Quercetin improves the apoptotic index and oxidative stress in post-thaw dog sperm

Freeze storage of ejaculated sperms is a crucial technique for the semen preservation of valuable pet animals such as dogs. The current study was conducted to investigate if quercetin (QRN) may ameliorate apoptosis and oxidative stress in post-thaw dog sperm. Herein, we evaluated the post-thaw apoptosis and oxidative stress after treatment with QRN (control, 25, 50, and 100 μM) in the freezing of dog semen. Reactive oxygen species levels were significantly affected (p < 0.05) between the various concentrations of QRN and the control (17.56 ± 1.02, 7.54 ± 0.48, 5.66 ± 0.80, and 10.41 ± 0.69), respectively. The apoptosis index was 9.1 ± 1.34, 6.66 ± 0.58, 6.77 ± 0.66, and 5.38 ± 0.86 in the control, and 25, 50, and 100 μM QRN treatment groups, respectively (p < 0.05). The effects of ameliorated cryo-induced damage by QRN on post-thaw sperm quality were also observed through improved structural and functional tests. Sperm treated with 50 μM QRN showed significantly higher motility (51.8 ± 2.1% vs. 43.1 ± 1.4%, P < 0.05), survival rates (46.9 ± 0.7% vs. 43.9 ± 0.4%, P < 0.05), and mucus penetration than control group, respectively. Results also indicated that higher concentrations of QRN (100 μM) were not effective on sperm quality and parameters when compared with the medium levels (50 μM). In conclusion, supplementation of freezing buffer with 50 μM QRN reduced oxidative damage and improved the quality of post-thaw dog sperm.


Introduction
Artificial insemination using frozen dog semen is associated with a sluggish progress compared with that of other animal species (England 1993). Identifying the best cryoprotectant for preventing cryodamage-induced molecular toxicity in dog sperm post-thaw is a focus for several research groups, including our group (Farstad 2009;Qamar et al. 2020). Reactive oxygen species (ROS) or free radicals are resulted from physiological cellular metabolic processes that lead to cellular early aging and apoptosis (Redza-Dutordoir and Averill-Bates, 2016). Living organisms have diverse and complex systems to balance and maintain harmless intracellular ROS levels to protect phospholipids, proteins, and DNA from the adverse effects of ROS (Schieber and Chandel, 2014).
In semen cryopreservation, seminal plasma is usually discarded and therefore sperms lack extracellular antioxidants that defeat ROS (Alahmar 2019, Fraser et al. 2011, Iwasaki and Gagnon 1992, Kashou et al. 2013, Papas et al. 2019, Sabeti et al. 2016). Consequently, cell cryopreservation increases intracellular ROS, promotes the oxidation of phospholipids in the cell membrane, and leads to DNA fragmentation and cell membrane damage (Bansal andBilaspuri 2010, El-Said et al. 2014;Su et al. 2019). This effect is propagated in case of gametes cryopreservation and compromises the fertility quality of post-thaw sperm (Baumber et al. 2000;Sariözkan et al. 2009). Therefore, supplementation with antioxidants has been applied to scavenge ROS, reduce its effect on cell components, and retain the fertility quality of sperm, such as motility and viability (Barciszewski et al. 2000;Liu et al. 2021;Snezhkina et al. 2019).
Interestingly, externalization and translocation of phosphatidylserine (PS) from the inner leaflet of sperm membranes to the external leaflet is considered as an early marker of apoptosis in spermatozoa (Martin et al. 2005;Shiratsuchi et al. 1997). ROS production is significantly related to the activity of anti-apoptotic Bcl2 and pro-apoptotic BAX proteins (Setyawan et al. 2016). DNA integrity is also a concern, as cryopreservation alters the properties of the mitochondrial membrane and increases the generation of free radicals that affect DNA oxidation and lead to single-and double-strand DNA breaks Lingner 2020, Ricci et al. 2002).
Antioxidant-supplemented sperm showed reduced lipid peroxidation, enabling the plasma membrane to maintain normal physiological and metabolic activity, ultimately resulting in enhanced viability (Alvarez and Storey 1989, Bansal and Bilaspuri 2010, Malo et al. 2010Qamar et al. 2020;Setyawan et al. 2016;Yoshimoto et al. 2008). However, finding an appropriate species-specific antioxidant is the target of several research groups to alleviate the cryodamage and maintain the functional integrity of spermatozoa during the freezing process (Bansal and Bilaspuri 2010). For instance, α-linoleic acid was shown to suppress ROS generation by stabilizing the plasma membrane during the cryopreservation of boar sperm (Qamar et al. 2020). In canine species, several antioxidants and ROS scavenger supplements were used for sperm cryopreservation, including rosemary and spermine (Setyawan et al. 2016;Vieira et al. 2018).
Quercetin is a flavonol from the flavonoid group of polyphenols found in many fruits, vegetables, and seeds (Formica and Regelson 1995). Quercetin prevents peroxidation in several organisms; displays anti-cancer, antibacterial, and anti-inflammatory effects; reduces nanoparticles toxicity; and improves oocyte in vitro maturation (Bungau et al. 2019;Ezzati et al. 2020;Grewal et al. 2021;Han et al. 2021;Hussein et al. 2016;Kang et al. 2016;Kim et al. 2020;Moodi et al. 2021;Russo et al. 2012). Quercetin can modulate the mitochondrial membrane potential by restoring ATP levels, blocking caspase-3, and minimizing DNA unpacking (Bali et al. 2014). Electron transport chain and cytochrome c were reported to be the molecular targets of quercetin, preventing H 2 O 2 production and protecting mitochondrial function and integrity (Carrasco-Pozo et al. 2012;Tanga et al. 2021). Quercetin supplementation in human and bovine sperm caused significant improvements in frozen/ thawed spermatozoa motility, viability, and DNA integrity, and prevents apoptosis (Azadi et al. 2017, Diao et al. 2019, Moon and Morris 2007, Tvrdá et al. 2016, Zribi et al. 2012. Conversely, quercetin can be a pro-oxidative in the longterm uses (Ashida et al. 2000) and its action is dose-dependent, particularly in the cell culture conditions (Fukuda and Ashida 2008). Therefore, determining the optimum concentration and conditions for the use of quercetin is vital for its application.
Therefore, we hypothesized that supplementing the freezing extender with quercetin could preserve sperm fertility in dogs by reducing free radical production, oxidative stress, and subsequent apoptosis. We examined the effects of supplementing various concentrations of quercetin on ROS, oxidative stress, and sperm apoptosis as well as some structural and functional analysis of post-thaw dog sperms as indicators of fertility quality.

