Ameliorative effects of date palm (<i>Phoenix dactylifera</i> L.) fruit extract on sperm quality, DNA integrity, and oxidative stress in a busulfan-induced model in rats

Hakimeh Akbari

doi:10.5653/cerm.2025.08732

Clin Exp Reprod Med > Epub ahead of print

Akbari: Ameliorative effects of date palm (Phoenix dactylifera L.) fruit extract on sperm quality, DNA integrity, and oxidative stress in a busulfan-induced model in rats

Original Article

Clinical and Experimental Reproductive Medicine 2026;cerm.2025.08732.

Published online: August 4, 2026

DOI: https://doi.org/10.5653/cerm.2025.08732

Ameliorative effects of date palm (Phoenix dactylifera L.) fruit extract on sperm quality, DNA integrity, and oxidative stress in a busulfan-induced model in rats

Hakimeh Akbari

Cellular and Molecular Research Center, Gerash University of Medical Sciences, Gerash, Iran

Corresponding author: Hakimeh Akbari Cellular and Molecular Research Center, Gerash University of Medical Sciences, Gerash, Iran
Tel: +98-9177810816 E-mail: anaakbari91@gmail.com

^*This study was financially supported by the Gerash University of Medical Sciences, Gerash, Iran (grant number: IR.GERUMS.RE.1398.0010).

Received September 23, 2025 Revised November 26, 2025 Accepted December 15, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

This study examined the potential protective role of date palm fruit extract (DPFE) and its underlying mechanisms against busulfan-induced sperm damage and infertility.

Methods

Sixty male Wistar rats were allocated to control, busulfan alone (10 mg/kg), DPFE alone (4 mg/kg), and busulfan+DPFE co-treatment groups for a period of 56 days. Semen parameters were evaluated along with DNA fragmentation, assessed using the sperm chromatin dispersion test, and protamine deficiency, detected by chromomycin A3 staining. Testicular oxidative stress was assessed by measuring malondialdehyde (MDA) levels as well as superoxide dismutase (SOD) and catalase (CAT) activities.

Results

Phytochemical analysis confirmed the high polyphenolic and flavonoid content of DPFE. Busulfan significantly induced testicular oxidative stress, as evidenced by increased MDA levels and reduced SOD and CAT activities. These alterations were accompanied by significant declines in sperm motility and viability, deterioration of sperm morphology, and marked increases in protamine deficiency and DNA fragmentation. Co-administration of DPFE significantly counteracted these adverse effects by normalizing testicular oxidative status, reflected by decreased MDA levels and restoration of SOD and CAT activities. Consequently, DPFE improved sperm motility and morphology and, importantly, reduced the proportion of sperm exhibiting high DNA fragmentation and protamine deficiency.

Conclusion

Antioxidant-rich DPFE exerts a significant protective effect against busulfan-induced testicular toxicity, with its mechanism strongly linked to attenuation of testicular oxidative injury, thereby improving conventional sperm quality and, critically, preserving sperm chromatin structure and DNA integrity.

Keywords: Antioxidant; Busulfan; Chemoprotection; Date palm fruit extract; Oxidative stress; Protamine deficiency; Sperm DNA fragmentation

Introduction

Infertility represents a serious global health problem and affects an estimated 15% of couples worldwide, with male factors contributing as a primary or causative element in approximately 50% of cases [1]. A critical determinant of male fertility is the production of functionally competent spermatozoa with intact DNA, as environmental contaminants, therapeutic regimens such as chemotherapy, and oxidative stress can profoundly disrupt spermatogenesis and compromise genetic integrity [2,3]. The chemotherapeutic agent busulfan, a prototypical alkylating compound used in conditioning regimens for bone marrow transplantation, is a well-established experimental model for inducing testicular injury and sterility in rodents due to its selective cytotoxicity toward proliferating germ cells [4]. This model therefore provides a robust system for investigating putative protective or reparative agents. Consequently, there has been increasing interest in natural phytochemicals with antioxidative activity as promising strategies to prevent such iatrogenic damage [5]. Phoenix dactylifera L., commonly known as date palm fruit, is a rich source of phenolic compounds, flavonoids, and natural sugars and has demonstrated potent antioxidant, anti-inflammatory, and anti-apoptotic activities across multiple models of oxidative stress-mediated injury [6]. Investigating its reparative potential in the context of busulfan-induced spermatogenic damage therefore holds substantial promise for the development of adjunctive therapeutic strategies aimed at preserving male fertility in patients undergoing gonadotoxic treatments.

