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Article

Extraction, Characterization, and Biological Evaluation of Atranorin Against Diabetes-Induced Reproductive Dysfunction Through Modulation of Oxidative Stress, Inflammatory Pathways and Key Reproductive Enzymes

by
Walaa I. El-Sofany
1,
Ahlam F. Alshammari
1,
Mona Zaheed Alshammari
1,
Hissah Khashman Alshammari
1,
Nawal S. Alshammari
1,
Najat Masood
1 and
Khaled Hamden
2,3,*
1
Department of Chemistry, College of Science, University of Ha’il, Ha’il 81451, Saudi Arabia
2
Biolival Laboratory, Monastir University, Monastir 5000, Tunisia
3
Higher School of Health Sciences and Technology of Sfax, Sfax University, Sfax 3064, Tunisia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(5), 2416; https://doi.org/10.3390/ijms27052416
Submission received: 26 January 2026 / Revised: 20 February 2026 / Accepted: 22 February 2026 / Published: 5 March 2026
(This article belongs to the Special Issue Advanced Research on Plant-Derived Products in the Service of Immune Regulation)
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Abstract

Male fertility is declining worldwide, with notable reductions in sperm counts, emphasizing the need for new therapeutic interventions. Atranorin (ATR), a lichen-derived secondary metabolite, exhibits strong antioxidant and anti-inflammatory activities. This study assessed the protective effects of ATR on type 1 diabetes (T1D)-induced reproductive dysfunction in rats. T1D was induced in male Wistar rats via a single intraperitoneal injection of alloxan at 150 mg/kg body weight (bw). ATR significantly ameliorated T1D-related reproductive damage. At 170 mg/kg bw, ATR reduced hyperglycemia by 66% and attenuated seminal inflammation, decreasing leukocyte infiltration (−51%) and myeloperoxidase (MPO) activity (−68%). Oxidative balance improved, as evidenced by increased total antioxidant status (TAS) (+203%) and decreased thiobarbituric acid reactive substances (TBARS) (−73%), hydrogen peroxide (H2O2) (−45%), and total oxidant status (TOS) (−70%). Steroidogenesis was restored through enhanced 3β-hydroxysteroid dehydrogenase (3β-HSD) (+65%) and 17β-hydroxysteroid dehydrogenase (17β-HSD) (+102%) activities, resulting in a 90% recovery of testosterone levels. Seminal plasma function improved, with increased fructose levels (+71%), normalized pH (7.4), and enhanced hyaluronidase (HYAL) (+71%), adenosine triphosphatase (ATPase) (+71%), and prostatic acid phosphatase (PAP) (+79%) activities. Fertility biomarkers, such as adenosine deaminase (ADA) (+148%) and lactate dehydrogenase-C4 (LDH-C4) (+62%), increased, and essential minerals Zn2+ (+72%), Ca2+ (+96%), Mg2+ (+84%), and Se (+57%) were restored. Consequently, sperm density (+87%), viability (+69%), and motility (+189%) improved, while abnormalities declined (−46%). Histological findings confirmed the restoration of spermatogenesis and epididymal maturation. ATR effectively counteracts diabetes-induced reproductive dysfunction by reducing oxidative and inflammatory stress while improving hormonal and seminal parameters.

1. Introduction

Over the past fifty years, male fertility has shown a worrying global decline. Several large meta-analyses have reported a reduction of more than 50% in sperm concentration between 1973 and 2018, dropping from around 100 million/mL to less than 50 million/mL, a threshold already considered subfertile by the WHO [1,2,3]. Although declining birth rates in many countries are multifactorial (aging populations, socioeconomic choices, reduced desire for children), deteriorating sperm quality clearly contributes to the rising prevalence of male infertility [4]. In addition, synthetic food additives, particularly artificial colorants like tartrazine (E102), have been linked to testicular oxidative stress, disrupted spermatogenesis, and reduced sperm motility in animal studies, suggesting a potential contribution to the decline in male fertility [5]. This alteration in sperm quality induced by various chemical agents is largely mediated through the induction of inflammatory responses and the overproduction of reactive oxygen species. Among the diseases associated with a decline in male fertility, diabetes mellitus represents a major contributing factor. Diabetes-induced oxidative stress triggers a cascade of cellular damage and apoptosis in key testicular cell types, including germ cells, Sertoli cells, and Leydig cells, which are essential for spermatogenesis and hormonal regulation [6,7,8]. In parallel, these pathological processes compromise the integrity of seminal plasma, the physiological microenvironment required for sperm survival and function. Persistent oxidative and inflammatory damage results in reduced sperm concentration, motility, and normal morphology, while also suppressing testosterone production and disrupting the activity of key enzymes involved in sperm motility, viability, viscosity, and overall fertilizing capacity [9].
Faced with this alarming trend, male fertility emerges as a major global public health concern, requiring urgent reduction in exposure to environmental and dietary pollutants, improvement in lifestyle habits, and greater awareness of risks linked to age and environmental factors. In this context, the use of natural compounds, particularly bioactive pigments, has gained increasing attention as promising alternatives to synthetic food additives and colorants [10,11,12]. Among natural pigments, ATR a polyphenolic secondary metabolite derived from the lichen Parmotrema hypoleucinum has attracted increasing interest due to its dual role as a natural colorant and a bioactive compound. Structurally, ATR possesses a distinctive chromophore system that enables selective absorption of light at specific wavelengths, resulting in a stable yellow coloration that is desirable for natural dye applications. Beyond its coloring properties, ATR has been reported to exhibit a range of biological activities, including antioxidant, anti-inflammatory, anticancer, and antimicrobial effects. These properties are largely attributed to its polyphenolic structure, which allows it to scavenge reactive oxygen species, modulate inflammatory pathways, and interfere with microbial growth and cancer cell proliferation [13,14,15,16,17].
To date, no studies have investigated the effects of ATR on male fertility-related enzymes or sperm parameters. Accordingly, this study aims, for the first time, to evaluate the impact of ATR ingestion on key enzymatic activities involved in male reproductive function, including enzymes related to sperm motility, metabolism, seminal plasma quality, and sperm–oocyte interaction, in T1D rats. In addition, the effects of ATR and T1D on inflammatory markers, antioxidant capacity of seminal plasma, and major sperm functional parameters—such as density, motility, viability, and morphology—are assessed.

2. Results

2.1. Extraction Yield of ATR

The extraction yield of ATR from 100 g of dried P. hypoleucinum Lichen powder was found to be 1.42%. After applying the extraction process, 1.42 g of ATR was obtained. This result demonstrates a high extraction efficiency for this heterocyclic compound, indicating that the method used was effective in isolating ATR from the lichen.

