Antioxidant activity of Opuntia sp. fruit extracts on human sperm quality and cryopreservation cycle

Opuntia sp. contain antioxidant phytochemicals resistant to ROS damage, whose excess negatively affect fertilization. We investigate the activity of fruit extracts of O. dillenii and O. ficus indica (cv red and yellow) on sperm quality and cryopreservation. In the first experiment, we exposed the samples to extracts (50 μl) for 1 hour to then evaluate semen parameters (vitality, motility, acrosome reaction, DNA fragmentation and oxidative stress). The results showed a significant increase in the motility (86%±0.19 for OFI cv yellow, 82%±0.15 for OFI cv red and 90%±0.08 for O. dillenii) compared to the control (80%±0.17). Moreover, we noted a reduction of DNA fragmentation on treated (3%±0.03 in OFI cv yellow, 7%±0.09 in OFI cv red and 5%±0.07 in O. dillenii) than the control (40%±0.14). Furthermore, the oxidative stress was reduced after exposure to solutions (3.15mV in the control and 2.94mV in the treated). In the second experiment, 50 μl of solutions were added to the Freezing medium. After thawing, we observed an improvement in vitality and the number of intact acrosomes. Our results suggest that Opuntia sp. fruit extracts improve sperm quality, both before and after cryopreservation, optimizing the potential of fertilization of sperm cells.


Introduction
In the last few years, natural phytochemicals of plants such as Opuntia ficus indica (L.) Mill. (OFI) and Opuntia dillenii Haw. (OD) have represented one of the research fields around which the scientific community developed a great interest for their protective effects on human health. It is known that the excess of highly unstable molecules like reactive oxygen species (ROS) and other free radicals is linked to the degradation of organic molecules (proteins, lipids, polysaccharides, DNA and RNA), stimulating, on the contrary, biochemical processes involved in the aetiology of diseases like cancer, diabetes, and ischemia [1,2]. fruit extracts of OFI cv red (F2) and yellow (F1) and OD to assess the potential antioxidant effect, capable to bring benefits to sperm cells and optimizing the gametic quality. Moreover, we evaluated the extract supplementation on a cryopreservation medium because we hypothesized that the sucking substances reduce the ice crystal formation, protecting sperm cells from deterioration during the thawing.

Extract preparation of Opuntia sp.:
In this study, clones of OD and OFI were used. The fruits, collected in July -October 2019 in Sicily (Italy), were harvested from a germplasm collection of CNR-ISAFOM, the section of Catania (Italy), grown under the same environmental conditions and with the same applied agronomic practices. The two cultivars of OFI used in this study were characterized with fruits red (F2) and yellow (F1) colours. For each accession, five fruits at the same ripeness stage were randomly collected. Juices were prepared according to Serra et al. (2013) [35]. The fruits were washed under tap water, spikes were removed with a brush and peel take out from the fruit manually. The pulps (containing seeds) were grounded and passed through a strainer to remove seeds. The juices were preserved under frozen storage (-80°C) until analysis. Each sample (1 g) was added to 10 ml aqueous methanol (70%), sonicated for 20 min and centrifuged at 5000 rpm for 10 min. The supernatants were filtered through syringe filters (0.45 µm, nylon) before analysis.

Total phenolic content:
The Total Phenolic Content (TPC) was performed using the protocol of Dewanto et al. (2002) [36] with some modifications. Aliquots (125 µl) of each extract were mixed with 625 µl of Folin Ciocalteau, previously diluted with ultrapure water (1:5, v/v). After 6 min, 1.25 ml of 7% Na2CO3 aqueous solution and 1 ml of ultrapure water were added. The mixture was shaken and placed in the dark at room temperature for 1 h. Afterwards, the total content of phenolic compounds was measured at 760 nm using the BioSpectrometer UV/Vis spectrophotometer (Eppendorf, Hamburg, Germany). Gallic acid standard solution (25-200 mg L -1 ) was used for the calibration curve (R 2 = 0.9987). All measurements were performed in triplicate. The results were expressed as mg of gallic acid equivalent 100 g -1 of juice (mg GAE 100 g -1 ).