Busulfan induction is a standard experimental approach in reproductive toxicology that involves administering the chemotherapeutic agent busulfan to laboratory animals, primarily rodents, to generate a reproducible model of impaired spermatogenesis and infertility [7]. As a bifunctional alkylating agent, busulfan selectively depletes spermatogonial stem cells, thereby disrupting the entire spermatogenic process and resulting in azoospermia, reduced testicular weight, and Leydig cell dysfunction [8]. The importance of this model lies in its ability to closely mimic the testicular damage observed in cancer patients undergoing chemotherapy, thus providing a controlled framework for screening potential protective interventions [8]. In contrast, date palm fruit extract (DPFE) comprises a complex mixture of bioactive compounds, including flavonoids such as quercetin, phenolic acids such as ferulic and coumaric acids, and carotenoids, all of which are well recognized for their potent free-radical scavenging activity [9]. Previous studies have established that DPFE can mitigate oxidative stress in multiple tissues. Specifically, it has demonstrated hepatoprotective and nephroprotective effects in toxin-induced injury models, largely through the upregulation of endogenous antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), and the inhibition of lipid peroxidation, as reflected by reduced malondialdehyde (MDA) levels [10,11]. However, its application in the specific context of busulfan-induced testicular dysfunction, its direct effects on the spermatogenic epithelium and sperm DNA, and its modulatory influence on testicular oxidative stress markers such as MDA, SOD, and CAT remain insufficiently explored and require further mechanistic elucidation. Comprehensive assessment of male fertility potential extends beyond simplistic evaluation of sperm count and instead requires a multi-parameter analysis encompassing sperm count, motility, morphology, and, critically, DNA integrity [12]. Sperm motility and concentration remain fundamental semen quality indicators, with the World Health Organization (WHO) establishing strict reference thresholds for normality; reductions in either parameter are strongly associated with diminished fertilization potential [13,14]. Normal sperm morphology, reflecting proper spermatogenesis and post-testicular maturation, represents another essential determinant, as structural abnormalities can impede sperm–oocyte interaction. Beyond these conventional parameters, sperm DNA integrity has emerged as a more powerful predictor of assisted reproductive technology outcomes, given its association with fertilization failure, impaired embryonic development, and increased risk of pregnancy loss [15,16]. Under physiological conditions, DNA integrity is preserved during spermatogenesis through the orderly replacement of histones with protamines, which tightly compact and protect nuclear chromatin. Protamine deficiency therefore renders spermatozoa more susceptible to oxidative insult and DNA strand breaks [17]. In addition, the spermatogenic process is tightly regulated by testicular hormones, particularly testosterone produced by Leydig cells and follicle-stimulating hormone, both of which are essential for germ cell proliferation, differentiation, and Sertoli cell function [18]. Accordingly, any intervention aimed at ameliorating testicular damage should demonstrate efficacy not only in restoring hormonal balance but also in improving seminal parameters and epigenetic indicators to ensure the production of genetically competent spermatozoa [18].

Despite substantial evidence supporting the antioxidant capacity of date palm extract, a clear research gap persists regarding the specific mechanistic actions of DPFE in spermatogenic recovery and DNA protection within this context. Prior investigations of natural antioxidants have largely focused on general oxidative stress markers, such as MDA and SOD, without adequately addressing protamine replacement efficiency, chromatin condensation, or broader implications for epigenetic regulation [19]. Furthermore, existing literature on DPFE is predominantly oriented toward its effects on somatic tissues, particularly the liver and kidneys, while comparatively few studies have examined its bioavailability within the testicular microenvironment or its direct interactions with spermatogonial stem cells and Sertoli cell function [20]. In addition, DPFE dosing strategies and therapeutic windows, particularly with respect to preventive versus restorative administration following busulfan exposure, remain insufficiently standardized and continue to be a subject of debate [21]. Moreover, although some studies have reported improvements in conventional sperm parameters, they often lack concurrent and rigorous evaluation of the DNA fragmentation index [22]. Addressing these limitations is therefore essential, not only to confirm observed improvements in sperm quality but also to elucidate the cellular and molecular mechanisms through which P. dactylifera L. fruit extract protects sperm DNA and nuclear chromatin against busulfan-induced damage. Accordingly, the primary objective of the present study was to systematically investigate the ameliorative effects of date palm (P. dactylifera L.) fruit extract on sperm quality parameters, testicular hormone concentrations, and, most importantly, sperm DNA integrity in a rat model of busulfan-induced testicular toxicity.