2.2. ATR FTIR Analysis

FTIR analysis of ATR confirmed the presence of characteristic functional groups consistent with its chemical structure. The spectrum revealed absorption bands indicative of carbonyl (C=O), aromatic CC, ester/ether CO, and aromatic CH functional groups, in agreement with previously reported molecular features of ATR (Figure 1A).

2.3. UV Analysis of ATR

UV–Vis analysis of ATR revealed two major absorption bands characteristic of its molecular structure. These bands correspond to electronic transitions within the conjugated aromatic system and carbonyl functional groups, consistent with previously reported spectral features of ATR (Figure 1B).

2.4. Effect of ATR on Blood Glucose Levels in T1D Rats

A single intraperitoneal injection of alloxan induced severe pancreatic β-cell damage, leading to a marked decrease in insulin activity and a 201% increase in blood glucose levels compared with healthy control rats. Oral administration of ATR for 30 days significantly attenuated hyperglycemia in a dose-dependent manner, with the most pronounced effects observed at doses of 170 and 200 mg/kg bw, resulting in 61% and 66% reductions in blood glucose levels, respectively, relative to untreated T1D rats (Figure 2).

2.5. Effect of T1D and TAR on Plasma Seminal Inflammation

This study demonstrates that T1D is associated with a significant increase in leukocyte infiltration into semen, triggering inflammatory responses as reflected by elevated MPO activity. ATR supplementation markedly attenuated this inflammatory state in a dose-dependent manner, with the most effective dose (170 mg/kg bw) reducing leukocyte counts by 51.4% compared with untreated T1D rats. This reduction in leukocyte infiltration was accompanied by a 68% decrease in MPO activity, highlighting the potent anti-inflammatory effect of ATR (Figure 3).

2.6. Effect of T1D and ATR on α-Amylase and Proteases Activities in Plasma Seminal

The results of this study show that T1D induces significant alterations in sperm quality, evidenced by marked reductions in seminal digestive enzymes, including α-amylase and proteases. Furthermore, ATR supplementation enhanced sperm metabolism in a dose-dependent manner, with maximal effects observed at 170 mg/kg (Figure 4).

2.7. Effect of T1D and ATR on the Oxidative Quality of Seminal Plasma

The results indicate that T1D is associated with marked oxidative stress in seminal plasma, characterized by a significant decrease in TAS and a concomitant increase in TOS. Oral administration of ATR (170 mg/kg bw) markedly restored redox balance, as evidenced by a 203% increase in TAS and a 70% reduction in TOS compared with untreated T1D rats (Table 1).

2.8. Effect of T1D and ATR on Sperm Quality and Seminal Biochemistry

The results of this study demonstrate that T1D induces profound impairments in sperm quality, characterized by reduced activities of seminal digestive enzymes (α-amylase and proteases), depletion of essential minerals involved in sperm viability, motility, and fertilization (Zn2+, Ca2+, Mg2+, and Se), and alterations in physicochemical parameters, including decreased citric acid levels and disturbed seminal pH. These biochemical disturbances were accompanied by marked declines in sperm density, viability, and motility, along with an increased incidence of morphological abnormalities. Oral administration of ATR at 170 mg/kg body weight markedly ameliorated these alterations by restoring sperm metabolic activity, essential mineral balance, and seminal physicochemical properties. These improvements translated into a substantial recovery of sperm functional parameters, including enhanced density, viability, and motility, together with a notable reduction in abnormal spermatozoa. In addition, ATR supplementation improved both progressive and non-progressive sperm motility in a dose-dependent manner, with maximal effects observed at 170 mg/kg (Table 1).

2.9. Effect of T1D and ATR on Key Steroidogenesis Enzymes and Testosterone Level

Figure 5 shows that T1D induces a marked reduction in the activity of steroidogenic enzymes and testosterone levels in seminal plasma. Oral administration of ATR significantly counteracted this effect, enhancing 3β-HSD and 17β-HSD activities by 65 and 102%, respectively, and restoring testosterone levels with a 90% increase compared to untreated T1D rats.

2.10. Impact of ATR on Key Enzymes of Sperm Motility

T1D was found to suppress the activity of essential enzymes regulating sperm motility. ATR administration significantly restored these activities in a dose-dependent manner, with the most effective effect observed at 170 mg/kg bw. At this dose, ATR increased ATPase activity by 71% and prostatic acid phosphatase (PAP) activity by 79%, compared to untreated T1D rats (Figure 6).

2.11. Effect of ATR Administration on Seminal Plasma Quality: Fructose, pH, and HYAL Activity

ATR supplementation in T1D rats markedly improved seminal plasma quality by elevating fructose levels by 71% and normalizing pH to 7.4. It also restored HYAL activity, increasing it by 71%, thereby reducing seminal viscosity and bringing values closer to those of healthy controls (Table 1, Figure 6).

2.12. Effect of ATR on the Activity of Key Fertility Enzymes in Sperm of T1D

Our findings show that T1D alters the activity of fertility-related enzymes, particularly ADA and LDH-C4. Oral administration of ATR significantly and dose-dependently increased their activities, with the most effective dose (170 mg/kg bw) enhancing ADA by 148% and LDH-C4 by 62%, compared to untreated T1D rats (Figure 7).

2.13. Effect of T1D and ATR on Testicular Tissue

T1D caused severe histological alterations in the testes, including decreased sperm density in the seminiferous tubules, indicative of impaired spermatogenesis, as well as pronounced lymphocyte infiltration reflecting testicular inflammation. Treatment with ATR for 30 days alleviated these damages in a dose-dependent manner, with the most effective dose (170 mg/kg bw) restoring sperm density within the tubules and markedly reducing inflammatory cell infiltration, thereby improving overall testicular structure and function (Figure 8).

2.14. Effect of T1D and ATR Supplementation on Epididymal Tissue Architecture

Histological examination revealed that the epididymides of T1D rats exhibited reduced sperm density, along with damaged spermatozoa and cellular debris within the lumen of the epididymal tubules, indicating marked cellular injury compared to healthy controls. In contrast, oral administration of ATR for 30 days restored epididymal architecture in a dose-dependent manner, with the most effective dose (170 mg/kg bw) maintaining normal sperm density and eliminating cellular debris and inflammatory infiltration, similar to that observed in control rats (Figure 9).

