Oxidative stress is one of the leading causes of male infertility that is a consequence of an imbalance in reactive oxygen species (ROS) (1, 2). This effect has been previously demonstrated by different authors in some mammalian species, including rams (3–5), stallions (6), red deer (7) and bulls (8). Sperm cells are very susceptible to oxidative stress due to the high number of unsaturated lipids that compose their cell membranes (9); for this reason, lipid peroxidation is highly correlated with ROS content in sperm cells (5). Sperm preservation methods (cooling and cryopreservation) are the main cause of oxidative stress in domestic animals (10). Sperm cells suffer detrimental effects due to the drastic temperature differences, including the overproduction of ROS, reduced acrosome integrity or impairment of mitochondrial membrane potential (10). An imbalance in the redox system due to an increase in ROS levels triggers a decrease in sperm quality. Specifically, high ROS levels cause lipid peroxidation, DNA fragmentation, apoptosis, and consequently low fertility (11, 12). ROS include superoxide anions (O2), hydrogen peroxide (H₂O₂), proxyl (·ROO), and hydroxyl (·OH) radicals (5). Balanced ROS generation is essential for sperm capacitation, hyperactivation, the acrosomal reaction and fertilization (13–15). Moreover, seminal plasma contains different antioxidants that are very relevant for sperm protection, including enzymatic antioxidants [superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT)] and nonenzymatic antioxidants such as vitamins (A, C, and E) (2, 11). High levels of ROS can be compensated for the seminal plasma antioxidant system (11). Nevertheless, an imbalance caused by excessive ROS production and decreased levels of antioxidant enzymes provokes oxidative stress (13, 14).

Therefore, a key point for improving sperm quality analyses should focus on the redox status of sperm (11, 16). This status can be assessed by direct measurements (ROS production) or indirect measurements (malondialdehyde (MDA), high DNA fragmentation index (hDFI), and SOD and GPX levels) (11, 17). In recent decades, the amount of MDA present in sperm cells has been used as a measure of lipid peroxidation (11, 18) and is an indirect measure of ROS production and oxidative damage (18). The reaction of MDA with thiobarbituric acid (TBA) forms a product that can be identified colorimetrically or fluorometrically, and its signal is proportional to the amount of MDA present. Moreover, several authors have noted an association between a high DNA fragmentation index and oxidative stress (19). Different techniques have been used to evaluate DNA fragmentation, in particular Sperm Chromatin Structure Assay (SCSA^®) technique described by Everson (20) which allows the use of flow cytometry. In addition, there are other techniques used for the evaluation of DNA fragmentation including Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL assay), in situ nick translation assay (ISNT), Sperm Chromatin Dispersion (SCD) or Single-cell gel electrophoresis assay (Comet) (21).

In recent years, new approaches based on multiparametric analyses by flow cytometry have been developed as innovative technologies to evaluate ROS production in ram sperm (22). Flow cytometry is a useful tool for the evaluation of sperm quality. This technique allows the analysis of a large number of cells, capturing many features of each of them in a few seconds (23). Its ability to analyze multiple sperm characteristics allows for a better understanding of sperm functionality (24), which will enable the development of sperm quality analysis (25, 26). Multiparametric analysis by flow cytometry has been developed to ensure adequate sperm quality to perform fertility studies in different mammalian species, such as humans (27, 28), bulls (29), and stallions (30). Some oxidative stress measurement techniques have been developed for the evaluation of specific ROS and other oxidative species can be carried out by reagents that accumulate intracellularly and become fluorescent upon oxidation (23). These fluorochromes include MitoSOX Red probe used to identify superoxide anion and hydrogen peroxide in human sperm (31), 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) to detect hydrogen peroxide (32, 33) or 4,5-diaminofluorescein diacetate (DAF-FM) used for identify nitric oxide in stallion sperm (34).

Recently, novel CellROX fluorescent probes in different colors have been introduced as ROS markers: CellROX™ Deep Red, CellROX™ Orange and CellROX™ Green. CellROX™ Deep Red and Green can detect hydroxyl radicals and superoxide anions in sperm cells (35–37). On the other hand, CellROX™ Orange detects hydroxyl peroxide, hydroxyl radicals, nitric oxide, peroxide nitrile anions and superoxide anions in sperm cells (37, 38). Additionally, CellROX probes can be used in fluorescence microscopy and spectrophotometry, unlike previous probes (35). Therefore, these innovative probes allow for deep characterization and identification of intracellular ROS localization. In the case of sperm cells, this characterization is more interesting because these cells are composed of a head (where the nucleus is located), middle piece (where the mitochondria are placed), and tail (39). ROS can be localized to the mitochondria but also in the nucleus, where they promote DNA damage (40). For this reason, these novel fluorescent probes should be analyzed.