Methods

1. Chemicals

Dimethyl sulfoxide (DMSO; Sigma, D2650), busulfan (Sigma, B2635), chromomycin A3 (CMA3), and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich. Additionally, sodium dodecyl sulfate, ethylenediaminetetraacetic acid, tris(hydroxymethyl)aminomethane, and dithiothreitol were purchased from the same supplier. All chemicals used in the study were of analytical grade.

2. Plant material and extract preparation

Fresh fruits of P. dactylifera were procured from Bam city, located in Kerman Province in southeastern Iran. A voucher specimen (KF-1638) was deposited for botanical authentication at the Herbarium of the Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. The fruit cores were manually separated and discarded. Subsequently, 100 g of the fruit mesocarp was immersed in 1,000 mL of distilled water and macerated at room temperature for 48 hours. The resulting mixture was then homogenized using a mechanical blender and centrifuged at 4,000 rpm at 4 °C for 20 minutes. The supernatant was collected, designated as the aqueous extract, and stored at 4 °C until administration via gavage [23].

3. Phytochemical analysis of the aqueous extract

The phytochemical composition of the aqueous DPFE, prepared according to the above-mentioned protocol, was quantified in the Department of Pharmacology. In brief, total phenolic content, determined using the Folin–Ciocalteu method, was 12.5±0.8 mg gallic acid equivalents (GAE)/g of dry extract. Total flavonoid content, evaluated by the aluminum chloride colorimetric method, was 4.2±0.3 mg quercetin equivalents (QE)/g of dry extract.

Additionally, high-performance liquid chromatography analysis was performed for the identification and quantification of selected phenolic compounds. Based on this analysis, several bioactive phenolics were identified, including gallic acid (1.8±0.1 mg/g), caffeic acid (0.9±0.05 mg/g), ferulic acid (1.2±0.1 mg/g), and p-coumaric acid (0.7±0.05 mg/g).

4. Busulfan solution preparation

A busulfan solution (Sigma, B2635) was prepared by initially dissolving the compound in DMSO (Sigma, D2650). An equal volume of sterile water was subsequently added to obtain a final busulfan concentration of 5 mg/mL [24].

5. Animals

Sixty adult male Wistar rats, weighing approximately 220–240 g, were used in this study. All experimental procedures were approved by the Gerash University of Medical Sciences Animal Ethics Committee (Approval No. 1401.002) and were conducted in accordance with its guidelines for the care and use of laboratory animals. The animals were housed under standard laboratory conditions at a controlled room temperature of 22±2 °C and a 12-hour light/dark cycle. All rats were provided ad libitum access to standard laboratory chow and tap water.

6. Experimental design

A randomized controlled experimental design was employed. Sample size calculation was based on prior studies with comparable endpoints [25] and was confirmed by power analysis using G*Power software (ver. 3.1.9.7), assuming an effect size of 0.4, an alpha error of 0.05, and a power of 0.8, which yielded a minimum requirement of 12 rats per group. A group size of n=15 was selected to account for potential attrition. To evaluate the effects of DPFE against busulfan-induced toxicity, male rats were randomly assigned to four groups using a computer-generated random number sequence (n=15 per group). The total treatment duration was 56 days [25]. The experimental groups were defined as follows.