3. Discussion

Alloxan injection caused severe destruction of pancreatic β-cells, leading to a progressive reduction in circulating insulin and a marked increase in blood glucose levels. At the seminal plasma level, T1D was associated with a significant increase in leukocyte counts, triggering an inflammatory response characterized by elevated MPO activity. This was accompanied by excessive ROS production, with increased H2O2 and TOS levels and a concomitant reduction in TAS, indicating pronounced oxidative stress. This redox imbalance was confirmed by elevated TBARS levels, reflecting enhanced lipid peroxidation and membrane damage. The resulting inflammatory and oxidative insults compromised the integrity of spermatozoa as well as Sertoli and Leydig cells. In parallel, steroidogenic enzyme activities were markedly reduced, leading to impaired testosterone biosynthesis and spermatogenesis. T1D also altered seminal biochemical and enzymatic properties, as evidenced by decreased fructose levels and reduced activities of HYAL, ATPase, PAP, LDH-C4, and ADA. Collectively, these alterations significantly impaired sperm quality, resulting in reduced sperm density, motility, viability, progressive motility (PR), and increased morphological abnormalities. These results are in agreement with the study by Delbarba et al. [18], which demonstrated that in patients, T1D is associated with a marked impairment of sperm quality, particularly in terms of volume, density, and motility. Another study by Khosravi et al. [19] reported that type 1 diabetes induces testicular inflammation, as evidenced by increased testosterone levels and elevated testicular inflammatory markers, including tumor necrosis factor alpha and interleukin 6. In addition, type 1 diabetes was associated with marked oxidative stress, evidenced by altered antioxidant defenses, including changes in carbonyl protein content, glutathione levels, and increased activities of SOD, CAT, and GPx, together with elevated malondialdehyde. This condition was also characterized by neutrophil infiltration, leukospermia, and increased MPO activity. Consistently, the deleterious effects of T1D on sperm quality observed in our study are in agreement with the findings of Simas et al. [20] who reported that T1D increases lipid peroxidation in the testis and epididymis. It also induces sperm DNA fragmentation, chromatin alterations, reduced mitochondrial mass, and impaired acrosome integrity. Together, these changes lead to a marked decline in male fertility.
In contrast, in T1D rats treated with ATR, the present study demonstrates for the first time that ATR effectively protects pancreatic islets of Langerhans against cellular damage, as confirmed by histological analysis. This protective effect was dose-dependent, with maximal efficacy observed at 170 mg/kg. These findings were supported by a significant reduction in blood glucose levels, approaching those of healthy control rats. The anti-diabetic action of ATR may be attributed to its strong anti-inflammatory and antioxidant properties. Consistently, AlRashidi et al. [21] reported that natural pigments exert protective effects through the suppression of neutrophil infiltration, attenuation of inflammation, and enhancement of pancreatic antioxidant capacity.
The beneficial effects of ATR were also observed at the seminal plasma of T1D rats. ATR treatment reduced leukocyte levels and MPO activity, indicating a decrease in inflammation. This improvement was associated with lower oxidative stress, as shown by reduced H2O2 and TOS levels and decreased TBARS. In addition, ATR enhanced the antioxidant capacity of seminal plasma, reflected by increased TAS levels. Although the effects of ATR on sperm quality have not been previously reported, our findings are consistent with earlier studies describing the anti-inflammatory and antioxidant properties of this natural compound [22]. The anti-inflammatory and antioxidant effects of ATR may result from its ability to limit inflammatory signaling and oxidative damage. ATR likely reduces the production of inflammatory mediators, such as tumor necrosis factor-α and interleukin-6, thereby attenuating inflammatory responses. At the same time, it may strengthen the body’s antioxidant defenses by increasing glutathione levels and enhancing key antioxidant enzymes, which helps neutralize excess reactive oxygen species. In addition, ATR can directly scavenge free radicals, reducing lipid and protein oxidation and preserving cellular integrity. It may also support mitochondrial function, limiting excessive oxidative stress at the cellular level. Through these combined actions, ATR exerts broad protective effects against inflammation and oxidative stress. Our results are in agreement with the study by Aladaileh et al. [23], which demonstrated the beneficial effects of phenolic compounds such as Diosmin on male reproductive function in diabetic rats. These effects are attributed to the ability of Diosmin to counteract oxidative stress, as evidenced by a reduction in malondialdehyde and protein carbonyl levels, along with an increase in reduced glutathione and normalization of catalase and superoxide dismutase activities. Moreover, Diosmin exhibits strong anti-inflammatory activity in the testes by suppressing the expression of NF-κB p65 and pro-inflammatory cytokines. In addition, it restores the Sirt1/Nrf2/HO-1 signaling pathway, leading to improvements in sperm parameters and testosterone levels.
Moreover, our study shows that ATR protects seminal plasma from T1D-induced nutritional deterioration by restoring essential minerals, fructose levels, pH balance, and key enzymes such as α-amylase and proteases. These factors are crucial for sperm function. Zn2+ supports chromatin stability and antioxidant defense, Ca2+ regulates motility and the acrosome reaction, Mg2+ supports ATP-dependent metabolism, and Se preserves sperm structure and antioxidant protection. Fructose provides the main energy source for sperm motility, while physiological pH maintains membrane stability. Previous studies have shown that these elements are essential for spermatogenesis, sperm maturation, capacitation, and motility. Trace elements such as zinc, selenium, manganese, and copper also reduce lipid peroxidation and protect sperm membranes [24], whereas calcium and magnesium contribute to osmotic balance and accessory gland function. Their deficiency markedly impairs sperm quality and male fertility [25]. Accordingly, ATR-mediated restoration of these components promotes spermatogenesis and improves sperm viability, motility, and fertilizing capacity. In addition, our findings are consistent with those of Sari et al. [26], who reported that proteases in seminal plasma are essential for semen liquefaction and sperm function. Reduced protease activity in T1D rats leads to increased semen viscosity and impaired fertilization potential. Conversely, ATR supplementation restored protease activity, normalized semen liquefaction, and enhanced overall sperm functionality.
α-amylase plays an essential role in carbohydrate metabolism and seminal viscosity, while proteases are required for protein remodeling and sperm maturation. The improvement in these enzymes by ATR is in agreement with the findings of Ferramosca et al. [27] who reported that plant extracts rich in phenolic compounds improve seminal enzymatic activity, and with the preservation of testicular and epididymal integrity observed in similar models by Abdella et al. [9]. ATR also appears to enhance seminal plasma quality by improving mineral availability, stabilizing cell membranes, and supporting endocrine signaling involved in steroidogenesis and Leydig–Sertoli communication, in agreement with Ibrahim et al. [28]. Consistent with studies by Liu et al. [29] and Aldaddou et al. [30], showed that phenolic compounds were shown to increase the activity of 3β-HSD and 17β-HSD, leading to higher testosterone levels, likely through improved enzyme efficiency and restoration of essential minerals (Zn2+, Mg2+, Se, and Ca2+).
Moreover, ATR restored sperm metabolism in T1D rats through the induction of key metabolic enzymes. In particular, the increased activity of LDH-C4, which provides energy for sperm motility and capacitation via glycolysis, and ADA, which regulates purine metabolism, protects against oxidative stress and inflammation, and supports sperm viability, contributed to improved sperm motility, viability, and overall fertilizing potential. These metabolic improvements were accompanied by the maintenance of a seminal microenvironment favorable to reproduction [31]. In parallel, ATR-treated diabetic rats showed marked protection against T1D-induced histopathological damage. Seminiferous tubules were largely preserved, with improved germinal epithelium organization, restoration of the spermatogenic series, and increased Leydig cell density. ATR also reduced Sertoli cell vacuolization and epithelial disintegration. Similarly, epididymal structure was preserved, with maintained epithelial integrity, higher luminal sperm density, and fewer degenerative changes, indicating improved sperm maturation and storage. Together, these findings confirm the ability of ATR to counteract T1D-induced testicular and epididymal damage, sustain spermatogenesis, and preserve reproductive potential, likely through its strong anti-inflammatory and antioxidant properties.
Overall, the beneficial effects of ATR on the physicochemical, nutritional, inflammatory, antioxidant, steroidogenic, and metabolic status of seminal plasma were reflected in a marked improvement in sperm quality. ATR significantly increased sperm density, viability, and especially progressive motility, a key indicator of male fertility, while reducing sperm abnormalities. These results clearly demonstrate the protective potential of this phenolic pigment. To date, no study has comprehensively evaluated the role of ATR in reducing oxidative stress and inflammation in this context. Its effects likely result from combined antioxidant and anti-inflammatory actions, together with stimulation of enzymes involved in seminal plasma viscosity, metabolism, sperm function, and fertilizing capacity. This original work highlights the need for further studies to clarify the molecular and enzymatic mechanisms underlying the anti-diabetic and reproductive effects of ATR. Future research should also explore its impact on sperm quality at the genetic level, assess its stability under gastric conditions, and evaluate its therapeutic potential as a dietary supplement, pharmaceutical agent, or natural food colorant.