The objective of this work was to validate the efficiency of the fluorescent probes CellROX™ Deep Red (CRDR) and CellROX™ Green (CRG) in identifying the presence of ROS [the hydroxyl radical (OH) and superoxide anion (O₂)], including their intracellular location and concentrations in ram sperm. In addition, the relationships between the fluorescent probes and different parameters of sperm quality were studied. For this purpose, motility and kinetic parameters, multiparametric flow cytometry analyses (viability and apoptosis), hDFI and, MDA measurements were correlated with the novel fluorescent probes. This evaluation could improve semen preservation protocols in rams.

Redox balance is essential for living systems, including sperm cells (46). The balance between electron loss (oxidation) and electron gain (reduction) is crucial for adequate cellular functionality (11, 12, 47). Therefore, oxidative stress occurs when ROS generation exceeds the protective capacity of the antioxidant mechanisms (11, 12, 46). This can be due to a lack of antioxidant protection or the overproduction of ROS (11, 12, 46). Excessive ROS production may be cytotoxic to sperm, causing a decrease in sperm quality (4, 48). Furthermore, it should be noted that ROS are necessary for the normal physiological functions of sperm and are involved in important signal transduction pathways (40). Moreover, ROS are required for sperm chromatin compaction, providing protection against oxidative DNA damage (49).

Recently, some fluorescent probes, such as CellROX™ Deep Red and CellROX™ Green, have been described as markers of sperm damage, as they can evaluate the presence of specific ROS in different mammalian species (35, 38, 44). CRDR has been considered an ROS marker in ram (35, 50), bull (38, 51) and stallion sperm (44, 52–54). However, contradictory results have been published concerning their correlation with sperm quality in rams (42, 55, 56) and stallions (57, 58). These results could suggest that there may be a species-specific effect, which we have decided to study in depth.

New markers based on redox status, including deep characterization of ROS probes, could be very useful to analyze sperm quality to optimize ram sperm preservation protocols (cooling and freezing). For this purpose, four experimental groups with different initial sperm qualities were established. These experimental groups included fresh (BS and NBS), frozen-thawed and positive control samples (as a damage control). Afterward, multiparametric analyses of the sperm, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters (VCL and ALH), were applied to corroborate the differences in quality of the previously established experimental groups. As expected, after analyzing the quality of the semen from the different experimental groups, two were of high quality with low levels of oxidative stress (fresh samples in the BS and NBS groups). In addition, two groups with low sperm quality and a high level of oxidative stress (thawed samples provided in the NBS and positive damage control samples) were obtained. However, Mendoza et al. (59) previously described increases in apoptotic markers, such as phosphatidylserine translocation, mitochondrial membrane potential or caspase activation, in samples from the nonbreeding season compared with those from the breeding season. Apoptosis appears to be triggered by oxidative stress, which leads to the activation of ROS generation (60). In addition, we chose the experimental groups with more damage (frozen-thawed and positive control) because they were considered the most suitable to experience oxidative stress and therefore an imbalance in ROS. On the one hand, frozen-thawed samples are subject to oxidative stress due to the drastic temperature changes during the cryopreservation process or the high dilution rates to which they are subjected (61, 62). As previously demonstrated, caspases 3–7 are activated, triggering apoptosis as a consequence of drastic temperature change (cryopreservation and thawing process) as a result of the generated ice crystals, which could result in damage to the sperm (42, 61, 63). On the other hand, the positive control samples were incubated with hydrogen peroxide as described by Soliman et al. (16). Oxidative stress induced by hydrogen peroxide causes a drastic decrease in sperm viability, as several authors have previously demonstrated (16, 64). Furthermore, Pujianto et al. (65) demonstrated that a high concentration of hydrogen peroxide is responsible for oxidative stress as a consequence of excessive ROS production in sperm that causes a decrease in sperm quality, including an increase in cell apoptosis. As expected, our results revealed that the fresh BS samples were of the highest quality among the experimental groups. The BS samples showed the highest viability and motility and kinetic parameters (TM, PM, VCL, ALH) and the lowest level of apoptosis (Figures 1A–F) compared to the group in which oxidative stress was induced (positive control samples). Some authors previously described this detrimental effect in oxidative stress-induced samples (16). With respect to the fresh samples inside and outside the reproductive season, while apoptosis significantly increased in the NBS group compared to the BS group, the viability and kinetic parameters increased significantly in the BS compared to the NBS samples (Figures 1A–F). This was reported previously by Kafi et al., who described lower viability in winter than in summer in Karakul rams located in Shiraz (Iran) (66). Moreover, we included cryopreserved sperm samples as a group with intermediate quality that gave values between the fresh and peroxide-treated samples for most of the analyzed parameters. As previously described by several authors and according to our results, cryopreservation induces detrimental effects to sperm quality parameters, including motility and viability (16, 42). To improve sperm quality analyses and confirm the initial differences among the experimental groups in terms of ROS production, the MDA concentration was used as a traditional marker for the assessment of lipid peroxidation and therefore oxidative stress (16). In our results, contrary to previous studies in rams, lipid peroxidation increased significantly in samples assessed after incubation with hydrogen peroxide for 24 h (positive damage control) compared to fresh samples (Figure 2C) (16). This may be because, in contrast to our study, Peris et al. (16) used synthetic oviduct fluid (SOF) as a diluent for both the fresh and peroxide-treated samples. However, we did not observe any differences in lipid peroxidation between the fresh and thawed samples, which is in agreement with the results of Soliman et al. (16). In accordance with previous studies in rams, a similar trend was observed for hDFI, which increased significantly in the positive control samples compared to the fresh samples (16). However, the absence of a difference between the fresh and thawed samples could be explained by the low DNA fragmentation index in rams compared to the other species (67). These results indicate that more accurate ROS tests should be performed to discriminate between fresh samples and cryopreserved samples in rams.