1) Control group: Rats in the control group served as vehicle controls and received a daily intraperitoneal injection of 1 mL of distilled water. To ensure consistency with solvent exposure in the other treatment groups, these animals also received a single intraperitoneal injection of DMSO.
(2) Busulfan group (Bus): Rats in this group received a single intraperitoneal injection of busulfan at a dose of 10 mg/kg to induce experimental testicular damage. In addition, to standardize handling and injection volume across groups, they were administered a daily intraperitoneal injection of 1 mL of distilled water.
(3) DPFE group: Animals in this group were administered DPFE by daily oral gavage at a dose of 4 mg/kg to assess the independent effects of the extract. These rats also received a daily intraperitoneal injection of 1 mL of distilled water to match the injection procedures applied in the other groups.
(4) Busulfan plus DPFE group (Bus+DPFE): Rats in the co-treatment group received both the toxicant and the proposed protective agent. This consisted of a single intraperitoneal injection of busulfan (10 mg/kg), followed by daily oral gavage administration of DPFE (4 mg/kg) throughout the 56-day experimental period.

7. Sample collection

At the end of the 56-day treatment period, rats were euthanized and final body weights were recorded. Blood samples were collected for the measurement of serum testosterone levels. Semen analysis, including evaluation of sperm motility, concentration, and morphology, was performed for all experimental groups in accordance with WHO guidelines. Sperm DNA integrity was assessed using the sperm chromatin dispersion (SCD) assay, while protamine deficiency was evaluated using CMA3 staining.

8. Hormone assay

Blood samples were obtained via cardiac puncture immediately following euthanasia. Samples were centrifuged at 2,500 rpm for 30 minutes, and the separated plasma was stored at −20 °C until analysis [26]. Serum testosterone levels were measured using a commercially available radioimmunoassay kit (Coat-A-Count RIA, IMMUNOTECH) with a sensitivity of 0.025 ng/mL, following the manufacturer’s instructions.

9. Assessment of sperm parameters

Semen analysis was conducted in accordance with WHO guidelines. Individual sperm parameters were evaluated using a computer-aided semen analysis (CASA) system (HFT 6.5) [27].

10. Sperm collection and processing

Sperm samples were collected from the left distal portion (1 cm) of the vas deferens. Samples were immediately transferred into a Petri dish containing 2.5 mL Ham’s F-10 medium supplemented with bovine serum albumin (8 mg/mL) and gently agitated at 37 °C for 3 minutes to allow sperm dispersion.

11. Sperm parameters: measurement of sperm count, motility, and morphology

Sperm concentration was determined using a hemocytometer. Diluted samples were loaded into the counting chamber, and sperm heads were counted in four large squares. The mean count was multiplied by a dilution factor of 10 to calculate sperm concentration per milliliter.

Sperm motility was graded and categorized into four groups based on movement characteristics [27].

(1) Grade A: Sperm exhibiting rapid progressive motility with straight-line movement.
(2) Grade B: Sperm exhibiting progressive motility with a non-linear or curved trajectory.
(3) Grade C: Sperm displaying non-progressive motility without forward progression.
(4) Grade D: Sperm showing complete absence of motility.

Sperm morphology was classified as normal or abnormal, with abnormalities further categorized into head, neck/midpiece, and tail defects. Morphological evaluation was performed using Eosin Y staining. A minimum of 200 spermatozoa per animal were assessed across 10 microscopic fields [16].

12. DNA fragmentation assay

Sperm DNA integrity was assessed using the SCD test. Briefly, sperm chromatin and DNA were denatured through sequential incubation in two types of solutions, namely a lysis buffer followed by an acid solution. This treatment facilitates the extraction of nuclear proteins and the denaturation of DNA. Following deproteinization and denaturation, the DNA strands dispersed, forming halos of varying diameters around the sperm heads upon staining.

Based on the observed patterns of chromatin dispersion, spermatozoa were categorized into four types:

(1) Type A: Sperm exhibiting a large halo, considered indicative of intact DNA.
(2) Type B: Sperm exhibiting a medium-sized halo, considered indicative of minimal DNA fragmentation.
(3) Type C: Sperm exhibiting an extremely thin or weak halo, considered indicative of high DNA fragmentation.
(4) Type D: Sperm in which no halo was observed, considered indicative of extensive DNA fragmentation [2].