4. Materials and Methods

4.1. Collection and Drying of Parmotrema hypoleucinum Lichen

Parmotrema hypoleucinum lichen was collected from the Tabarka region in northern Tunisia, dried at room temperature in the absence of light, and then ground into a fine powder to increase the surface area for solvent contact. The extraction of Parmotrema hypoleucinum was performed using a cold maceration procedure with acetone. The solvent was evaporated under vacuum, and the resulting dry residues were stored at −20 °C for further studies, including compound isolation and identification. All chemicals and solvents used in this study were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

4.2. Isolation of ATR from Parmotrema hypoleucinum Lichen

ATR was isolated from 100 g of Parmotrema hypoleucinum lichen by extracting the dried parts with 150 mL of chloroform using a Soxhlet apparatus. The extract was filtered and stored at 4 °C for 24 h to allow the precipitation of ATR. The precipitate was purified by column chromatography with silica (70–230 mesh) using a chloroform/hexane (5/1) solvent system. After purification, 1.42 mg of ATR was obtained, corresponding to a yield of 1.42% (w/w). The isolated ATR was stored at −80 °C to ensure its stability. For biological assays, ATR was dissolved in DMSO at a concentration of 10 mg/mL, and serial dilutions were prepared. At the maximum concentration of 100 μg/mL, the DMSO concentration was 0.01% [32].

4.3. ATR FTIR Identification

The FT-IR spectrum of ATR was recorded using an FT-IR spectrometer (PerkinElmer, Shelton, CT, USA) with a scanning range in the mid-infrared region (MIR) from 450 to 4000 cm−1 and a resolution of 1.0 cm−1. This setup ensured precise identification of absorption bands corresponding to the functional groups in the ATR molecule, providing detailed insights into its chemical structure.

4.4. UV Analysis of ATR

The UV-Vis spectral analysis of ATR was performed to confirm its structural characteristics and assess its absorption properties. The ATR solution, prepared in DMSO, was scanned in the wavelength range of 200–400 nm using a UV-Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

4.5. Induction of T1D and Experimental Design

A total of 36 male Wistar rats (3 months old; 293 ± 9 g) were housed in the animal facility of the Faculty of Science of Tunis under standard laboratory conditions of temperature, humidity, and a 12 h light/dark cycle. Diabetes was induced in rats by a single intraperitoneal injection of alloxan at 150 mg/kg body weight, dissolved in citrate buffer. One week after alloxan injection, blood glucose levels were measured using a glucometer from tail vein blood, and only rats with blood glucose ≥2 g/L were considered diabetic and subsequently selected for inclusion in the experimental groups. The rats were randomly assigned to eight experimental groups (6 rats per group) as follows: Group 1 (C), healthy control rats fed a standard diet; Group 2 (T1D), diabetic rats without treatment; Groups 3–5 (T1D-ATR50, T1D-ATR170, and T1D-ATR200), diabetic rats receiving 50, 170, and 200 mg/kg body weight of ATR, respectively, daily for 30 days via oral gavage; and Group 6 (T1D-MET), diabetic rats treated with 10 mg/kg of Metformin daily via gastric gavage for 30 days [33]. All compounds were homogenized in physiological saline and administered every morning, while control rats received an equivalent volume of 0.9% NaCl solution to ensure comparable physiological conditions. At the end of the treatment period, all rats were weighed and euthanized by decapitation. Blood samples were collected and stored at −80 °C for subsequent serum biochemical analyses. The epididymides were immediately harvested, and sperm was collected and diluted in PBS buffer (pH 7.4) for evaluation of sperm parameters. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University (approval number A547-2024), and animals were euthanized following anesthesia induced by intraperitoneal injection of pentobarbital sodium at a dose of 30 mg/kg body weight [33].

4.6. Tissue Histology and Morphological Assessment

Immediately following euthanasia, testes and epididymides were carefully removed, rinsed in cold physiological saline (0.9% NaCl) to eliminate residual blood, and fixed in 10% neutral buffered formalin (NBF) at room temperature for 48 h to preserve tissue architecture. After fixation, the tissues were washed, dehydrated through a graded ethanol series (70% to 100%), cleared in xylene, and embedded in paraffin wax. Paraffin blocks were sectioned at 4–5 µm thickness using a rotary microtome, and the resulting sections were mounted on poly-L-lysine-coated slides and allowed to dry overnight at 37 °C. Sections were then subjected to hematoxylin and eosin (H&E) staining, involving deparaffinization, rehydration, staining with hematoxylin for 5 min, differentiation in acid alcohol, counterstaining with eosin for 2 min, dehydration, clearing in xylene, and mounting with DPX medium. Histological analysis was performed under a light microscope at magnifications ranging from 100× to 400× to evaluate structural integrity and pathological changes in the seminiferous tubules and epididymal ducts.

