatids
6	No spermatozoa, no late spermatids, few early spermatids
5	No spermatozoa or spermatids, many spermatocytes
4	No spermatozoa or spermatids, few spermatocytes
3	Spermatogonia only
2	No germinal cells, Sertoli cells only
1	No seminiferous epithelium

qRT-PCR

Bax, caspase-3, and Bcl-2 gene expression were analyzed via qRT-PCR using SinaSyber Blue HF-qPCR mix (CinnaGen, Iran) on a StepOne system (Applied Biosystems, USA), with 18SrRNA as the reference (Table 7). The experiment was carried out in biological form for each group. The cycle conditions included 35 cycles at 94 °C, 55 °C, and 72 °C, which were preceded by initial denaturation at 95°C. In order to calculate the relative expression levels of the genes in the samples, expression was determined using a formula provided: The relative expression can be measured using the formula 2^{−(SΔct-CΔct)}, where sΔct is obtained by subtracting the cycle threshold (ct) of the reference gene from that of the target gene and CΔct is the ct values from the internal control samples. The values were then transformed logarithmically with base 10 and analyzed to determine the normal distribution of the relative expression values⁵².

Table 7.

Nucleotide sequences and product size of primers used in reverse transcription-polymerase chain reaction.

Gene name	Primer	Band size
Bcl-2	Forward: 5-CTCGTCGCTACCGTCGTCACT TCG-3 Reverse: 5-CAGATGCCGGTTCAGGTACTCAGTC-3	242 bp
Bax	Forward: 5-ACC AGC TCTGAACAGATC ATG-3	424 bp
	Forward: 5-TGG TCT TGGATCCAGACAAG-3
caspase-3	Forward: 5-GTACAGAGCTGGACTGCGGTATTG-3	84 bp
	Reverse: 5-AGTCGGCCTCCACTGGTATCTTC-3
18SrRNA	Forward: 5-GCAATTATTCCCCATGAACG-3 Reverse: 5-GGCCTCACTAAACCATCCAA-3	123 bp

Statistical analysis

The data analysis was carried out using SPSS (version 26.0; IBM, USA). The normality of the data was verified by the Shapiro–Wilk test, while the Levene test was used to ensure the homogeneity of the variance. A one-way ANOVA was performed for comparison between groups, followed by Tukey’s post-hoc analysis. Nonparametric data were compared using the Kruskal–Wallis test and then with Dunn’s post-hoc analysis. Statistical significance was set at p ≤ 0.05.

Abbreviations

A

Allicin

HSD

Hesperidin

Ca²⁺

Calcium

TM

Total motility

PM

Progressive motility

VCL

Curvilinear velocity

VSL

Straight-line velocity

VAP

Average path velocity

STR

Straightness

LIN

Linearity

ALH

Amplitude of lateral head displacement

BCF

Beat-cross frequency

PMF

Plasma membrane functionality

TAC

Total antioxidant capacity

MDA

Malondialdehyde

GPx

Glutathione peroxidase

SOD

Superoxide dismutase

AO

Acridine orange

ROS

Reactive oxygen species

H&E

Hematoxylin and eosin

CAT

Catalase

NO

Nitric oxide

Author contributions

Tohid Mohammadi and Golsa Behnejad contributed to the conception, design, data collection, statistical analysis, and manuscript drafting. Golsa Behnejad, Tohid Mohammadi, and Ali Soleimanzadeh contributed to the conception, design, supervision of the study, and drafting of the manuscript. All authors approved the final version for submission.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All protocols were approved by the Faculty of Veterinary Medicine’s Committee on the Ethics of Animal Experiments at the Islamic Azad University. Every procedure was carried out by the relevant laws and standards. The study was conducted in compliance with the ARRIVE standards. The owner(s) of the animals gave their informed consent for us to use them in the study.

Competing interests

The authors declare no competing interests.

Declaration of generative AI in scientific writing

While preparing this work the author(s) used ChatGPT to make the text antive. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the publication’s content.