The presence of a large or medium halo (types A and B) is characteristic of intact DNA strands, whereas the presence of a small halo or the absence of a halo (types C and D) reflects extensive DNA strand breaks.

13. Protamine deficiency testing

Protamine deficiency was assessed using CMA3 staining. CMA3 is a fluorochrome that preferentially binds to cytosine-guanine–rich regions of DNA and competitively inhibits protamine binding, thereby serving as an indirect index of chromatin protamination status.

The assay was performed as follows. Semen samples were washed with PBS and centrifuged three times at 3,000 rpm for 5 minutes. The resulting pellet was fixed in Carnoy’s solution (three parts methanol and one part glacial acetic acid) for 5 minutes at 4 °C. Fixed samples were smeared onto clean glass slides. Each smear was stained with 100 µL of a 0.25% CMA3 solution and incubated in the dark for 2 minutes. The slides were then washed, mounted with distyrene, plasticizer, and xylene (DPX), and examined using a fluorescence microscope (Nikon Eclipse 600) equipped with a 460–470 nm filter at 100× magnification. Approximately 200 spermatozoa were evaluated per sample. Spermatozoa exhibiting bright yellow–green fluorescence in the head region (CMA3-positive) were classified as protamine-deficient, whereas those lacking fluorescence (CMA3-negative) were considered to possess normal protamination [16]. The findings were presented as a percentage of CMA3-positive spermatozoa for each sample.

14. Assessment of testicular oxidative stress markers

The right testis from each rat was homogenized in chilled PBS (pH 7.4). The homogenate was centrifuged at 12,000 ×g for 15 minutes at 4 °C, and the resulting supernatant was used for the assessment of oxidative stress parameters. Lipid peroxidation was evaluated by measuring MDA levels using the thiobarbituric acid reactive substances method and expressed as nanomoles of MDA per milligram of protein [28]. SOD activity was determined by measuring its ability to inhibit the autoxidation of pyrogallol and expressed as U/mg protein [29]. CAT activity was assessed based on the rate of hydrogen peroxide decomposition at 240 nm and expressed as U/mg protein [30]. Protein concentrations in the homogenates were determined using the Bradford method and used to normalize enzyme activity values across samples.

15. Randomization and blinding

Animals were allocated to the four experimental groups using a computer-generated randomization sequence to minimize selection bias. Due to the nature of the interventions, which involved different administration routes and substances, investigators responsible for daily injections and oral gavage were not blinded to group allocation. However, to minimize ascertainment bias during outcome assessment and data analysis, all subsequent evaluations were conducted by personnel blinded to the treatment groups. These evaluations included CASA, sperm morphology assessment, scoring of SCD and CMA3 assays, hormone level measurements, and all biochemical assays related to oxidative stress.

16. Statistical analysis

Data are expressed as mean±standard error of the mean. Statistical comparisons among groups were performed using one-way analysis of variance (ANOVA), followed by the Tukey’s post hoc multiple comparison test. The data satisfied the assumptions of normality and homogeneity of variance required for ANOVA. All statistical analyses were conducted using SPSS software ver. 21 for Windows (IBM Corp.). A p-value <0.05 was considered statistically significant.

Results

The analysis revealed that DPFE contained a total phenolic content of 12.5±0.8 mg GAE/g and a total flavonoid content of 4.2±0.3 mg QE/g of dry extract. These findings confirm that the extract used in this study is rich in polyphenolic antioxidants. The effects of busulfan and DPFE on sperm parameters are presented in Table 1. Analysis of sperm viability demonstrated a significant treatment effect, with the highest viability observed in the DPFE-only group (p<0.000). Marked differences in sperm motility patterns were also observed among the experimental groups. Sperm motility was classified by grade, with grade A (rapid progressive motility) being the most favorable for fertility potential. Busulfan treatment exerted a pronounced negative effect, significantly reducing the proportion of sperm exhibiting grade A motility compared with the control group (p<0.000). Consequently, the Bus group exhibited the highest proportion of immotile sperm (grade D).