4.7. Biochemical Analysis

4.7.1. Collection and Preparation of Rat Epididymal Seminal Plasma for Biochemical Assays

Following euthanasia, seminal plasma was collected from the epididymides. The abdominal cavity was opened, and the epididymides were carefully excised. Each epididymis was incised, and the contents were gently expressed into PBS buffer (pH 7.4). The resulting suspension was centrifuged at low speed (1000–1500× g) to separate spermatozoa from the seminal plasma. The collected seminal plasma was aliquoted and stored at −80 °C for subsequent enzymatic and biochemical analyses.

4.7.2. Assessment of Epididymal Sperm Parameters in Rats

Following euthanasia, the epididymides were carefully excised. Each epididymis was incised, and the released contents were diluted in 10 mL of physiological buffer (PBS, pH 7.4). The resulting sperm suspension was further diluted in PBS, and sperm concentration was determined using a hemocytometer under a light microscope. Sperm motility, a critical parameter of male fertility, was evaluated in accordance with WHO guidelines [34]. Sperm motility was classified into three categories: progressive motility (PR), in which sperm move actively either in a straight line or in a large circular pattern; non-progressive motility (NP), in which sperm exhibit movement but fail to make forward progress; and immotile (IM), in which no movement is observed. Sperm morphology was evaluated microscopically to assess the structural integrity of the head, midpiece, and tail, following WHO criteria. Sperm viability was determined using the Nigrosin-Eosin staining method. Briefly, 100 µL of seminal plasma was mixed with an equal volume of 1% Eosin Y and 10% Nigrosin solution and incubated for 30 s at room temperature. Smears were prepared on clean glass slides, air-dried, and examined under a light microscope. Live spermatozoa remained unstained due to intact plasma membranes, whereas dead spermatozoa absorbed the dye and appeared pink or red [34].

4.7.3. Assessment of Leukospermia and MPO Activity

Leukocyte counting was performed using a hemocytometer under light microscopy at 1000× magnification. An aliquot of the sperm sample was mixed with 0.4% (w/v) Trypan Blue vital stain and incubated for 10 min at room temperature to facilitate leukocyte visualization. The stained sample was then loaded into the hemocytometer chamber, and leukocytes were counted within the designated grid area to estimate their concentration per milliliter of semen. MPO activity in seminal plasma was measured spectrophotometrically based on the oxidation of tetramethylbenzidine in the presence of H2O2. Briefly, aliquots of seminal plasma were mixed with a reaction buffer containing TMB and H2O2, and the reaction was allowed to proceed at room temperature for a defined period (e.g., 5–10 min). The enzymatic reaction produces a colored product, the intensity of which is proportional to MPO activity. The absorbance of the reaction mixture was measured at 460 nm using a microplate reader or spectrophotometer. MPO activity was expressed in units per milliliter of seminal plasma, calculated from a standard curve prepared using purified MPO or a known concentration of hydrogen peroxide [35].

4.7.4. Determination of Enzymes Critical for Sperm Function and Fertility

ATPase activity in seminal plasma was determined spectrophotometrically. Briefly, 200 µL of seminal plasma was incubated with 200 µL of 5 mM KCl, 1300 µL of 0.1 M Tris-HCl buffer, and 40 µL of 50 mM ATP for 30 min at 37 °C. The reaction was terminated by adding 1 mL of distilled water and ammonium molybdate, followed by the addition of a freshly prepared 9% ascorbic acid solution. The mixture was then placed on ice for 10 min. The inorganic phosphate released, which directly reflects ATPase activity, was quantified by measuring the absorbance at 660 nm [36]. The activity of 3β-HSD in seminal plasma was measured following the method described by Talalay [37] which quantifies the enzymatic conversion of Δ5-steroids to Δ4-ketosteroids as an indicator of steroidogenic activity. 3β-HSD activity in seminal plasma was assessed by mixing 100 µL of seminal plasma with 25 µL of 100 mM sodium pyrophosphate buffer (pH 8.9), 10 µL of ethanol containing 0.3 mM DHEA, and 40 µL of 25% BSA. Subsequently, 50 µL of 0.5 mM NAD+ was added at room temperature (25 °C), and the enzymatic conversion was monitored by measuring the increase in absorbance at 340 nm, corresponding to the reduction of NAD+ to NADH. Similarly, 17β-HSD activity was determined by incubating 100 µL of seminal plasma with 25 µL of 100 mM sodium pyrophosphate buffer (pH 10.2), 10 µL of ethanol containing 0.3 mM testosterone, and 40 µL of 25% BSA, followed by the addition of 50 µL of 0.5 mM NAD+. The enzymatic reaction was monitored spectrophotometrically at 340 nm, reflecting the reduction of NAD+ to NADH as an indicator of 17β-HSD activity. β-HSD [38]. LDH-C4 activity in seminal plasma was measured enzymatically by monitoring the oxidation of NADH in the presence of lactate. Semen samples were centrifuged and incubated with lactate, NAD+, and buffer at 37 °C. The increase in absorbance at 340 nm was recorded, and enzyme activity was calculated using Beer’s Law. Results were expressed in U/mL, where one unit represents the conversion of 1 µmol NAD+ to NADH per minute at 37 °C [39]. Prostatic acid phosphatase activity in seminal plasma was measured spectrophotometrically using p-nitrophenyl phosphate (pNPP) as a substrate. Seminal plasma was incubated with 2 mM pNPP in buffer at 37 °C for 15–30 min. The reaction was stopped with an alkaline solution, and the formation of p-nitrophenol was quantified by measuring absorbance at 405 nm [40]. Adenosine deaminase (ADA) activity in seminal plasma was measured enzymatically using adenosine as the substrate. Seminal plasma was incubated with adenosine at 37 °C for 30–60 min, allowing ADA to convert adenosine into inosine. The reaction was stopped by adding a reagent that reacts with the released ammonia to form a colored complex, and absorbance was measured at 405 nm. Activity was expressed in U/mL, where one unit corresponds to the production of 1 µmol of ammonia per minute at 37 °C [41]. Hyaluronidase activity in seminal plasma was measured by incubating 0.2 mL of plasma with 0.8 mL of hyaluronate solution (1.25 g/L, pH 4) at 37 °C. The reaction stopped with 0.1 mL of 0.8 M tetraborate and heating at 100 °C for 3 min. After adding 3 mL of DMAB reagent, the mixture was incubated at 37 °C for 20 min to develop color. Absorbance was measured at 585 nm, and enzyme activity was calculated from the color intensity [42]. The α-amylase activity in seminal plasma was determined using the Amylase CNPG3 kit (Biolabo, ref. 80106, Maizy, France) at 405 nm and 37 °C, expressed in U/mL after correction. Total protein concentration was measured by the Biuret method (Biolabo, ref. 80008, France) at 546 nm and expressed in g/L. Fe2+, Ca2+, and Mg2+ levels were quantified using Biolabo kits (Biolabo, refs. 98212, 92108, 80004). Protease activity was assessed using a colorimetric commercial kit (Clinisciences, NB-22-40834-96, Nanterre, France) based on chromogenic substrate hydrolysis, measured at 405 nm and expressed in U/mL. Se level was determined by ICP-MS following acid digestion (HNO3/H2O2, 30 min predigestion, microwave-assisted digestion), using matrix-matched standards, certified reference materials, and recovery tests, in accordance with U.S. EPA 6020B and ISO 17294-2:2003, expressed in µg·L−1 or µg·g−1 protein [43]. Finally, seminal plasma pH was measured directly with a calibrated pH meter (Mettler Toledo, SevenCompact, Columbus, OH, USA) at room temperature, with the electrode immersed in the sample, and values expressed in pH units (0–14).

