All procedures involving animals in this study were conducted in strict accordance with internationally accepted guidelines for the ethical use of animals in scientific research. The experimental protocol complied with the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes, and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Ethical approval for the use of animals and semen samples was obtained from the Animal Welfare Office of Justus-Liebig-University Giessen, Germany, under approval number kTV 11–2018, which was renewed on October 01, 2023.

All dogs included in the study were privately owned animals presented for routine reproductive evaluation. Only clinically healthy and fertile male dogs were enrolled after a comprehensive clinical examination to ensure normal reproductive health and the absence of systemic disease. Semen collection was performed using manual stimulation, a non-invasive, routinely applied procedure in canine reproductive medicine that does not cause pain or distress to the animals. No surgical procedures, pharmacological interventions, or experimental manipulations were applied to the animals during semen collection.

Written informed consent was obtained from all dog owners before participation in the study. Owners were informed about the purpose of the research, the procedures involved, and the intended scientific use of the collected semen samples. All procedures were conducted by trained veterinarians under controlled clinical conditions to ensure animal welfare and minimize stress during handling and semen collection.

The study design followed the principles of the 3Rs (Replacement, Reduction, and Refinement) to ensure responsible animal use in research. The number of animals was kept to the minimum required to obtain scientifically valid results, and all experimental procedures were conducted in accordance with standard veterinary practices to ensure the highest level of animal welfare throughout the study.

The experimental work took place at the Clinic for Reproductive Medicine and Neonatology at Justus-Liebig University of Giessen, Germany, from November 15, 2023, to February 20, 2024.

Semen samples were collected from healthy, fertile male dogs to evaluate their breeding suitability. Ejaculates were obtained by manual stimulation from 10 clinically healthy, fertile male dogs (mean age: 5.0 ± 2.26 years), representing 8 different breeds. Two breeds, Collie and German Wirehaired Pointer, were represented by two dogs each, while the others, Cane Corso Italiano, English Bulldog, Whippet, Jack Russell Terrier, Great Dane, and Hovawart, were each represented by one dog. This diverse-breed sampling was intentionally chosen to reduce the influence of individual-dog effects and prevent breed-specific bias, not to compare specific breeds. All dogs underwent a clinical examination before semen collection and were only included if they had normal testicular morphology, normal body temperature, and no apparent systemic or reproductive issues. Semen was collected at room temperature (20°C–22°C) in a quiet setting, following established guidelines [37]. Ejaculates were obtained via manual stimulation with an estrous teaser bitch or a female phantom present. Three different ejaculate fractions were collected into sterile, prewarmed glass containers. One ejaculate per dog was used for the study, and only the second sperm-rich fraction was selected for further analysis.

After collection, the semen was carefully kept in a water bath at 37°C during the initial phase of the experiment. Next, each ejaculate was evaluated individually; samples showing >70%–75% progressive motility and normal morphology were selected for inclusion in this study and subsequent cryopreservation. Following the initial semen evaluation, each ejaculate (experimental unit) was divided into four equal aliquots, which were then randomly assigned to different experimental treatments. All aliquots were diluted with a Tris–fructose–egg yolk extender, prepared by dissolving 3.025 g of Tris (hydroxymethylaminomethane), 1.7 g of citric acid, and 1.25 g of fructose in 100 mL of double-distilled water. The solution pH was adjusted to 7.0, and the osmolality was set to 320 mOsm/kg. The extender was then supplemented with fresh, filtered chicken egg yolk (20% (v/v)), and glycerol was added in a single step to reach a final concentration of 5.0% (v/v) at 37°C during the dilution process [38].