Antioxidant activity:
The antioxidant activity was evaluated using the method proposed by Brand-Williams et al. (1995) [37], with some modifications, through the evaluation of the free radical-scavenging effect on the 1,1-diphenyl-2-picrylhydrazine (DPPH) radical. 100 µl of supernatant was mixed with 2 ml of 0.1 mM DPPH in methanol (freshly prepared). After incubating at room temperature for 30 min in the dark, the absorbance of the mixture was measured at 517 nm. Trolox was used as a reference (calibration range 10-200 µmol L -1 ; R 2 = 0.9992). All samples were analyzed in triplicates and the results are expressed as µmol Trolox equivalent (TE) g -1 of juice.

Identification and quantification of phenolic compounds by HPLC:
The phenolic acids and flavonoids profiles in the Opuntia sp. pulp extracts were determined using a liquid chromatography system Dionex UltiMate 3000 (Thermo Fisher Scientific, Waltham, MA, USA), including quaternary pump with an integrated fourchannel degasser, thermostated column compartment, autosampler and a four wavelength UV-Vis detector, all managed by the Chromeleon software. Separation was performed on a column Dionex Acclaim 120, C18, 3 µm (4.6×150 mm) (Thermo Fisher Scientific, Waltham, MA, USA) with a gradient program at a flow rate of 1 ml min -1 . The column temperature was maintained at 30°C and the injection volume was 20 µl. The mobile phases consisted of water and formic acid (95:5, v/v) (eluent A) and acetonitrile, water, and formic acid (80:15:5, v/v/v) (eluent B). The gradient started with 3% B up to 19 min, to reach 13% at 30 min, to keep 13% up to 38 min and then to reach 14% at 55, 30% at 65 min and 35% at 68 min, and then return to the initial conditions. Chromatograms were recorded simultaneously at 280, 320 and 360 nm. Identification of individual phenolic acids and flavonoids was carried out using the retention times and UV spectra, comparison with commercial standards, and running the samples after the addition of pure standards. The quantification of phenolic compounds was performed using the external standard method. The calibration curves showed a good linear correlation with R 2 > 0.999. Relative standard deviations (RSD%) were ≥ 0.5%, showing good stability and reproducibility of the analysis.

Preparation of semen samples:
Semen samples were collected at M.A.P. (Medically Assisted Procreation) centre, MEDI. SAN. "Clinica del Mediterraneo", Ragusa, and A.S.T.E.R. "Centro di Diagnosi e Cura della Sterilità", Catania, from normospermic patients with a concentration higher than 60 million/ml and motility higher than 50%. The samples were produced after ipsation and then they were cryopreserved and transported to the Laboratory of Reproductive Biotechnology at the University of Catania. Furthermore, before the cryopreservation, we added 50 µl of the filtered and diluted extracts on some samples and finally the Freezing Medium. During the transport, the samples were placed in 2 ml vials in a container with liquid nitrogen at the temperature of -196 °C. Once in the laboratory, we transferred the vials into a cryogenic container.

Thawing of semen samples and exposure to extracts:
The vials were removed from the liquid nitrogen for the thawing, following the standard procedure, described by WHO Laboratory Manual for the Examination and Processing of Human Semen (2010) [38]: ten minutes at room temperature and then ten minutes in a water bath in an incubator at 37 °C with CO2 at 5%. After thawing, the vials were slightly shaken, and the seminal liquid was aliquoted in different Eppendorf tubes, to which we added a washing medium (Gems) with a 1:1 ratio. The solutions were centrifugated at 2.000 rpm for 10 min. The supernatant was discarded, and the pellet was resuspended with the medium in the controls (CTRL) and with medium enriched with 10% of the three extracts (50 µl), (OFI F1 and F2, and OD), previously diluted with 1:4 ratio, in the other samples. They were incubated at 37 °C with an inclination of 45° to facilitate the swim-up (ascent of the sperm) for 1 hour. Then we evaluated the different semen parameters.