4.7.5. Seminal Plasma Oxidative and Hormonal Assessment

Testosterone levels were quantified using a radioimmunoassay kit with antibodies from Paris (Compiègne, France), with intra-assay and inter-assay coefficients of variation of 4.6% and 7.5%, respectively [44]. Lipid peroxidation was assessed by measuring TBARS rate using the method of Buege and Aust [45]. H2O2 level in seminal plasma were measured using the method described by Dingeon et al. [46], which quantifies H2O2 based on its reaction with a specific chromogenic substrate to produce a colored compound detectable spectrophotometrically at 410 nm. Fructose levels were determined using the method described by Gavella [47]. In this method, a known volume of seminal plasma is mixed with an enzyme solution containing fructokinase, ATP, and magnesium ions. This mixture catalyzes the phosphorylation of fructose to fructose-1-phosphate. The absorbance or fluorescence was measured at a specific wavelength, usually around 340 nm, and compared to a standard curve generated from known concentrations of fructose. TAS level in seminal plasma was measured using the ABTS assay, where 3 mg of ABTS was dissolved in phosphate buffer (pH 7.4) and reduced with 0.2 mM H2O2 to produce a blue color. A total of 100 µL of seminal plasma was incubated with the ABTS-H2O2 solution for 30 min, and absorbance was read at 734 nm [46]. Total oxidant status (TOS) was assessed by incubating 100 µL of seminal plasma with phosphate buffer, allowing oxidation of ferrous ions by sample oxidants. The reaction was treated with 2,4,6-tripyridyl-s-triazine, and TOS levels were expressed as µmol H2O2 equivalents, reflecting oxidative stress [48].

4.7.6. Statistical Analysis

Data are expressed as mean ± standard deviation (SD), with six animals per experimental group. Data were analyzed using multiple-ANOVA (MANOVA), followed by two-way and one-way ANOVAs when significant effects were detected. Fisher’s post hoc test was applied for group comparisons. A p-value ≤ 0.05 was considered statistically significant.

5. Conclusions

Our study demonstrates, for the first time, that ATR exerts potent protective effects against T1D-induced reproductive dysfunction in male rats. ATR preserved pancreatic β-cell integrity, reduced hyperglycemia, and mitigated oxidative stress and inflammation in seminal plasma. It restored essential minerals, fructose, pH balance, and key enzymes such as α-amylase, proteases, HYAL, ATPase, and PAP, which are critical for sperm metabolism, motility, and fertilization capacity. ATR also stimulated steroidogenic enzymes 3β-HSD and 17β-HSD, enhancing testosterone biosynthesis and supporting spermatogenesis. Histological analyses confirmed improved testicular and epididymal architecture. Collectively, these effects resulted in enhanced sperm density, viability, progressive motility, and reduced abnormalities. The underlying mechanisms likely involve antioxidant, anti-inflammatory, metabolic, and enzymatic regulatory actions of this phenolic pigment. This study underscores ATR’s potential as a therapeutic agent to improve male reproductive health in T1D. Future investigations should explore molecular pathways, genetic interactions, and the stability of ATR for potential applications as a dietary supplement, pharmaceutical product, or natural food colorant.

Author Contributions

W.I.E.-S., A.F.A., M.Z.A. and N.M.: Writing—Original Draft, Formal Analysis. H.K.A., N.S.A. and K.H.: Writing—Review & Editing, Formal Analysis. W.I.E.-S. and K.H.: Writing—Review & Editing, Formal Analysis, Investigation, Conceptualization, Supervision, Project Administration, Methodology, Funding Acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been funded by Scientific Research Deanship at University of Ha’il-Saudi Arabia through project number <<RG-24 039>>.

Institutional Review Board Statement

Animal experimentation was conducted in strict accordance with ethical standards and animal welfare protocols. Ethical approval for this study was granted by the National Ethics Committee (TNAPO-06-B-071) under approval number TNC#2024-3122. All animal procedures were performed in compliance with the ARRIVE guidelines and in accordance with the U.K. Animals (Scientific Procedures) Act, 1986, EU Directive 2010/63/EU on the protection of animals used for scientific purposes, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data sets obtained and analyzed during the current study are available in the manuscript. Further inquiries can be directed to the corresponding author.

Acknowledgments

This research has been funded by Scientific Research Deanship at University of Ha’il-Saudi Arabia through project number <<RG-24 039>>.

Conflicts of Interest

The authors declare no conflicts of interest.

Correction Statement

This article has been republished with a minor correction to Acknowledgments. This change does not affect the scientific content of the article.