Conversely, DPFE treatment appeared to counteract these adverse effects. The DPFE-treated group demonstrated a significant increase in the percentages of sperm with grade A and grade B motility, accompanied by a corresponding reduction in sperm exhibiting non-progressive (grade C) and immotile (grade D) motility patterns (p<0.000).

The results presented in Table 2 indicate a significant difference in protamine deficiency among the experimental groups (p<0.001), with the Bus group exhibiting the highest levels and the DPFE group exhibiting the lowest. Administration of DPFE was associated with a reduction in both abnormal sperm morphology and protamine deficiency. Notably, sperm head deformities were significantly less frequent in the Bus+DPFE co-treatment group than in the Bus group (p<0.001), while the DPFE-only group demonstrated the lowest incidence of this abnormality.

To further assess the influence of DPFE and busulfan on spermatogenic function, serum testosterone levels were measured using a radioimmunoassay (Coat-A-Count RIA kit). The results demonstrated that the Bus+DPFE group exhibited a significantly higher testosterone level than the Bus group (p=0.000). In parallel, evaluation of body weight revealed that the DPFE-only group showed the greatest increase in body weight (p=0.003) (Table 3).

Sperm DNA fragmentation was evaluated using the SCD test. The findings indicated that DPFE treatment significantly increased the proportions of spermatozoa exhibiting large (SCD1) and medium (SCD2) halos, which are indicative of intact DNA. Conversely, a reduction was observed in the proportions of spermatozoa exhibiting small (SCD3) or absent (SCD4) halos, which correspond to fragmented DNA, in both treated groups (Table 4).

To explore the potential mechanisms underlying the protective effects of DPFE, key biomarkers of oxidative stress were evaluated in testicular tissue. As shown in Table 5, busulfan administration induced a pronounced oxidative stress state, characterized by a significant increase in MDA levels (p=0.000) and marked reductions in the activities of the antioxidant enzymes SOD (p=0.000) and CAT (p=0.000) compared with the control group. Treatment with DPFE alone did not significantly alter these oxidative stress parameters relative to the control. Importantly, co-treatment with DPFE in the Bus+DPFE group substantially attenuated busulfan-induced oxidative damage, as evidenced by a significant reduction in MDA levels (p=0.000 vs. Bus group) and significant restoration of SOD (p=0.000 vs. Bus group) and CAT (p=0.001 vs. Bus group) activities toward normal levels (Table 5).

Discussion

The mechanism underlying these salutary effects appears to be multifactorial, involving the dense combination of antioxidant and anti-inflammatory compounds present in the extract, including the phenolics and flavonoids quantified in our analysis, which collectively counteract the oxidative stress and apoptotic cascades induced by the alkylating action of busulfan on germ cells. Our findings provide direct biochemical evidence supporting this mechanism. The significant elevation in testicular MDA levels following busulfan treatment confirms the induction of severe lipid peroxidation, while the concomitant decrease in SOD and CAT activities reflects a compromised antioxidant defense system. The ability of DPFE co-treatment to significantly normalize MDA levels and restore the activities of these critical antioxidant enzymes provides compelling evidence that its protective effects are mediated, at least in part, through attenuation of oxidative stress and enhancement of testicular antioxidant capacity. These results therefore offer solid empirical support for this mechanistic interpretation.

The results of the present study demonstrate that oral administration of P. dactylifera L. fruit extract significantly alleviates the deleterious effects of busulfan on sperm quality and, critically, on sperm DNA and chromatin integrity in a rat model. The recovery of key sperm parameters, including motility, viability, and morphology, in the co-treatment group underscores the restorative potential of the extract. Most importantly, the SCD test revealed a significant reduction in the DNA fragmentation index, while CMA3 staining demonstrated a concomitant decrease in protamine deficiency. These findings indicate that bioactive compounds in date fruit not only improve conventional semen quality but also directly protect sperm genetic material [31], a crucial determinant of successful fertilization and normal embryonic development [32].

The mechanism underlying these positive effects is likely multifold and attributable to the dense mixture of antioxidant and anti-inflammatory constituents within the extract, such as flavonoids, phenolics, and natural sugars, which collectively mitigate oxidative stress and apoptotic cascades triggered by the alkylating action of busulfan on germ cells. These results therefore provide solid empirical evidence supporting the concept that P. dactylifera fruit extract functions as a bioactive dietary component with the potential to prevent chemotherapy-induced gonadal toxicity.