Semen analysis:
2.8.1. Motility: Motility is the fundamental activity that allows the spermatozoa to go up the female genital tract to reach the oocyte. We measured the motility, according to the procedure of WHO (2010) [38], dividing spermatozoa into three categories: progressive motility (PR), non-progressive motility (NP) and immotility (IM). The analysis was made by placing 10 µl of sample on a slide and observing under an optical microscope at x400 magnification. We counted 100 spermatozoa at least.

Vitality:
We investigated the vitality through Eosin Y (0.5%) that dyed dead spermatozoa in pink due to the loss of membrane integrity, compared to live spermatozoa that maintain their original colouring. The procedure involves the positioning of 10 µl of sample on a slide in which we added 10 µl of the dye. Then, we placed a coverslip. Finally, the slides were observed under an optical microscope Leica DMLB at x400 magnification. At least 100 spermatozoa were counted.

Acrosome Reaction:
The acrosome reaction (AR) is the process that induces the release of lytic enzymes through which spermatozoa open a passage on the space between zona pellucida and the plasmatic membrane of the oocyte. The test implies the use of two dyes, Trypan Blue, and Giemsa (eosin and azurII). First, one drop of the sample (10 µl) and one drop of Trypan blue (10 µl), with a 1:1 ratio, were placed on a slide and were faintly crawled. After the air drying, the slides were dipped in a fixative solution (HCl 1 N, formaldehyde at 37% and azocarminium) for 5 minutes. Then, we washed the sample with distilled water, and we immersed it in Giemsa, composed of the dye at 7,5% dissolved in distilled water, and left in the incubator at 37 °C for 2 hours. We proceeded with a wash in tap water and distilled water. Finally, the slides were mounted with Entellan (Bio-Optica) and they were observed under an optical microscope Leica DMLB at x400 magnification. This protocol distinguishes dead spermatozoa (blue) from live ones (pink), but it also allows to determine the state of the acrosome. The intact acrosome is dyed in violet, while the damaged or lost acrosome is in lavender. For this test, we counted at least 100 sperm cells.

DNA fragmentation:
To assess the fragmentation of DNA, we used the Halosperm (HT-HS10), a kit of Halotech. Following the manufacturer instruction, we melted the agar (100 µl) stored in an Eppendorf tube, at 90-100 °C, and then we aliquoted it in two different Eppendorf tubes, 50 µl in each one. We added 25 µl of sample in 50 µl of agar. Then, we withdraw 25 µl of this mix and placed it in a slide covered with a coverslip. After 5 min in the fridge at 4 °C, the coverslip was removed, and the slide was incubated with denaturant solution (DAsolution) for 7 min. The DA-solution was token off using the lysis solution (LS-solution) that acted for 25 min. We proceeded with the dehydration through a series of increasing ethanol solutions (70°,80°,100°) for 2 minutes each, letting the slides air dry once finished. Finally, we coloured the slides with Eosin Y for 5 min and with Methylene-Blue for 2 min. Once dry, the slides were read under a bright-field optical microscope with a 1000x objective. The protocol distinguishes spermatozoa with non-fragmented DNA that have halos around the head and sperm cells with fragmented DNA, without halos.

Oxidative stress:
To evaluate oxidative stress, we used the MiOXSYS System. It is based on the measurement of the Oxidation Reduction Potential (sORP), an overall index of the electron passage to which a biological component is subjected. sORP is an integrated measure of all oxidants and reducing agents, evaluating the oxidative damage that spermatozoa are undergoing due to free radicals. The test is carried out by placing a small aliquot (30 µl) of seminal fluid on a sensor previously inserted in the analyser that applies a low voltage current. The electron activity is measured in millivolts (mV).