Dilution was performed to achieve a final sperm concentration of 50–60 × 10⁶ sperm/mL. MitoQ (BYTORB1105367, Biorbyt Ltd., Cambridge, UK) was administered to the treatment groups at final concentrations of 100 nM, 200 nM, or 400 nM, while the control group received an equal volume of vehicle solution without the supplement. MitoQ was initially supplied as a 200 mg/mL solution in a 1:1 ethanol–water mixture (v/v), corresponding to an approximate stock concentration of 295 mM, based on its molecular weight of 678.82 g/mol. Upon delivery, the stock solution was aliquoted to reduce freeze–thaw cycles, stored at −80°C under light-protected conditions, and used within 3 months. Working solutions at millimolar concentrations (1–50 mM) were prepared by diluting the stock solution with an ethanol–water mixture (1:1, v/v) according to the manufacturer’s instructions. These intermediate solutions were further diluted into the experimental extender to achieve the desired final concentrations. All groups, including the control, contained the same final solvent concentration to ensure uniform conditions. The study included a control group and multiple MitoQ-supplemented treatment groups, each replicated 10 times.

The diluted samples were loaded into 0.25-mL straws [39] and sealed. Samples were cooled from 20°C–22°C to 5°C over 1.5 h using passive cooling in a refrigerator, without active temperature control. Following equilibration, the straws were frozen in liquid nitrogen (LN₂) vapor, positioned 4–5 cm above the liquid nitrogen surface on a rack for 10 min in a standard LN₂ container, and then plunged into liquid nitrogen. Frozen straws were stored in LN₂, for 4 weeks. Straws were thawed individually in a 37°C water bath for 1 min for evaluation. After thawing, the samples were immediately processed for downstream analyses. Samples were maintained at 37 °C during post-thaw handling and assessments, and all assays were initiated within a consistent time frame following thawing.

Sperm motility characteristics were assessed using a computer-aided sperm analysis system (CASA; AndroVision™, Minitüb GmbH, Tiefenbach, Germany) following the manufacturer’s guidelines for canine semen evaluation. The image sequences were recorded at 25 frames per second. The CASA output included total motility (TM) [%], progressive motility (PM) [%], average path velocity (VAP) [μm/s], straight-line velocity (VSL) [μm/s], curvilinear velocity (VCL) [μm/s], straightness (STR) [%], linearity (LIN) [%], amplitude of lateral head displacement (ALH) [μm], beat cross frequency (BCF) [Hz], and wobble (WOB) [%]. For each replicate, 5 μL of semen was placed in a prewarmed chamber slide (20-μm depth) and covered with a glass coverslip. Thresholds for TM and PM, as well as particle size and brightness parameters, were set according to the default canine-specific configuration provided by the AndroVision™ software. A minimum of 10 microscopic fields was examined to ensure that at least 200 spermatozoa were analyzed per sample. All CASA measurements were performed by a single trained operator who was blinded to the experimental treatments.

The percentage of viable sperm was determined using a modified eosin-B staining method (eosin-B, Merck KGaA, Darmstadt, Germany; sodium citrate, Merck KGaA) [40]. For each sample, 5 μL of diluted sperm was mixed with 10 μL of eosin solution on a prewarmed slide (final ratio 1:2) and allowed to dry at 25°C. Duplicate smears were prepared for each sample, and a single operator, blinded to the treatment groups, evaluated the slides. Under a 400× magnification microscope (Olympus, Japan), sperm with pink or red-stained heads were classified as dead, while those with unstained (white) heads were classified as viable. A total of 200 spermatozoa were counted per sample, and the results were expressed as the percentage of viable sperm.

MDA levels in sperm samples were measured as an indicator of lipid peroxidation (LPO) using the thiobarbituric acid (TBA) reaction method [41]. For each assay, 1 mL of the prepared sperm suspension was mixed with 2 mL of a TCA-TBA-HCl solution containing thiobarbituric acid, trichloroacetic acid (Merck KGaA), and 0.25 N hydrochloric acid. The mixture was boiled in a water bath at 100°C for 15 min, then cooled to 20°C–22°C. After centrifugation at 1200× g for 15 min, the supernatant’s absorbance was measured at 535 nm using a Hitachi U-2001 spectrophotometer (Hitachi Ltd., Tokyo, Japan). MDA levels were calculated using the specific extinction coefficient (1.56 × 10⁵ L/mol/cm) instead of a standard curve and were expressed as nmol per mL of semen. Blanks containing only the TCA-TBA-HCl reagent were included to correct for background absorbance.