Animals and ethics
Four healthy beagles (age, 2-4 years old; weight, 8-12 kg) were used in the current study. Formulated food and water were available ad libitum to the beagles and were maintained in a comfortable facility and isolated from external stresses.

Semen collection and freezing
Semen was collected two times per week using digital manipulation for a total of eight times. The pooled semen was washed, diluted, and analyzed using a computer-aided sperm analysis software (MICROTOPIC CASA System; SCA class analyzer, Josep Tarradellas, Barcelona, Spain). Samples of more than 100 × 10 6 sperms/mL, 70% motility, and 80% viability were selected, pooled, and processed. Cell debris was discarded after centrifugation at 100 × g for 1 min (at 25 °C); then, the supernatant was used. The supernatant was mixed with buffer 1 (1v/1v, buffer 1: Tris (hydroxymethyl) aminomethane (198.11 mM), citric acid (72.87 mM), fructose (44.39 mM), and kanamycin sulfate (0.25 mM)) and centrifuged at 700 × g for 2 min at 25 °C to collect the pellets . Sperm pellet was resuspended in buffer 1 to attain a 200 × 10 6 sperm/mL and was then adjusted to 100 × 10 6 sperms/mL by adding a freezing extender (freezing extender) which was prepared using 54% buffer 1 (v:v), 40% egg yolk (v:v), and 6% glycerol (v:v)) that contained 0 (control), 25, 50, or 100 μM quercetin. After dilution, the samples were loaded into 0.5-mL straws (Minitube, Tiefenbach, Germany) and cooled for 45-60 min at 4 °C. Sperm freezing was performed through keeping the straws about 2-4 cm above the level of liquid nitrogen (LN 2 ) for 10-15 min. The frozen straws were stored in an LN 2 container (− 196 °C). For thawing, straws were maintained at 37 °C for 30 s and semen was sequentially diluted with buffer 1 for further evaluation within 5-10 min after thawing.

Computer-aided sperm analysis (CASA) of thawed sperms
Five microliters of post-thaw sperm samples was transferred onto a clean glass slide to assess the motility and motion characteristics using a CASA imaging system. For the analysis, five fields of more than 200 sperms for each semen sample were monitored for 1 s at 25 Hz. The proportions of total and progressive motility were analyzed. The recorded motion characteristics were curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), straightness, linearity, and amplitude of lateral head displacement (ALH).