Statistical analysis:
Statistical analysis was carried out using the CoSTAT software program. Data were submitted to Barlett's test for the homogeneity of variance and then analyzed using the ANOVA test, followed by Tukey's. Means were statistically separated based on the Student-Newman-Keuls test. Significance was accepted at p≤ 0.05 level and all data are presented as mean ± standard deviation (SD) [39].

Total phenolic content and antioxidant activity:
The total phenolic content and the antioxidant activity of Opuntia sp. juices are reported in Table 1.
Total phenolic content showed an average value of 110.28 mg GAE 100 g -1 of juice, ranging from 76.89 of F1 to 150.77 mg GAE 100 g -1 of O. dillenii.
The antioxidant activity showed a similar trend of total phenolic content, with the mean value of 3.15 µmol TE g -1 of juice, varying significantly (p≤ 0.05) between 1.00 in F1 and 6.64 µmol TE g -1 in OD. Even then, the OD showed considerably higher contents than OFI, and also, as reported by Abdel-Hameed et al. (2014), the antioxidant capacity of red O. ficus indica (F2) was higher than the yellow cultivar (F1) [40].

Determination of phenolic compounds by HPLC:
A total of five different phenolic acids and five flavonoids were identified. The qualitative and quantitative profile of identified compounds is shown in Table 2 and indicates that the OD genotype exhibits the highest content of total measured polyphenols (7.717 mg L -1 ), about twice compared to OFI genotypes (4.147 mg L -1 in F1 and 4.420 mg L -1 in F2). The analysis showed significant differences in the content of polyphenols between the tested genotypes, in agreement with Medina et al. (2007), who found that the chemical composition of OD and OFI fruits were clearly different [41].
Kaempferol was found only in OD (0.025±0.005 mg L -1 ) and in traces, contrary to Kuti (2004), who found it in all the samples analyzed (green-skinned, purple-skinned and redskinned of Opuntia sp.) [42]. These differences may be due to cultivar and genetic factors, environment, stress and agrotechnical processes, as well as to growth conditions, harvesting time, degree of ripeness, fruit processing and determination methods [43].

Semen parameters of examples exposed to fruit extract after thawing:
After 1 h of exposure, we observed lower motility on the control group (80%±0.17) compared to the treated, in which it corresponds to 86%±0.19 for OFI F1, 82%±0.15 for OFI F2 and 90%±0.08 for OD. The results are statistically significant with p<0.05. The best result was obtained with OD.
The analysis of vitality, through the Eosin test, highlighted a high number of dead spermatozoa on the controls in which the viability was 78%±0.19, while it was 86%±0.22, 76%±0.41 and 85%±0.22, respectively on samples exposed to OFI cv yellow, OFI cv red and OD (Figure 1). The viability of the control was comparable with that of OFI cv red, while better results were obtained in the samples exposed to OD and OFI cv yellow.
The Trypan-blue test showed the acrosome that appeared damaged on the control compared to the samples enriched with the three extracts. The percentage of intact acrosome is equal to 19%±0.14 in the controls while, between the treated, the best appeared the OFI cv red (31%±0.20), followed by OFI cv yellow (26%±0.26) and OD (17%±0.16) (Figure 2).
The Halosperm test highlighted an elevated number of sperm cells with nonfragmented DNA, and therefore with large halos, in the samples enriched with OFI F1 and OD, slightly less in the samples exposed to OFI F2, and a certain number of fragmented DNA, free of halos, in spermatozoa of the controls (p<0.05) (Figure 3). Indeed, the DNA fragmentation was evident in the control (40%±0.14), while it is almost absent in the exposed: 3%±0.03 in OFI cv yellow, 7%±0.09 in OFI cv red and 5%±0.07 in OD.
Oxidative stress was examined with the MiOXIS System through which we obtained the following result. The control groups showed a higher current passage (3.15 mV) than the exposed (2.94 mV). Therefore, the evidence indicates how the extracts reduce the oxidant amount, compared to the control groups.