Sperm plasma membrane integrity was assessed using the hypo-osmotic swelling test (HOST). The hypo-osmotic solution was prepared to 100 mOsm, as previously described [42]. For the assay, 5 μL of sperm suspension was mixed with 50 μL of hypo-osmotic solution and incubated at 37°C for 30 min. Two drops of the mixture were placed on a prewarmed slide and covered with a coverslip after incubation. Positive HOST reactions were scored based on tail swelling: sperm with coiled or swollen tails were considered intact, while those with straight tails were considered damaged. At least 200 spermatozoa were evaluated per sample under a light microscope at 400× magnification. All slides were assessed by a single trained operator blinded to the treatment groups.

The acrosome integrity was assessed using a modified formalin–citrate solution (containing formaldehyde and sodium citrate, Merck KGaA) [43]. In this method, 10 μL of semen was mixed with 100 μL of formalin–citrate solution and placed on a microscope slide beneath a coverslip. A portion of the mixture was examined using a phase-contrast microscope (Hund Wetzlar H500, Helmut Hund GmbH, Wetzlar, Germany) equipped with a 100× oil immersion objective and a phase-contrast condenser, under Koehler illumination at 1000× magnification. Spermatozoa were classified as having intact acrosomes if the acrosomal cap appeared smooth, uniform, and clearly outlined, while swollen, irregular, or missing acrosomes were considered damaged. At least 200 sperm were counted from a minimum of three different microscopic fields for each sample. Results are expressed as the percentage of sperm with intact acrosomes.

The percentage of apoptotic sperm was determined by flow cytometry using phosphatidylserine translocation as a marker. Initially, the sperm were washed twice with cold BioLegend cell staining buffer (Cat. No. 420201, BioLegend Inc., San Diego, CA, USA) and resuspended in annexin V-binding buffer (Cat. No. 422201, BioLegend Inc.) at a concentration of 1 × 10⁶ cells/mL. Subsequently, a 100 μL aliquot was incubated with 5 μL FITC-Annexin V (Cat. No. 640905, BioLegend Inc.) for 15 min at 20°C–22°C in the dark, followed by an additional incubation with 5 μL propidium iodide (PI, Cat. No. 421301, BioLegend Inc.) for 15 min. Before analysis, samples were diluted with 400 μL binding buffer, and at least 10,000 events were acquired per sample.

Flow cytometry was conducted on a CytoFLEX flow cytometer (BG49443, Beckman Coulter, Krefeld, Germany) at a low flow rate. To maintain consistent performance, the instrument was calibrated daily with standardized fluorescent beads. Fluorescence compensation controls were prepared using unstained, single-stained FITC, and single-stained PI samples to correct for spectral overlap between fluorophores. FITC was excited at 488 nm and measured on the 525/40 nm channel, while PI was excited at 561 nm and measured on the 610/20 nm channel. All data were acquired and analyzed with CytExpert software (Version 2.7 and Manufacturer: Beckman Coulter GmbH, Krefeld, Germany). Gating was systematically performed to identify distinct sperm subpopulations. Forward scatter (FSC) and side scatter (SSC) were initially used to gate singlet sperm populations, excluding debris and aggregates. The fluorescence intensity was then analyzed to classify cells into three categories: viable sperm (Annexin−/PI−), early apoptotic sperm (Annexin+/PI−), and late apoptotic or dead sperm (PI+). This strategy enabled accurate quantification of apoptotic cells across all treatment groups. Additionally, intracellular ROS levels were measured using DCFH-DA (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 10 μM in 25 μL of buffer. Appropriate unstained and single-stained controls were included to establish baseline fluorescence and ensure accurate interpretation of ROS signals.

Rhodamine 123 (R123; 83702, Sigma-Aldrich, St. Louis, MO, USA) and PI (0.01 mg/ml, 537060, Sigma-Aldrich) were used to assess mitochondrial activity [31]. The initial semen sample, 300 μl, was treated with 10 μl of Rh123 (0.01 mg/ml) and incubated for 20 min at 20°C–22°C in the dark. The semen samples were then centrifuged at 500 g for 3 min and resuspended in 500 μl of Tris buffer. Finally, 10 μl of PI (1 μg/ml) was added before flow cytometry analysis, where sperm cells that showed a positive signal for Rh123 and a negative signal for PI were counted as having active mitochondria.