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Figure 1. FTIR (A) and UV (B) characterization of ATR.
Figure 1. FTIR (A) and UV (B) characterization of ATR.
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Figure 2. Effect of ATR on alloxan-induced pancreatic β-cell inflammation, damage, and death leading to T1D-associated hyperglycemia. ATR administration effectively protected pancreatic β-cells against alloxan-induced injury, thereby reducing blood glucose levels in T1D rats. These findings suggest that ATR exerts protective effects through attenuation of β-cell inflammation and preservation of their structural and functional integrity. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
Figure 2. Effect of ATR on alloxan-induced pancreatic β-cell inflammation, damage, and death leading to T1D-associated hyperglycemia. ATR administration effectively protected pancreatic β-cells against alloxan-induced injury, thereby reducing blood glucose levels in T1D rats. These findings suggest that ATR exerts protective effects through attenuation of β-cell inflammation and preservation of their structural and functional integrity. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
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Figure 3. Effect of T1D and ATR on seminal leukocyte count and MPO activity. ATR administration inhibits seminal inflammation by reducing leukocyte recruitment and suppressing inflammatory enzyme activity in a dose-dependent manner. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
Figure 3. Effect of T1D and ATR on seminal leukocyte count and MPO activity. ATR administration inhibits seminal inflammation by reducing leukocyte recruitment and suppressing inflammatory enzyme activity in a dose-dependent manner. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
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Figure 4. Effect of T1D and ATR on seminal plasma key digestive enzyme activities (α-amylase and proteases). T1D markedly suppressed the activity of key digestive enzymes in seminal plasma, including α-amylase and proteases, leading to impaired carbohydrate and protein catabolism and consequently reduced sperm motility and functionality. In contrast, oral administration of ATR significantly restored the activity of these enzymes, thereby promoting both carbohydrate and protein metabolism within the seminal plasma. This enzymatic stimulation contributes to improved seminal fluid liquefaction, reduced viscosity, and enhanced sperm motility, ultimately supporting better sperm viability and fertilizing potential. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
Figure 4. Effect of T1D and ATR on seminal plasma key digestive enzyme activities (α-amylase and proteases). T1D markedly suppressed the activity of key digestive enzymes in seminal plasma, including α-amylase and proteases, leading to impaired carbohydrate and protein catabolism and consequently reduced sperm motility and functionality. In contrast, oral administration of ATR significantly restored the activity of these enzymes, thereby promoting both carbohydrate and protein metabolism within the seminal plasma. This enzymatic stimulation contributes to improved seminal fluid liquefaction, reduced viscosity, and enhanced sperm motility, ultimately supporting better sperm viability and fertilizing potential. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
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Figure 5. Effect of T1D and ATR on seminal steroidogenesis enzymes (3β-HSD and 17β-HSD) and testosterone levels. ATR significantly prevents T1D-induced decline in enzyme activities and restores testosterone levels in seminal plasma. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
Figure 5. Effect of T1D and ATR on seminal steroidogenesis enzymes (3β-HSD and 17β-HSD) and testosterone levels. ATR significantly prevents T1D-induced decline in enzyme activities and restores testosterone levels in seminal plasma. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
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Figure 6. Impact of T1D and ATR on seminal plasma HYAL, PAP, and ATPase activities. T1D significantly reduced the activity of HYAL, PAP, and ATPase in seminal plasma, resulting in impaired sperm penetration ability, altered seminal liquefaction, and diminished flagellar energy supply, respectively. Oral administration of ATR effectively restored the activities of these key enzymes toward normal levels, thereby improving seminal plasma functionality. Restoration of HYAL activity facilitates degradation of hyaluronic acid surrounding the oocyte and reduces seminal viscosity; PAP activation promotes semen liquefaction and sperm dispersion, and normalization of ATPase activity ensures adequate ATP hydrolysis to fuel progressive motility. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
Figure 6. Impact of T1D and ATR on seminal plasma HYAL, PAP, and ATPase activities. T1D significantly reduced the activity of HYAL, PAP, and ATPase in seminal plasma, resulting in impaired sperm penetration ability, altered seminal liquefaction, and diminished flagellar energy supply, respectively. Oral administration of ATR effectively restored the activities of these key enzymes toward normal levels, thereby improving seminal plasma functionality. Restoration of HYAL activity facilitates degradation of hyaluronic acid surrounding the oocyte and reduces seminal viscosity; PAP activation promotes semen liquefaction and sperm dispersion, and normalization of ATPase activity ensures adequate ATP hydrolysis to fuel progressive motility. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg; β p < 0.05 vs. T1D + ATR 200 mg/kg.
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Figure 7. Effect of T1D and ATR on key seminal plasma fertility enzymes, including ADA and LDH-C4. T1D markedly decreased ADA and LDH-C4 activities in seminal plasma, leading to impaired purine metabolism, reduced antioxidant protection, compromised glycolytic energy supply, and diminished sperm motility. Oral administration of ATR significantly restored ADA and LDH-C4 activities toward normal values, thereby improving sperm energy metabolism, progressive motility, and overall fertilization potential. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg.
Figure 7. Effect of T1D and ATR on key seminal plasma fertility enzymes, including ADA and LDH-C4. T1D markedly decreased ADA and LDH-C4 activities in seminal plasma, leading to impaired purine metabolism, reduced antioxidant protection, compromised glycolytic energy supply, and diminished sperm motility. Oral administration of ATR significantly restored ADA and LDH-C4 activities toward normal values, thereby improving sperm energy metabolism, progressive motility, and overall fertilization potential. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D + ATR 50 mg/kg; α p < 0.05 vs. T1D + ATR 170 mg/kg.
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Figure 8. Histological analysis of testicular tissue. ATR administration preserves seminiferous tubule architecture, maintains germ cell layers, and reduces cellular debris and lymphocyte infiltration in T1D rats, compared to untreated T1D rats.
Figure 8. Histological analysis of testicular tissue. ATR administration preserves seminiferous tubule architecture, maintains germ cell layers, and reduces cellular debris and lymphocyte infiltration in T1D rats, compared to untreated T1D rats.
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Figure 9. Histological analysis of epididymal tissue. ATR treatment restores sperm density in the epididymal lumen, reduces morphological abnormalities, and prevents the accumulation of cellular debris, supporting normal sperm maturation and storage.
Figure 9. Histological analysis of epididymal tissue. ATR treatment restores sperm density in the epididymal lumen, reduces morphological abnormalities, and prevents the accumulation of cellular debris, supporting normal sperm maturation and storage.
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Table 1. Effect of T1D and ATR supplementation on sperm quality. T1D significantly impaired sperm quality, as evidenced by reduced sperm density, viability, total motility, progressive motility (PR%) and non-progressive motility (NP%), together with an increased percentage of abnormal sperm. ATR supplementation effectively protected against these impairments in a dose-dependent manner, improving all sperm quality parameters. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D rats treated with ATR at dose 50 mg/kg; α p < 0.05 vs. T1D rats treated with ATR at dose 170 mg/kg; β p < 0.05 vs. T1D rats treated with ATR at dose 200 mg/kg.
Table 1. Effect of T1D and ATR supplementation on sperm quality. T1D significantly impaired sperm quality, as evidenced by reduced sperm density, viability, total motility, progressive motility (PR%) and non-progressive motility (NP%), together with an increased percentage of abnormal sperm. ATR supplementation effectively protected against these impairments in a dose-dependent manner, improving all sperm quality parameters. Values are expressed as mean ± SD. Statistical significance: * p < 0.05 vs. normal group; @ p < 0.05 vs. untreated T1D group; # p < 0.05 vs. T1D rats treated with ATR at dose 50 mg/kg; α p < 0.05 vs. T1D rats treated with ATR at dose 170 mg/kg; β p < 0.05 vs. T1D rats treated with ATR at dose 200 mg/kg.
Group/ParameterConT1DT1D-ATR50T1D-ATR170T1D-ATR200T1D-Met
Density (×106/mL)32.2 ± 0.814.8 ± 0.42 *19.3 ± 0.52 *@27.9 ± 0.52 *@#28 ± 0.6 *@#20.9 ± 0.43 *@αβ
Viability (%)78.4 ± 2.841.9 ± 2.3 *51.7 ± 1.9 *@71.2 ± 3.4 *@#71 ± 4.1 *@#44.8 ± 1.7 *#αβ
Total motility (%)71.2 ± 2.522.4 ± 0.67 *38.4 ± 2.1 *@65 ± 3.6 *@#64.2 ± 3.5 *@#41.2 ± 1.3 *@αβ
PR (%)58.4 ± 1.95.5 ± 0.8 *23.4 ± 1.6 *@51.7 ± 3.3 *@#50.1 ± 3.8 *@#24.1 ± 1.1 *@αβ
NP (%)12.8 ± 1.816.9 ± 0.6 *15.0 ± 0.63 *@13.1 ± 0.8 *@#14.1 ± 1.7 *@#α17 ± 0.94 *@#αβ
Anomaly (%)24.8 ± 0.452.8 ± 1.9 *44.6 ± 2.3 *@28.4 ± 2 *@#31.2 ± 2.4 *@#α44.8 ± 1.4 *@#αβ
Zn2+ (mmol/L)3.4 ± 0.31.69 ± 0.2 *1.74 ± 0.08 *@2.92 ± 0.13 *@#2.78 ± 0.13 *@#α2.1 ± 0.09 *#αβ
Ca2+ (mmol/L)1.9 ± 0.20.9 ± 0.02 *1.12 ± 0.04 *@1.81 ± 0.09 *@#1.74 ± 0.08 *@#0.9 ± 0.06 *#αβ
Mg2+ (mmol/L)1.1 ± 0.20.52 ± 0.04 *0.63 ± 0.02 *@0.9 ± 0.03 *@#0.89 ± 0.04 *@#0.7 ± 0.03 *#@αβ
Se (mmol/L)0.12 ± 0.080.07 ± 0.03 *0.1 ± 0.006 *@0.11 ± 0.03 *@#0.10 ± 0.04 *@#α0.09 ± 0.02 *#@αβ
Fruct level (mM/mL)168 ± 789.4 ± 5.4 *109 ± 8.4 *@152.9 ± 6.5 *@#144.3 ± 4.2 *@#131.2 ± 3.9 *@#αβ
Prot levels (mg/mL)49.8 ± 0.1326.4 ± 1.32 *31.5 ± 1.3 *@44.2 ± 2.5 *@#43.2 ± 2.5 *@#38.4 ± 2.3 *#αβ
pH of semen7.4 ± 0.066.8 ± 0.13 *6.9 ± 0.08 *@7.3 ± 0.08 *@#7.34 ± 0.2 *@#7.13 ± 0.23 *@#αβ
TOS level (U/mL)0.3 ± 0.0011.34 ± 0.09 *1.09 ± 0.04 *@0.39 ± 0.04 *@#0.37 ± 0.06 *@#α0.87 ± 0.03 *@#αβ
TBARS (U/mL)0.38 ± 0.011.7 ± 0.07 *1.17 ± 0.04 *@0.46 ± 0.02 *@#0.74 ± 0.03 *@α0.74 ± 0.07 *@#α
TAS level (U/mL)2.6 ± 0.120.8 ± 0.003 *1.16 ± 0.09 *@2.49 ± 0.01 *@#2.53 ± 0.013 *@#α1.32 ± 0.07 *@#αβ
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MDPI and ACS Style