Semen parameters of samples exposed to extracts before cryopreservation:
The addition of the extracts in the Freezing medium caused an improvement in motility after thawing, as this parameter was found higher in the exposed samples compared to the control. The percentage of motility corresponds to 70% in the control, a much lower value than of the treated: 98%±0.2 for OFI cv yellow, 97%±0.15 for OFI cv red and 95%±0. 19 for OD.
After thawing, viability appeared exceptionally high in the treated samples in which a high number of intact spermatozoa was found, suggesting that antioxidants, contained in the extracts, may protect the sperm cells from oxidative damage due to the change in membrane fluidity during cryopreservation and to the production of ROS. Indeed, the control had 37%±0.22 of live spermatozoa, while among the treated, the samples exposed to OD had 80%±0.18 of viable spermatozoa, followed by OFI cv red with 76%±0.20 and OFI cv yellow with 69%±0.22 (Figure 4).
The use of extract led to an improvement in the condition of the acrosome. It appeared more damaged in the control, in which only 16%±0.24 of spermatozoa was characterized by an intact acrosome. However, in the treated there was a very high percentage of normal and unaltered acrosomes (50%±0.19 in OFI cv red, 55%±0.25 in OFI cv yellow and 70%±0.15 in OD) ( Figure 5).
The exposure of spermatozoa to Opuntia sp. extracts protected sperm cells from DNA breaks, as our results highlight. The control showed a higher number of spermatozoa with fragmented chromatin (30%±0.16), compared to the treated ones (10%±0.18 in OFI cv red, 9%±0.16 in OFI cv yellow and 3%±0.14 in OD) ( Figure 6). From the results obtained through the MiOXIS, it was evident a greater passage of current in the control (3.20 mV) compared to the treated (2.50 mV), in which the values are superimposable.