To measure hydrogen peroxide (H₂O₂) levels in sperm, semen samples were diluted with phosphate-buffered saline (PBS; Sigma-Aldrich) to a concentration of 5 × 10⁶ sperm/mL. Then, 25 μL of the fluorescent dye 2′,7′-dichlorofluorescein diacetate (Sigma-Aldrich) was added to 1 ml of the sperm suspension, and the mixture was incubated at 20°C–22°C for 40 min. After centrifugation to remove the supernatant, the sperm pellet was resuspended in PBS to the same concentration and incubated at 20°C–22°C for 10 min. Before flow cytometric analysis, a 2 μL addition of PI (1 μg/mL) was added to the semen samples [31].

Data normality was assessed using the Shapiro-Wilk test, confirming that all variables were normally distributed (p > 0.05). Statistical analyses were conducted with the General Linear Model procedure in SAS (version 9.1; SAS Institute Inc., Cary, NC, USA). To account for repeated measures from multiple ejaculates per dog, a dog was included as a random effect in a mixed model. The model was specified as: Yij = μ + Ti + Dj + Eij, where Yij is the observed value, μ is the overall mean, Ti is the fixed effect of treatment, Dj is the random effect of dog, and Eij is the residual error. Orthogonal contrasts were used for mean comparisons, including comparisons of each MitoQ concentration versus the control, and linear and quadratic trends across concentrations were evaluated. Multiple comparisons were handled with the Tukey-Kramer adjustment. Statistical significance was set at p < 0.05, and results are shown as mean ± standard deviation (Mean ± SD).

The normality of all data was evaluated using statistical tests, and all parameters adhered to a normal distribution (p > 0.05). Normality was confirmed for the following primary outcomes: TM (p = 0.72), membrane integrity (p = 0.49), PM (p = 0.56), curvilinear velocity (VCL, p = 0.68), average path velocity (VAP, p = 0.61), and straight-line velocity (VSL, p = 0.59). Secondary outcomes, including early and late apoptosis (p = 0.66 and 0.57, respectively), lipid peroxidation (LPO, p = 0.62), acrosome integrity (p = 0.54), viability (p = 0.58), and intracellular ROS-H₂O₂ (p = 0.68), also followed a normal distribution, supporting the use of parametric statistical methods.

Table 1 summarizes the effects of different concentrations of MitoQ (100, 200, and 400 nM) on the primary outcomes TM and membrane integrity, as well as on secondary outcomes. TM was significantly higher in the 200 nM MitoQ group (38.40 ± 2.94%, p < 0.05) compared to the control group (33.30 ± 2.63%), the 100 nM group (33.71 ± 2.82%), and the 400 nM group (34.55 ± 3.35%).

PM showed a tendency to improve at 200 nM (33.34 ± 2.71%, p > 0.05), surpassing the values in the 100 nM (28.74 ± 2.34%) and 400 nM (28.75 ± 2.97%) groups, while remaining comparable to the control group (32.79 ± 2.45%).

Among the velocity parameters, curvilinear velocity (VCL) was significantly higher at 200 nM (41.73 ± 2.55 μm/s, p < 0.05) compared to the control (37.67 ± 1.82 μm/s), 100 nM (37.06 ± 2.08 μm/s), and 400 nM (37.63 ± 2.44 μm/s) groups. In contrast, average path velocity (VAP) and straight-line velocity (VSL) showed no significant differences among the groups (p > 0.05). Similarly, other CASA parameters, including straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), BCF, and wobble (WOB), did not differ significantly (p > 0.05). Overall, TM, PM, and VCL tended to improve at 200 nM MitoQ, although the functional significance of these changes remains to be determined.