Flow cytometric analysis of ROS and PS translocation index
Flow cytometry analysis was performed through BD Accuri™ C6 plus (Becton Dickinson, BD Biosciences, Franklin Lakes, NJ, USA). The flow cytometer was fitted with blue (488 nm, solid state, 20 mW) and diode red (640 nm, 14.7 mW) excitation lasers. The fluorescent probes used in this experiment were 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Inc., OR, USA), annexin V-FITC (apoptosis detection kit I (BD Biosciences), and propidium iodide (Malo et al. 2010) were excited using a 488-nm blue-solid state laser. Live spermatozoa stained with H2DCFDA and annexin V were detected using a filter detector 533/30 BP (wavelength range 511-543 nm). The signal from dead sperm stained with propidium iodide was detected using a filter detector 586/42 BP (wavelength range 565-607 nm). The flow rate of the flow cytometer was medium (35 μL/min, 16-μm core). Sperm populations were divided into regions and quadrants. The data were analyzed using BD Accuri™ C6 Plus Flow cytometer software. Intracellular ROS levels were detected using H2DCFDA according to the experimental protocol of Guthrie and Welch (Guthrie andWelch 2010, Mahfouz et al. 2009). The level of ROS was evaluated by calculating the percentage of sperm stained with H2DCFDA from the total percentage of live sperm (stained negatively with PI). The PS translocation status was assessed through the annexin V-FITC detection kit. Briefly, spermatozoa were pelleted twice using PBS at 300 × g for 5 min and then diluted in 1 mL of 1 × annexin buffer (5 × 10 6 sperm/mL). From this suspension, 100 μL was collected in new 1.5 mL tubes and mixed with 5 μL annexin-FITC stain and 5 μL propidium iodide (Malo et al. 2010). The mixture was maintained in the dark at room temperature (25 °C) for 15 min. Thereafter, 400 μL of 1 × annexin buffer was mixed into the tubes and analyzed by flow cytometry.

Sperm plasma membrane and acrosome integrity
Hypo-osmotic swelling (HOS) assay was used to assess the sperm plasma membrane integrity. In brief, approximately 200 sperms were incubated for 30 min in the HOS solution and then examined under a phase-contrast microscope (Eclipse Ts2; Nikon, Minato-Ku, Tokyo, Japan) within 5-10 min. Coiled sperm tails showing swelling indicate an intact sperm plasma membrane. Acrosome integrity was examined through using fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) stain (Almubarak et al. 2021;Kang et al. 2020). Briefly, smears were prepared and fixed with methanol. After washing with PBS, 30 μL of FITC-PNA (100 μL/mL in PBS) was added to the fixed smear and incubated in a humid dark place for 30 min. Stained spermatozoa (n > 200) were examined through an epifluorescence microscope (1000 × magnification; Eclipse Ts 2, Nikon) and categorized according to the presence or absence of fluorescence staining (i.e., acrosome-intact spermatozoa emitted intense green fluorescence on the anterior sperm).

Measuring sperm mitochondrial activity and chromatin integrity
Mitochondrial activity was measured according to our previous method . In brief, 1 mg rhodamine 123 (R123) fluorescent stain was mixed with post-thaw diluted sperms (20 × 10 6 sperm/mL) and incubated at 37 °C for 15 min in a dark chamber. Ten microliters of PI was mixed with the mixture and was kept at 37 °C for 10 min. Sperms were retrieved by centrifugation (500 × g for 5 min at room temperature) and the pellets were resuspended in phosphate-buffered saline (PBS). One drop of sperm mixture was placed on a glass slide, covered with a coverslip, and examined through an epifluorescence microscope (Eclipse Ts2, Nikon, Japan) to detect the functional mitochondriacontaining sperms with green fluorescence color. Sperm chromatin integrity was estimated by the acidic aniline blue staining method (Kazerooni et al. 2009). In brief, semen samples were smeared and buffered glutaraldehyde (3:100 w/v) was added for fixing the smears for 30 min. Acidic aniline blue was added for 7 min to stain the smears, rinsed with purified water, and air-dried. Sperms were examined under the oil immersion lens and the blue-stained heads were considered as abnormal chromatin, while the non-stained heads were considered as normal ones.

Sperm mucus penetration test
With the aid of modified synthetic oviductal fluid on flat capillary tubes (80 ± 0.5 mm long, 1.25 ± 0.05 mm wide; Hilgenberg GmBH, Stutzerbach, Germany), the sperm penetration test was evaluated. After sealing one periphery of the flat capillary tube, it was filled with mucus and left to stand for 15 min. Thereafter, the open periphery of the flat capillary tubes was immersed in a 100 μL of semen samples and left horizontally at 25 °C for 2 h. The numbers of spermatozoa that reached the marks of 1 and 3 cm were counted.