Consequently, after analyzing the differences in sperm quality among the experimental groups, we performed a specific study of two commercial ROS detection probes (CRG and CRDR) for ram sperm quality evaluation.

Our results revealed that CRDR and CRG gave opposite labeling patterns. The number of CRDR-positive cells was higher in fresh BS samples than in frozen-thawed and positive control samples (Figure 2A). Because of this, some significant positive correlations were found between CRDR-positive cells and positive markers of sperm quality, including motility and viability (Figure 3). Furthermore, attending to the viable sperm population, a higher expression of CRDR-positive cells was observed in fresh samples compared to peroxide-treated samples (Figure 5A). In accordance with our results, some authors have previously hypothesized that CRDR is a positive quality marker in stallion sperm (57, 58). Davila et al. (57) have previously included the CRDR probe in their studies, where they demonstrated that it measures superoxide production. Their studies showed that an increased concentration of superoxide anion in mitochondrial sperm was associated with intense mitochondrial activity. Moreover, these authors also observed positive correlations with motility and membrane integrity in stallion sperm. This could be explained by the fact that the blocking electron transfer caused a disruption in production of superoxide anion, which is the target molecule of CRDR. In addition, other studies have shown that 1% to 2% of superoxide anion (O₂) used in the electron transport chain is not completely reduced, generating the O₂ (68). These observations are in accordance with our results, where sperm samples affected by cryopreservation or peroxide treatment suffered a decrease in superoxide anion production, triggering a decrease in CRDR labeling. For this reason, considering that the CRDR probe mainly detects this anion, an increase in the O₂ is associated with high mitochondrial activity rather than oxidative stress. On the other hand, CRG has been described as a ROS marker in humans (69) and bulls (36, 70). Unlike CRDR, the number of CRG-positive cells was higher in the frozen-thawed and positive control samples than in the breeding season samples (Figure 2B). Moreover, some significant positive correlations were found between CRG-positive cells and negative sperm quality markers, such as apoptosis and MDA (Figure 3). Likewise, attending to the viable sperm population, a higher expression of CRG-positive cells was also observed in peroxide-treated samples compared to fresh samples (Figure 5B). Our results were in accordance with previous works that demonstrated an increase in the percentage of CRG-positive cells after exposure to an oxidative environment (36). Moreover, de Castro et al. demonstrated that this probe was a very sensitive marker in bull sperm (36). They observed a dose-dependent effect, and CRG fluorescence intensity increased with increasing concentrations of hydrogen peroxide (36). However, the CRG probe has not yet been studied in ram sperm. According to the manufacturer's instructions, and as described by Riley et al. (69) in their study, the CRG probe has weak basal fluorescence that increases as the sample becomes oxidized (Supplementary Figure 4). Based on our results, it is necessary to include all the available techniques for CellROX characterization, including intracellular location, which is a crucial factor (40). In addition to the opposite labeling pattern, our studies revealed different intracellular locations of the CellROX fluorescent probes as studied by confocal microscopy. CRDR fluorescence was located in the middle piece of the sperm (Figure 4C) as previously published by Rodrigues et al. (35). These authors found that only sperm subjected to oxidative stress showed red fluorescence. Contrarily, our study only detected red fluorescence in fresh samples (35). This inconsistency may be due to the solvent used for the oxidative stress induction agent since Rodrigues et al. (35) used TALP medium, which has antioxidants in its composition and could had had some interaction during samples incubation.