El-Sofany, W.I.; Alshammari, A.F.; Alshammari, M.Z.; Alshammari, H.K.; Alshammari, N.S.; Masood, N.; Hamden, K. Extraction, Characterization, and Biological Evaluation of Atranorin Against Diabetes-Induced Reproductive Dysfunction Through Modulation of Oxidative Stress, Inflammatory Pathways and Key Reproductive Enzymes. Int. J. Mol. Sci. 2026, 27, 2416. https://doi.org/10.3390/ijms27052416

AMA Style

El-Sofany WI, Alshammari AF, Alshammari MZ, Alshammari HK, Alshammari NS, Masood N, Hamden K. Extraction, Characterization, and Biological Evaluation of Atranorin Against Diabetes-Induced Reproductive Dysfunction Through Modulation of Oxidative Stress, Inflammatory Pathways and Key Reproductive Enzymes. International Journal of Molecular Sciences. 2026; 27(5):2416. https://doi.org/10.3390/ijms27052416

Chicago/Turabian Style

El-Sofany, Walaa I., Ahlam F. Alshammari, Mona Zaheed Alshammari, Hissah Khashman Alshammari, Nawal S. Alshammari, Najat Masood, and Khaled Hamden. 2026. "Extraction, Characterization, and Biological Evaluation of Atranorin Against Diabetes-Induced Reproductive Dysfunction Through Modulation of Oxidative Stress, Inflammatory Pathways and Key Reproductive Enzymes" International Journal of Molecular Sciences 27, no. 5: 2416. https://doi.org/10.3390/ijms27052416

APA Style

El-Sofany, W. I., Alshammari, A. F., Alshammari, M. Z., Alshammari, H. K., Alshammari, N. S., Masood, N., & Hamden, K. (2026). Extraction, Characterization, and Biological Evaluation of Atranorin Against Diabetes-Induced Reproductive Dysfunction Through Modulation of Oxidative Stress, Inflammatory Pathways and Key Reproductive Enzymes. International Journal of Molecular Sciences, 27(5), 2416. https://doi.org/10.3390/ijms27052416

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