Dogs residing in high-temperature environments, particularly southern regions of China or during the intense summer months, are frequently exposed to elevated ambient temperatures, which pose a considerable risk of heat stress to their testes. This exposure poses a significant challenge to maintaining reproductive health in dogs. The testes, as the primary organ of the male reproductive system, play a crucial role in maintaining the reproductive capacity of dogs through their normal physiological functions (1, 2). However, studies have shown that high-temperature environments can cause spermatogenic dysfunction in the testes, resulting in reduced sperm quality and quantity, and ultimately affecting the reproductive performance of dogs (3). Therefore, exploring effective strategies to mitigate testicular heat stress in dogs, along with elucidation of the underlying mechanisms, holds immense theoretical and practical importance.

Epimedii Folium has been found to contain more than 379 compounds, among which are flavonoids, lignans, organic acids, terpenoids, dihydrophenanthrene derivatives, alkaloids, and other compounds. Flavonoid compounds, such as epimedin A, B, C, icariin, baohuoside I, icariside I, and icaritin, are acknowledged as the leading chemical and pharmacologically active components in Epimedii Folium (4, 5). In recent years, icariin, a compound derived from traditional Chinese medicine and the primary active ingredient in Epimedii Folium, has shown extensive potential in enhancing testicular function. Chen et al. reported that a dosage of 200 mg/kg of icariin significantly increased epididymal sperm count and testosterone levels in male rats by modulating the expression of specific genes, including peripheral benzodiazepine receptors (PBR) and steroidogenic acute regulatory protein (STAR) (6). Similarly, Zhao et al. found that the administration of 50 mg/kg body weight (BW) of icariin for 80 consecutive days significantly improved the reproductive performance of male dairy goats, manifesting as increased serum levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and testosterone, enhanced spermatogenesis and sperm motility, and improved testicular organ coefficients (7). Furthermore, icariin has demonstrated remarkable efficacy in ameliorating age-related declines in spermatogenic function, safeguarding the junctional function of testicular Sertoli cells, and upregulating ERα/c-fos signaling and the PKR pathway. Its mechanism of action is contingent upon the presence of these signaling molecules (8). Additionally, icariin exerts protective effects against testicular damage in diabetic rats by markedly improving the morphology of the seminiferous tubules, increasing the number of spermatogenic cells, and effectively inhibiting cell apoptosis (9).

While Epimedii Folium contains multiple bioactive compounds, icariin is its primary flavonoid component and has demonstrated significant effects in enhancing testicular function and spermatogenesis across rodent and caprine models (6, 7, 9). Given icariin’s dominant concentration and prior evidence of spermatoprotective effects, we focused on its specific mechanisms in mitigating canine testicular heat stress. Particularly under high-temperature conditions, the manner in which icariin regulates the testosterone synthesis pathway and alleviates spermatogenic dysfunction by influencing the expression of related proteins in testicular tissue warrants further elucidation. Consequently, the present study aims to investigate the therapeutic effects and underlying mechanisms of icariin in dogs with testicular heat stress. It is expected that this study will elucidate the molecular mechanisms by which icariin alleviates heat stress in canine testes and provide a scientific basis for the application of icariin in protecting the reproductive health of dogs.

All experimental procedures involving Beagle dogs were approved by the Animal Care and Welfare Committee of the Chinese Academy of Sciences (Ethical Approval No. ISA-2024-0031). The study strictly adhered to institutional guidelines for the humane use of animals, and all participating institutions obtained informed consent for the experimental protocols.