Statistical analysis
Data were analyzed by the Statistical Package for Social Sciences (SPSS, version 24.0 software, IBM, Armonk, NY, USA). Data were expressed as the mean ± standard error of the means (SEM). Data of motion characteristics and percentages of live sperms were analyzed with a one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. P < 0.05 was considered a statistical significance.

Effect of quercetin on ROS level
The Control group showed increased ROS levels (P < 0.05) when compared with the quercetin-supplemented groups (Fig. 1). ROS proportions were 17.56 ± 1.02, 7.54 ± 0.48, 5.66 ± 0.80, and 10.41 ± 0.69 in the control, and the 25, 50, and 100 μM quercetin treatment groups, respectively. ROS levels were statistically significantly different (p < 0.05) among the various concentrations of quercetin and the control (Fig. 1). Of the treatment groups, the 50 μM quercetin group was found to have the most significantly lower ROS level, which indicates that the optimal concentration for antioxidant activity is 50 μM.

Effect of quercetin on PS translocation index (apoptosis status)
The apoptosis index was measured by evaluating the PS translocation. The percentage of apoptotic spermatozoa (annexin V + /PI −) was calculated from the total live spermatozoa (PI −), and the data were considered as the PS translocation index. The apoptosis index values were 9.1 ± 1.34, 6.66 ± 0.58, 6.77 ± 0.66, and 5.38 ± 0.86 in the control, and the 25, 50, and 100 μM quercetin treatment groups, respectively; the control group showed significantly higher values than the quercetin-supplemented groups. However, no differences were observed between the quercetin-supplemented groups (Fig. 2).

Effect of quercetin on sperm motility and percentage of live sperm
The motility, motion characteristics, and viability results are shown in Table 1. Post-thaw semen in samples frozen with 50 μM quercetin (51.8 ± 2.1%) showed higher motility than control samples (43.1 ± 1.4%) (P < 0.05). Motion characteristics showed no difference between the control and 25 μM quercetin groups, while the values, except for ALH, tended to be reduced as quercetin concentration increased. In addition, the percentage of live sperms was increased in the samples frozen with 50 μM quercetin (46.9 ± 0.7%) when compared with the control samples (43.9 ± 0.4%) (P < 0.05). However, there was no difference between the control, 25 μM (45.7 ± 1.6) and 100 μM (43.6 ± 0.9) quercetin groups ( Table 2). The motility and viability of post-thaw sperm were significantly increased in sperms supplemented with 50 μM quercetin when compared with the other groups.

Effect of quercetin on sperm plasma membrane integrity, mitochondrial activity, and chromatin integrity
The HOS test showed a significant increase in plasma membrane integrity of the 50 μM QRN groups compared to the control group (54.0 ± 0.6% vs. 46.6 ± 0.7%, respectively). However, there was a significant decrease in the membrane integrity of sperm samples supplemented with 100 μM quercetin (45.2 ± 0.6%) ( Table 2). FITC-PNA staining showed that quercetin supplementation did not effectively protect the acrosomal integrity of the cryopreserved dog sperm, as no statistically significant differences were found between the quercetin-supplemented sperm samples and the control samples. Similar patterns were observed on mitochondrial activity and chromatin integrity and results showed the effectiveness of QRN at 50 μM when compared with the control and 100 μM groups ( Table 2).

Effect of quercetin on mucus penetration
Results showed that 50-μM quercetin-supplemented postthaw sperm penetrated the modified synthetic oviductal fluid more effectively than the control group (69.2 ± 1.3 vs. 51.5 ± 1.9 at 1 cm and 15.1 ± 1.6 vs. 27.0 ± 1.1 at 3 cm), respectively. The sperm counts for quercetin-supplemented sperm samples were significantly higher at both the 1-and 3-cm marks than those for the control sperm (Fig. 3). The motility-promoting effects of 25 and 100 μM quercetin were less than those of 50 μM quercetin at both 1 and 3 cm;  however, the effect of 100 μM was relatively higher than that of 25 μM quercetin at 1 cm (Fig. 3).