Figure 1 shows how MitoQ supplementation in extenders affects secondary outcomes, specifically sperm viability and lipid peroxidation (LPO), in frozen–thawed canine semen. Concerning viability, there were no significant differences (p > 0.05) between the control group (36.33 ± 2.43%) and the treatment groups: 100 nM (32.78 ± 2.65%), 200 nM (35.89 ± 2.13%), and 400 nM MitoQ (33.22 ± 1.67%). A tendency toward improvement was noticed, with the 200 nM group showing the highest viability, although these differences were not statistically significant.

LPO levels in the control group (110.66 ± 8.65 nmol/mL) were similar to those in the 100 nM (112.86 ± 7.23 nmol/mL) and 400 nM (115.87 ± 5.71 nmol/mL) groups (p > 0.05). The 200 nM MitoQ group (107.21 ± 7.11 nmol/mL) showed a significant reduction in LPO compared to the 400 nM group (p < 0.05), while no significant difference was found between the control and 200 nM groups (p > 0.05). These results suggest that 200 nM MitoQ may slightly improve sperm viability and reduce oxidative stress, indicating its potential effects on secondary outcomes, including apoptosis, LPO, mitochondrial activity, and intracellular ROS-H₂O₂.

Figure 2 shows how different MitoQ levels (100, 200, and 400 nM) affect secondary outcomes, specifically membrane and acrosome integrity, in frozen–thawed canine sperm. The control group had an average membrane integrity of 57.00 ± 2.13%, with no significant differences between the 100 nM (53.33 ± 2.45%, p > 0.05) and 200 nM (58.00 ± 2.67%, p > 0.05) groups. However, the 400 nM group showed a significant decrease in membrane integrity (50.00 ± 2.31%) compared with the 200 nM group (p < 0.05), suggesting that the highest concentration may have harmful effects.

Regarding acrosome integrity, the control (58.33 ± 2.67%) and 100 nM (58.00 ± 2.45%) groups showed no significant difference from the 200 nM group (63.78 ± 2.71%, p > 0.05). The 400 nM group had the lowest acrosome integrity (53.89 ± 2.52%) and was significantly lower than the 200 nM group (p < 0.05). These results suggest that 200 nM MitoQ may help maintain membrane and acrosome integrity, whereas higher concentrations (400 nM) could adversely affect these outcomes, highlighting the need for dose optimization.

Figure 3 shows the effects of different MitoQ concentrations (100, 200, and 400 nM) on secondary outcomes related to sperm viability, including percentages of live, apoptotic, and dead sperm in frozen–thawed canine semen. The percentage of live sperm was similar between the control (33 ± 1.87%) and 100 nM (33 ± 2.13%) groups (p > 0.05), whereas the 200 nM group had the highest live sperm percentage (38 ± 3.13%). Although this difference was not statistically significant compared to the control or 100 nM groups, the 200 nM group was significantly higher than the 400 nM group (32 ± 2.35%, p < 0.05).

For apoptotic sperm, the 200 nM group showed a distinct pattern compared to the other groups: control (34 ± 2.82%), 100 nM (34.67 ± 1.98%), 200 nM (33 ± 2.22%), and 400 nM (34.11 ± 2.15%), as shown in Figure 3.

Regarding dead sperm, the control (33 ± 1.6%) and 100 nM (32.33 ± 2.54%, p > 0.05) groups were similar, while the 200 nM group had the lowest percentage (28.33 ± 2.69%), which was significantly lower than both the control (p < 0.05) and 400 nM (33.89 ± 1.75%, p < 0.05) groups. Overall, MitoQ at 200 nM tended to improve sperm viability by increasing the number of live sperm and decreasing the proportion of dead sperm, highlighting its potential benefit on secondary outcomes in frozen–thawed canine semen.

Figure 4 illustrates the effects of various MitoQ concentrations (100, 200, and 400 nM) on secondary outcomes related to mitochondrial function and oxidative stress, specifically mitochondrial activity and intracellular ROS-H₂O₂ levels, in frozen–thawed canine spermatozoa. For mitochondrial activity, the control group exhibited the highest percentage (36.1 ± 1.53%), followed by the 200 nM group (35.6 ± 2.27%) and the 100 nM group (34 ± 2.67%). The 400 nM group showed the lowest mitochondrial activity (32.96 ± 1.29%). Despite these differences, statistical analysis revealed no significant differences among the groups (p > 0.05).