In mammals, prolonged exposure to hyperthermic environments disrupts systemic homeostasis by exceeding physiological thermoregulatory thresholds (12). The testes, which require a temperature of 2 to 8°C below core body temperature for normal spermatogenesis, are particularly vulnerable in dogs due to compromised scrotal cooling mechanisms (13, 14). To model this pathology, we subjected dogs to scrotal immersion in a 45°C water bath for 30 min, a protocol adapted from Henning et al. (11) that minimizes confounding stressors through non-invasive thermal exposure. Histopathological analyses revealed seminiferous epithelial thinning, disorganization of spermatogenic cell layers, and sloughing of spermatogonia in heat-stressed testes, concomitant with significant declines in sperm concentration (a 68.4% reduction compared to the control) and progressive motility (67.9% reduction). These morphological and functional impairments correlated with suppressed serum testosterone levels, suggesting partial mediation through the androgen signaling pathways. The observed reproductive dysfunction aligns with previous reports of heat-induced testicular atrophy and steroidogenic suppression in mammals (15). Plant saponins, including icariin, have shown promise in ameliorating high-temperature-induced spermatogenesis disorders in various animal models (14, 16, 17). Icariin, in particular, has demonstrated significant effects in regulating testosterone synthesis, protecting testicular tissue structure, and inhibiting cell apoptosis (6, 7, 9). Therefore, we selected icariin as the intervention in this study and, based on the metabolic body weight of the experimental animals (18), set the dietary icariin supplementation level between 0 and 1 g/kg to systematically evaluate its effects on canine sperm quality, testicular function, hormone levels, related protein expression, and testicular tissue structure.

Thermal stress induces mitochondrial oxidative damage through reactive oxygen species (ROS) overproduction, particularly superoxide anion (O₂⁻) and hydroxyl radical (OH⁻), which preferentially target electron transport chain complexes (19–21). This oxidative insult disrupts ATP synthesis, leading to substrate limitation for ATP-dependent membrane transporters. Specifically, Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities in testicular tissue decreased by 65.6 and 61.5%, respectively, in heat-stressed dogs. Given the critical role of these enzymes in maintaining membrane potential and calcium homeostasis, their functional impairment is likely to contribute to the observed deficits in sperm motility. Notably, icariin supplementation restored testicular ATPase activities in a dose-dependent manner (22.9 and 15.4% recovery at 0.5 g/kg; 33.2 and 24.3% at 1 g/kg for Na⁺-K⁺-ATPase and Ca²⁺-ATPase, respectively), paralleling improvements in sperm motility parameters. The dose-dependent efficacy of icariin (1.0 g/kg > 0.5 g/kg) aligns with its role in modulating testosterone synthesis and cellular resilience. Higher doses more robustly enhanced ATPase activity, restored blood–testis barrier integrity via ZO-1 upregulation, and suppressed apoptosis (reduced BAX/BCL-2 ratio), collectively explaining superior spermatogenic recovery in the Icariin-H group. These data suggest that icariin mitigates oxidative damage through ion transport modulation, potentially via direct ROS scavenging or upregulation of antioxidant enzymes, as demonstrated in murine models (22).

Hypothalamic–pituitary–gonadal (HPG) axis dysregulation has emerged as a key mechanism underlying heat-induced reproductive failure (23). Nevertheless, this axis can be perturbed by external stimuli, such as inflammation and oxidative stress, which can impair spermatogenesis. Our data corroborate previous reports of HPG axis dysregulation under thermal stress. Specifically, heat-exposed dogs exhibited suppressed hypothalamic GnRH release, resulting in decreased pituitary LH production and subsequent reductions in E2 synthesis. Post-treatment, serum GnRH and LH levels recovered to baseline values, while E2 levels showed a gradual increase. Heat stress activates the hypothalamic–pituitary–adrenal axis, leading to elevated cortisol levels. Cortisol exerts a negative feedback effect on hypothalamic GnRH synthesis, thereby reducing the synthesis of GnRH. Since GnRH is critical for pituitary LH production, this suppression consequently diminishes LH synthesis. The restoration of HPG axis function coincided with improved blood–testis barrier (BTB) integrity. Immunoblotting revealed that ZO-1 expression increased by 2.1-fold, while the BAX/BCL-2 ratio decreased from 2.4 to 0.6, suggesting dual mechanisms of junctional stabilization and apoptosis suppression. The restoration of these proteins’ expression levels is likely instrumental in the recovery of testicular physiological function (23–25). Moreover, our study revealed that, under heat stress conditions, the testes of the Model group exhibited considerable structural damage, manifested by the emergence of large gaps and morphological distortions in the seminiferous tubules. Western blot analysis validated that the expression levels of ZO-1 and BCL-2 were substantially decreased, while the level of BAX was significantly increased in the testicular tissue of the experimental dogs. Notably, following icariin intervention, the expression levels of ZO-1 and BCL-2 recovered to varying extents, suggesting a potential restoration of the BTB structure. These findings imply that icariin not only modulates testosterone secretion but also effectively ameliorates testicular heat stress injury by rejuvenating the integrity of the BTB (26). Speculatively, icariin may activate Nrf2/HO-1 signaling or directly scavenge hydroxyl radicals, as its flavonoid structure contains phenolic hydroxyl groups capable of donating protons to neutralize ROS. These mechanisms could synergize with ion transport regulation to reduce oxidative injury to spermatogenic cells. Notably, BAX and BCL-2 extend beyond apoptotic regulation. BCL-2 family proteins also modulate autophagy via interactions with Beclin-1, and BAX has been associated with mitochondrial dynamics and cellular metabolism (27, 28).