Our findings are consistent with a growing body of literature describing the reproductive benefits of natural antioxidants in toxin-induced infertility models. Comparable improvements in sperm parameters and oxidative stress markers have been reported with extracts derived from pomegranate, ginger, and olive leaf [16,33,34]. However, the present study extends existing knowledge by directly linking DPFE supplementation to enhanced sperm DNA integrity in a busulfan-induced model, a potent chemotherapeutic agent that has been less extensively investigated in this context than environmental toxicants such as cadmium. While previous studies have highlighted the beneficial effects of date pollen on sperm motility [35], our focus on the fruit itself, combined with rigorous assessment of DNA fragmentation and chromatin integrity, provides important new insights into its potential therapeutic utility for the prevention of iatrogenic infertility.

Despite these promising findings, several limitations should be acknowledged. First, the use of a whole fruit extract, selected to better reflect dietary consumption, entails a complex mixture of constituents, making it difficult to identify the specific phytochemicals responsible for the observed effects. Second, as this is an animal study, direct extrapolation of the results to human clinical practice requires further investigation. Third, although the present study established a clear association between DPFE-mediated protection and attenuation of oxidative stress, as reflected by changes in MDA, SOD, and CAT levels, the precise upstream molecular mechanisms underlying these effects were not elucidated. In particular, the involvement of apoptosis-related signaling pathways, such as alterations in the Bcl-2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio, was not examined [36]. Based on these considerations, future studies should incorporate histopathological evaluation of testicular tissue to visualize recovery of seminiferous tubule architecture and spermatogenic cell lineages. At the molecular level, further investigation into apoptotic signaling pathways, including Bax/Bcl-2 balance, caspase-3 activation, pro-survival signaling cascades, and key inflammatory regulators such as nuclear factor-κB and tumor necrosis factor-α, would provide deeper mechanistic insight into the protective actions of DPFE [36].

In conclusion, this study demonstrates that DPFE, rich in antioxidant phytochemicals, effectively preserves conventional sperm parameters and, more importantly, safeguards the genomic integrity of male gametes against busulfan-induced damage. These findings have broader implications by positioning DPFE as a potential protective adjunct during chemotherapy. For men undergoing treatment with busulfan or similar alkylating agents, dietary supplementation with date fruit extract may represent a nontoxic and cost-effective strategy for fertility preservation. This work contributes to the emerging paradigm of using food-derived antioxidants to mitigate oxidative stress–related pathologies and highlights the potential of such approaches to inform future nutritional guidelines and advances within the nutraceutical field.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

I would like to express my gratitude to the Department of Pharmacology at the Faculty of Pharmacy, Kerman University of Medical Sciences for their collaboration in preparing and processing the extract.

Table 1.

The impact of Busulfan (Bus) and Date Palm Fruit Extract (DPFE) on sperm viability and motility parameters in adult Wistar rats

Groups	Sperm viability (%)	Grade A motility (%)	Grade B motility (%)	Grade C motility (%)	Grade D motility (%)
Control	26.3±1.4^b)	31.4±1.3^b)	30.1±0.8^b)	17.0±1.2^b)	21.5±0.8^b)
Bus	18.8±1.4^a)	16.8±1.3^a)	18.2±0.8^a)	33.5±1.2^a)	31.5±0.8^a)
DPFE	30.0±1.4^b)	29.5±1.3^b)	29.8±0.8^b)	20.0±1.2^b)	20.7±0.8^b)
Bus+DPFE	24.8±1.4^b)	23.9±1.3^a),b)	23.1±0.8	27.8±1.2^a),b)	25.2±0.8^b)
p-value	<0.000	<0.000	0.005	<0.000	<0.000

Values are presented as mean±standard error of the mean from n=15 independent rats per group. Significance was p<0.05. In intergroup comparisons, one-way analysis of variance was applied, followed by Tukey's post hoc test for multiple comparisons.