Regarding intracellular ROS-H₂O₂ levels, the control group recorded 33 ± 2.41%, similar to the 100 nM (33 ± 2.11%) and 400 nM (32 ± 1.72%) groups. The 200 nM group exhibited the highest ROS-H₂O₂ level (38 ± 2.01%), which was significantly higher than that in the 400 nM group (p < 0.05), although no significant differences were seen between the control and either the 100 or 200 nM groups (p > 0.05). These results indicate that MitoQ at 200 nM may slightly increase intracellular ROS-H₂O₂ without significantly impacting mitochondrial activity, emphasizing the importance of dose optimization to maintain oxidative balance in frozen–thawed canine sperm.

Cryopreservation remains an essential method for long-term storage of genetic material in animal breeding and conservation efforts; however, it is often associated with damage to spermatozoa’s structure and function. These changes mainly result from cold shock, osmotic stress, and oxidative stress, which together damage plasma membrane integrity, acrosomal stability, and mitochondrial function [1, 2, 31, 36]. Mitochondria are especially vulnerable to cryo-induced injury because they are both a major source and target of ROS. Excessive mitochondrial ROS production during freezing and thawing can cause lipid peroxidation and DNA damage, and trigger apoptosis, ultimately decreasing sperm viability and function [31, 36]. As a result, there is ongoing interest in developing cryopreservation extenders containing antioxidants to protect mitochondrial function and reduce oxidative damage [1, 2, 31, 43].

Among mitochondria-targeted antioxidants, MitoQ has gained significant attention because it can specifically accumulate in the inner mitochondrial membrane and directly neutralize ROS, especially superoxide anions and hydrogen peroxide [31, 44–47]. Previous research in various species, including bovine, caprine, and avian models, has shown improvements in post-thaw sperm motility, membrane integrity, and acrosomal preservation after supplementing with low concentrations of MitoQ [30, 31, 44–47].

Supplementation with 200 nM MitoQ led to the most consistent improvements in post-thaw motility, acrosome integrity, and plasma membrane stability, along with a decrease in markers linked to cell death. However, it should be noted that ROS-H₂O₂ levels at 200 nM were not always lower than those at 400 nM, suggesting that oxidative protection at this concentration may not be solely reflected by a reduction in all ROS markers but rather by a balance between controlled ROS signaling and preventing excessive oxidative damage. Therefore, the improvements in sperm quality observed at 200 nM likely result from qualitative modulation of mitochondrial redox homeostasis rather than a uniform decrease in ROS production.

Although the overall increases in parameters such as TM and curvilinear velocity were small, these changes remain biologically significant in the context of cryopreserved semen, where post-thaw sperm quality is naturally compromised. Even modest improvements can greatly raise the proportion of sperm that are functionally competent. From a practical perspective, slight enhancements in motility and velocity could improve sperm transport, interaction with the zona pellucida, and fertilization success, especially when only a subset of sperm survives cryopreservation. However, because fertilization capability, pregnancy outcomes, and embryo development were not evaluated, any conclusions about whether these in vitro improvements translate to in vivo fertility should be drawn with caution.

These findings support the hypothesis that MitoQ can effectively reduce oxidative and structural damage caused by cryopreservation when used at an optimal concentration.

Notably, the beneficial effects observed at 200 nM MitoQ differ from our previous findings, where MitoQ supplementation alone at the same concentration did not lead to a statistically significant improvement in cryopreserved canine sperm quality. Beneficial outcomes were only seen when MitoQ was combined with AFP III. This discrepancy highlights that statistical significance alone does not fully capture biological efficacy and underscores the importance of considering modest yet consistent improvements within the broader physiological context of sperm cryotolerance.