The maintenance of a stable testosterone concentration within the seminiferous tubules is crucial for the differentiation of spermatogonia into mature spermatozoa (29–31). Heat stress significantly suppressed key steroidogenic enzymes (STAR: 70.7% reduction; 17βHSD: 69.9%; CYP450: 55.8%), with these reductions mirroring concomitant declines in testosterone levels, a pattern consistent with the findings from Ji et al. (2024) (32). Molecular docking studies revealed a strong binding affinity between icariin and 17βHSD, consistent with the observed upregulation of steroidogenic proteins post-treatment. Concomitant increases in STAR and CYP450 expression (key regulators of testosterone biosynthesis) suggest a coordinated enhancement of the steroidogenic pathway, where icariin’s binding to 17βHSD serves as a mechanistic trigger. These findings align with the restoration of serum testosterone levels (implied by GnRH/LH recovery) and testicular ATPase activities, collectively indicating that icariin modulates both enzyme structure and pathway dynamics to mitigate heat stress–induced steroidogenic dysfunction. Importantly, the high-dose icariin group (1 g/kg) exhibited complete restoration of sperm count (82.9 × 10⁶ vs. 94.5 × 10⁶/mL in controls). This finding was further corroborated by Western blot analysis, which showed a significant increase in the expression levels of STAR, 17βHSD, and CYP450 following icariin treatment. These results suggest that icariin may have a direct modulatory effect on the expression of these key proteins involved in testosterone biosynthesis. These findings align with murine studies demonstrating icariin’s safety profile at 200 mg/kg (6), while extending dose–response evidence to dogs. In the current study, we administered a higher dose of icariin (Icariin-H group: 1 g/kg) to experimental dogs, which was well within their maximum tolerable dose. Our therapeutic findings indicate that the effectiveness of icariin in mitigating heat stress-induced damage to the reproductive system increased with higher dosages. This dose-dependent response suggests that icariin is not only efficacious in protecting the reproductive system from heat stress-induced damage but also safe and devoid of side effects within the normal dosage range for dogs (33). Collectively, these preclinical data support further investigation of icariin as a therapeutic candidate for mitigating heat stress-induced reproductive dysfunction in dogs. The observed dose-dependent efficacy (up to 1 g/kg) and absence of adverse effects within the tested range warrant translational studies to establish clinical safety and optimal dosing regimens.

The study provides preliminary evidence suggesting that icariin may exert a multifaceted protective effect against heat stress-induced damage in canine testes. Specifically, our data indicate that it upregulates the expression of proteins crucial for testosterone synthesis, enhances ATPase activity, regulates hormone levels, promotes repair of the blood–testis barrier, and inhibits cell apoptosis. These findings contribute to the scientific basis for exploring the use of icariin in preserving canine reproductive health and highlight its potential therapeutic relevance for managing reproductive disorders associated with environmental heat stress.