While in our study we used hydrogen peroxide, Rodrigues et al. used ferrous sulfate and sodium ascorbate (35). This could mean that the medium used by Rodrigues et al. did not induce oxidative stress as effectively, since in our study CRDR fluorescence correlated with the most parameters of seminal quality and oxidative stress. This could be due to the antioxidant effect of Tyrode's Albumin Lactate Pyruvate (TALP) medium that could neutralize the effect of the agents used by Rodrigues et al. to induce oxidative stress. The components of the TALP medium contain pyruvate and lactate, components with an antioxidant effect and promoting mitochondrial functionality as previously described (71, 72). In addition, mitochondria are located in the midpiece of the sperm, which is the location of the highest metabolic activity in viable sperm (73). Therefore, the localization of CRDR fluorescence in the midpiece of the fresh samples could be a useful indication of proper sperm functionality. On the other hand, we observed that CRG fluorescence was located in the sperm nucleus in positive control samples (Figures 4D, 5H). In accordance with our results, CRG fluorescence was observed in the nuclei of bull sperm subjected to hydrogen peroxide treatment (36).

These results demonstrated that CRG and CRDR presented an opposite labeling pattern that was corroborated by fluorescence microscopy that showed different localization of the probes. While CRDR fluorescence was observed in the middle piece of the sperm from the fresh samples with superoxide anion production (intense mitochondrial activity sperms), CRG was found in the nuclei of the sperm in the positive control samples and was associated with DNA damage. These results could be explained by the fact that CRG is a DNA dye, and upon oxidation, it binds to DNA; thus, its signal is localized primarily in the nucleus and mitochondria. This fact was previously demonstrated by in a ROS localization study in Solea senegalensis sperm (40). In contrast, the signals from CRDR were localized in the cytoplasm. These results could hypothesize that in sperm, the localization of ROS is a crucial factor in sperm quality determination, as in previous studies performed in fish (40). Furthermore, we can conclude from our results that CellROX probes could be more discriminatory tests for the assessment of oxidative stress than lipid peroxidation measurements or DNA fragmentation. This is because, unlike the evaluation of lipid peroxidation, the use of CRDR and CRG allowed us to establish differences between the two experimental groups (frozen-thawed and peroxide-treated samples). Furthermore, we could consider that CRDR is a more accuracy test compared to CRG according to the highest correlations observed with the semen quality parameters studied (Figure 3). In addition, differences in the significance of both tests were also observed in viable sperm: CRDR (P < 0.05) shows a high level of significant differences between the two extreme groups compared to CRG (P < 0.001). This work allowed us to characterize CRG and CRDR with fluorescence assays for use as innovative and accurate tools in seminal assessment by focusing on the redox status of ram sperm cells. In this study, we observed the relationship between intracellular ROS location and the effect of ROS on sperm cells. CRDR fluorescence was located in the sperm mitochondrial region (middle piece) in the fresh sperm samples, which could be associated with high mitochondrial activity according to viability and motility analyses performed in this work. On the other hand, CRG fluorescence was mostly located in the cell nuclei in the positive control and frozen-thawed samples, which could be related with sperm damage, due to its positive correlation with detrimental sperm quality parameters such as MDA concentration.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by Animal Care and Use Committee of the University of León (Spain) ÉTICA-ULE-050-2022. The study was conducted in accordance with the local legislation and institutional requirements.

CP-M: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. LA-L: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Visualization, Writing – review & editing. MA: Conceptualization, Project administration, Resources, Writing – review & editing. MN-M: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, Writing – review & editing. RM-G: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, Writing – review & editing. CS-Ú: Conceptualization, Resources, Writing – review & editing. PP: Conceptualization, Resources, Writing – review & editing. LA: Conceptualization, Funding acquisition, Project administration, Resources, Writing – review & editing. MR: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – review & editing.