The divergence further indicates that the biological effect of MitoQ depends on multiple interacting factors, not just concentration alone. Variations in donor populations may play a significant role. Including multiple breeds with different seminal traits can greatly influence mitochondrial redox balance and the response to mitochondria-targeted antioxidants. Breed-related differences in membrane lipid composition, cholesterol-to-phospholipid ratios, endogenous antioxidant defenses, and seminal plasma composition affect susceptibility to oxidative stress and cryo-induced membrane destabilization [16, 31, 36]. These inherent differences may determine not only whether statistical significance is achieved but also whether the observed changes result in biologically meaningful improvements in sperm function.

Methodological differences between studies, such as subtle variations in extender composition, freezing rate, or storage conditions, may also affect MitoQ stability and bioavailability during cryopreservation.

The concentration-dependent dual activity of MitoQ is an important and biologically relevant factor to consider. At low concentrations, MitoQ functions as an effective mitochondrial antioxidant; however, at higher doses, it may engage in redox cycling and paradoxically increase ROS production [44–47]. Supplementing with 400 nM MitoQ was associated with higher levels of oxidative stress markers, consistent with previously reported biphasic effects of MitoQ in spermatozoa and somatic cells [31, 44–47]. Nonetheless, its role in activating adenylate kinase 2 in the sperm flagellum further clarifies its ability to improve motility parameters, while enhanced mitochondrial function and cell survival have been repeatedly observed across different species, supporting MitoQ’s effectiveness as a targeted mitochondrial antioxidant for semen preservation [48–50].

Although some of these differences reached statistical significance, their biological importance mainly lies in identifying a threshold beyond which mitochondrial protection diminishes and oxidative damage worsens, rather than in any functional advantage. This narrow therapeutic window underscores the need for precise dose optimization.

From a mechanistic perspective, the improvements observed at 200 nM MitoQ are likely linked to stabilization of mitochondrial membrane potential, protection of ATP production, and inhibition of apoptosis-related pathways. Sperm mitochondria control critical functions, including energy metabolism and calcium homeostasis, which are vital for motility and fertility [16, 31, 36]. Cryo-induced mitochondrial dysfunction promotes cytochrome c release and caspase activation, leading to apoptotic cell death and decreased sperm viability [36, 51–54].

The reduction in dead sperm and the trend toward improved mitochondrial activity at 200 nM indicate meaningful preservation of mitochondrial integrity, although statistical significance was not consistently observed across all endpoints. Overall, these results support the use of MitoQ as a mitochondria-targeted antioxidant to enhance certain post-thaw sperm parameters in dogs at moderate concentrations.

The biological significance of these findings comes from the cumulative effect of several small but consistent improvements across multiple sperm quality parameters, rather than from isolated statistically significant results. However, the absence of fertility-related functional endpoints is a major limitation, so conclusions about cryoprotective effectiveness should be limited to improvements in in vitro sperm quality rather than extended to reproductive performance. Additionally, the variability in responses among individuals and across studies indicates that MitoQ’s effectiveness depends on context and is influenced by factors such as dosage, sperm source, and experimental conditions. Therefore, more research is needed to refine dosing strategies and to explore whether combining MitoQ with other cryoprotective agents can enhance and stabilize its beneficial effects.

This study has several limitations that should be considered when interpreting the results. The relatively small sample size may have reduced the statistical power to detect subtle treatment effects, and some biologically relevant trends may therefore not have reached statistical significance. The exclusive use of canine sperm limits broader applicability to other species, and the inclusion of multiple breeds may have introduced biological variability, obscuring breed-specific responses. Functional endpoints directly related to reproductive success, such as fertilization capacity or embryo development, were not evaluated; thus, the biological significance of the observed improvements must currently be viewed as indicative rather than conclusive.

Additionally, the effects of long-term cryostorage were not evaluated, and only some ROS markers were measured, limiting a full understanding of redox regulation. Intermediate MitoQ concentrations between 200 and 400 nM were not tested, restricting detailed dose–response analysis. Future research should include functional fertility tests, longer storage assessments, a wider range of oxidative stress markers, and more precise dose trials. These methods will be crucial for more clearly connecting statistically significant findings to biologically relevant reproductive outcomes.