Discussion
Cryopreservation is a crucial tool for preserving spermatozoa and the genetic merits of valuable species for a long time. However, cryodamage due to freezing and thawing compromises the fertility of sperm post-thaw. Cryodamage at the molecular level due to freezing and thawing is also a concern during the long-term preservation of dog sperm (England 1993;Farstad 2009;Vieira et al. 2018). Cryodamage can be caused through the generation of ROS in sperm. However, supplementing antioxidants during sperm freezing to reverse the effect of ROS has been used to ameliorate these adverse effects (Majzoub and Agarwal, 2020). Herein, the observed reduction in oxidative stress by the application of quercetin in dog sperm coincides with the results found in the rats, red jungle fowl, and the roosters where supplementation with quercetin reduced oxidative stress (Najafi et al. 2020;Rakha et al. 2020;Yelumalai et al. 2019). The mechanism employed by quercetin to ameliorate oxidative stress in post-thaw sperm is thought to occur via an increase in the sperm total antioxidant capacity and the amelioration of lipid peroxidation (Papas et al. 2019). Quercetin has also caused improvements in conserving DNA integrity, owing to reduced ROS levels, as reported in bulls (Avdatek et al. 2018), which could have contributed to fertility. The lipid peroxidation reduction ability of quercetin (Yang et al. 2020) is believed to contribute to oxidative stress in postthawed sperm. Accordingly, we anticipate that further sperm quality criteria and the in vitro fertility test could be conducted to further validate the effectiveness of sperm cryopreservation. Evaluation of post-thaw sperm revealed significantly improved viability in the quercetin-supplemented sperm samples compared to control samples. Moreover, higher percentages of sperm with intact membranes were observed in the quercetin-supplemented sperm samples than in the control samples. The greater membrane integrity of quercetin-supplemented sperm is an indicator of preserved sperm structure and ultimately the enhanced survival rate of sperm (Ismail et al. 2020).
ROS generation during cryopreservation triggers the apoptosis of spermatozoa, eventually leading to sperm loss of function or death (Said et al. 2004(Said et al. , 2010. Moreover, under oxidative stress conditions, the mitochondrial membrane potential decreases and apoptosis is enhanced by ROS generation (Redza-Dutordoir and Averill-Bates, 2016). Quercetin displayed potent antioxidative properties, improved membrane integrity, improved mitochondrial activity, and chromatin integrity owing to its ROS scavenging activity (Mazzi et al. 2012). Quercetin antagonizes the enzymatic (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nicotinamide adenine dinucleotide (NADH)-dependent oxidoreductase) and non-enzymatic systems involved the ROS formation (Walczak-Jedrzejowska et al. 2013).
The reduced apoptotic index due to the application of quercetin is attributed to its ability to inhibit lipopolysaccharide-induced mRNA expression of tumor necrosis factor α (TNF-α) and interleukin (IL-1α) (Bureau et al. 2008). This effect of quercetin could be an additional mechanism for the reduced apoptosis and enhanced integrity of the sperm; however, further studies are needed to confirm this. Our results coincide with the recent results of Kawasaki et al. (2020) who observed that the skim milk-based extender supplemented with 5 μg/mL quercetin and 0.1% dimethyl sulfoxide improved the motility and fertility of cryopreserved dog spermatozoa. Similarly, the application of quercetin in stallion sperm cryopreservation caused improved postthaw sperm quality, such as protecting DNA fragmentation, Fig. 4 The proposed beneficial effects of supplementing quercetin during the dog sperm freezing-thawing process. Quercetin ameliorates reactive oxygen species (ROS) generation and increases the antioxidant capacity, thereby reducing the oxidative stress impacts on the sperms, such as phospholipid damage, mitochondrial membrane potential, and phosphatidylserine (PS) translocation, which are associated with apoptosis. Dashed line indicates suppression of the effects motility, and zona binding ability (Gibb et al. 2013;Seifi-Jamadi et al. 2016).
The current results revealed that supplementation of a freezing extender with 50 μM quercetin improved motility and survival as well as the proportion of total and progressively motile frozen-thawed dog sperm. This finding aligns with that of previous studies on the effect of quercetin on reducing oxidative stress in humans (15 μg/mL) (Zribi et al. 2012) and stallions (45 μg/mL) (Zribi et al. 2012). However, quercetin is a versatile flavonoid; after displaying its antioxidant effect, it changes to a toxic product that might affect sperm quality (Mazzi et al. 2012) as observed in the 100 μM quercetin group in this study. Therefore, determining the optimum concentration and cryopreservation conditions should be performed to enable the effective application of antioxidants in cryopreservation. The mucus penetration test also confirmed the improved motility of the quercetinsupplemented sperm sample. The effects of quercetin supplementation on canine sperm cryopreservation are summarized in Fig. 4.

Conclusions
The quercetin-supplemented freezing extender had a significant effect in ameliorating cryo-induced oxidative stress and apoptosis, and protected the key fertility parameters of postthaw dog sperm, such as mucus penetration, membrane integrity, and livability. Therefore, we recommended the addition of quercetin as a vital cryoprotectant supplement to maintain the survival and motility of post-thaw sperm (Collins andRyan 2011, Henkel et al. 2004). Based on our analysis, the optimum concentration of quercetin for protecting post-thaw dog sperm against oxidative stress-induced damage is 50 μM.