Bus, busulfan; DPFE, date palm fruit extract.

^a)Significantly different from the control group (p<0.05);

^b)Significantly different from the Bus group (p<0.05).

Table 2.

Effect of Bus and DPFE on protamine deficiency and sperm morphology deformity in adult Wistar rats

Group	Protamine deficiency (%)	Head deformity (%)	Neck deformity (%)	Tail deformity (%)
Control	21.4±1.0^b)	20.4±1.1^b)	24.5±1.0	28.4±1.2^b)
Bus	34.0±1.0^a)	38.3±1.1^a)	24.0±1.0	18.0±1.2^a)
DPFE	21.1±1.0^b)	17.5±1.1^b)	25.6±1.0	29.0±1.2^b)
Bus+DPFE	23.6±1.0^b)	23.9±1.1^b)	25.9±1.0	24.6±1.2^b)
p-value	<0.000	<0.000	0.628	<0.000

Values are presented as mean±standard error of the mean from n=15 independent rats per group. Statistical significance was assessed by one-way analysis of variance, post hoc analysis by Tukey's test; a p-value less than 0.05 was considered statistically significant.

Bus, busulfan; DPFE, date palm fruit extract.

^a)Significantly different from the control group (p<0.05);

^b)Significantly different from the Bus group (p<0.05).

Table 3.

Effect of Bus and DPFE on testosterone level and body weight in adult Wistar rats

Group	Mean testosterone level (ng/mL)	Mean body weight (g)
Control	8.36±0.12^b)	224.5±4.1
Bus	7.07±0.12^a)	208.2±3.8
DPFE	8.28±0.12^b)	238.6±4.2^a)
Bus+DPFE	8.38±0.12^b)	228.4±3.9
p-value	<0.000	0.003

Values are presented as mean±standard error of the mean from n=15 independent rats per group. Statistical analysis was done using one-way analysis of variance and post hoc Tukey's test to ascertain intergroup differences, which were significant at p<0.05.

Bus, busulfan; DPFE, date palm fruit extract.

^a)Significantly different from the control group (p<0.05);

^b)Significantly different from the Bus group (p<0.05).

Table 4.

Effect of Busulfan (Bus) and Date Palm Fruit Extract (DPFE) on sperm chromatin dispersion (SCD) in adult Wistar rats

Group	A: SCD1 (%)	B: SCD2 (%)	C: SCD3 (%)	D: SCD4 (%)
Control	29.4±1.4^a)	26.4±0.7	29.0±1.0	15.2±1.2
Bus	13.6±1.2	21.9±0.6	29.2±0.9	35.3±1.1^a)
DPFE	32.7±1.4^b)	27.7±0.7	20.6±1.1	19.0±1.3
Bus+DPFE	26.0±1.3^b)	24.4±0.7	20.6±1.0	29.0±1.2
p-value	<0.000	0.501	0.001	<0.000

Values are presented as mean±standard error of the mean from n=15 independent rats per group. Statistical significance was p<0.05. Statistical analysis was performed by one-way analysis of variance, and post hoc analysis was performed with Tukey's test.

Bus, busulfan; DPFE, date palm fruit extract; SCD, sperm chromatin dispersion.

^a)Significantly different from control group (p<0.05);

^b)Significantly different from the Bus group (p<0.05).

Table 5.

Effect of Bus and DPFE on testicular oxidative stress markers

Group	MDA (nmol/mg protein)	SOD (U/mg protein)	CAT (U/mg protein)
Control	1.52±0.15^b)	18.4±1.2^b)	32.1±2.5^b)
Bus	3.89±0.31^a)	9.1±0.8^a)	18.6±1.9^a)
DPFE	1.48±0.14^b)	19.1±1.4^b)	33.8±2.8^b)
Bus+DPFE	2.01±0.18^a),b)	15.3±1.1^a),b)	26.4±2.2^a),b)
p-value	<0.000	<0.000	<0.000

Values are presented as mean±standard error of the mean (n=15).

Bus, busulfan; DPFE, date palm fruit extract; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase.

^a)Significantly different from the control group (p<0.05);

^b)Significantly different from the Bus group (p<0.05).

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