Discussion
Phytochemicals are known for their protection of human health, especially for their antioxidant activity. Indeed, the excess of ROS is deleterious for the gametic quality and reproductive success when it is out of the physiological range. Furthermore, ROS level is an index of morphologic anomalies of spermatozoa and diseases like varicocele, metabolic syndrome, prostatitis, responsible for the poor sperm quality [44]. Our experiment provided a series of tests in which semen samples from normospermic donors were exposed to fruit extracts of O. ficus indica cv red (F2) and yellow (F1) and O. dillenii. In addition, we conducted another test, adding these extracts on cryopreservation medium to evaluate their output on the semen parameters after thawing Then, we investigate the motility, vitality, acrosome reaction, fragmentation of DNA and oxidative stress. The evidence indicates how the extracts reduce the oxidant amount, compared to the control groups. Probably, antioxidant impacts are linked with the presence of betalains, flavonoids and vitamins, whose scavenger activity is due to chemical ring structures, capable of stabilizing scattered electrons, blocking the activation of a chain reaction and the passage of electrons [45]. Our experiment showed that the OD has a greater antioxidant power (6.64±0.043 a µmol TE g -1 ) than the other two fruit. This could be explained by the high quantity of flavonoids in this variety (7.717 mg L -1 ) compared to the others. All the OFI genotypes showed lower contents than OD genotype in agreement to Medina et al. (2007), who reported for two species of prickly pear from Tenerife Island, total phenolic contents equal to 117.0 and 45.2 mg GAE 100 g -1 for OD and OFI respectively [41]. The most abundant compound is gallic acid, found in OD compared to OFI, in which rutin is present in greater quantities. These results are in agreement with Abdel-Hameed et al. (2014), who analyzed the concentration of phenolic compounds in the juice of two cactus cultivars (Opuntia ficus indica Mill.) growing in Taif (KSA), although he found gallic acid and catechin more present in yellow cultivar, rutin more abundant in red cultivar, and quercetin in traces in all samples [40]. Also, Mata et al. (2016) identified caffeic acid, rutin, quercetin and isorhamnetin among the 32 compounds detected in the juice of OFI fruit growing in Portugal. Several studies have shown that consumption of Opuntia spp. fruits may present important health benefits due to the presence of bioactive compounds, and in particular of those bioavailable as quercetin, isorhamnetin and their derivates, studied in many matrices [46]. Moussa-Ayoub et al. (2016) illustrated the superiority of OD as one of the most important and promising fruit-producing cactus Opuntia sp., as the results showed that OD fruit's juice is rich in different antioxidant compounds such as vitamin C, phenolic compounds, and betacyanins [47].
In the controls, in which a drop of semen parameters was found, there was also an increase in the sORP. The results are reflected in other studies in which high levels of sORP were paralleled with the poor gametic quality (count, viability, morphology, motility) [48,49,50]. After the exposure to the extracts, we have shown a general improvement in all semen parameters, underlining how oxidative stress is correlated with a decrease in the gametic quality and therefore in the fertilization potential. We observed low motility in the control group compared to the treated. Our findings agree with those obtained by Benkhalifa et al. (2008) that highlighted an enhancement of sperm motility in samples exposed to betalains [51]. The reduction of sperm motility in the control is due to lipid peroxidation and alteration of proteins involved in the electron transport chain (ETC) in the mitochondrial membrane, resulting in the less production of ATP, essential for flagellum movements, suggesting that the speed of sperm depends on the turnover of mitochondrial membrane components [52,53]. Therefore, antioxidants could avoid slowing turnover, ensuring the proper functionality of mitochondria. From our result, it is possible to deduce a greater vitality in the treated than in the controls, due to greater resistance against oxidative damage. The most interesting value was obtained from the samples treated with OD and OFI cv yellow. The cascade, activated by ROS, explains the higher mortality in the control compared to treated, since lipid peroxidation causes the release from mitochondria of cytochrome c, involved in programmed cell death [54]. The acrosome functionality is also improved by the presence of antioxidants. Indeed, the Trypan-blue test showed the acrosome that appeared damaged on the control compared to the samples enriched with the three extracts. ROS production plays a crucial role during capacitation. ROS, in fact, ensure the activation of an enzymatic cascade that ends with the phosphorylation of the residues of proteins involved in the acrosome reaction [55,56]. The study conducted by Griveau et al. (1994) has shown that the addition of catalase enzyme in semen fluid causes a 47% decrease in the number of spermatozoa that release lysis enzymes from acrosome [57]. However, free radical, produced in an unchecked manner, leads to an early reaction, causing a reduction in the fertilization rate. Concerning our findings, we decided to investigate a further aspect, the DNA fragmentation, through the Halosperm test that highlighted an elevated number of sperm cells with nonfragmented DNA in the samples exposed to extracts compared to the control, in which there is acrosome damage greater than 30% compared to the treated. Numerous studies in literature reported how the ROS increasing is correlated with DNA fragmentation [58] and how the latter is connected to reproductive failure even after in-vitro fertilization (IVF) and intracytoplasmic injection (ICSI) [59,60]. Indeed, DNA peroxidation can lead to chromatin cross-linking or double-stranded breaks [61,62], mainly associated with the formation of adducts such as 8-hydroxy-20-deoxyguanosine (8-OHdG) [63]. DNA anomalies put the embryo at death risk, promoting abortion. In this context, the antioxidant role becomes the epicentre of the balance of oxidants/antioxidants system to protect sperm cells from insults of free radicals [64].
The purpose of the second experiment is to demonstrate that the application of extracts to the Freezing medium can improve the parameters of semen after thawing. First, we investigate the motility, whose percentage is stackable in all samples treated (about 90%). Vitality and acrosome reaction were investigated by the acrosome reaction test. In this case, we highlighted a high amount of live sperm cells and with normal acrosome spermatozoa in the samples exposed to the three extracts compared to the control group. Our study showed that the use of Opuntia sp. extracts cause a decrease in the damage resulting from the thawing of the samples. Although cryobiology is essential for the conservation of spermatozoa, which can be used later, it determines a 50% reduction in the spermatozoa integrity and this is problematic, especially for patients with abnormal parameters [65,66,67]. The fortified resistance of spermatozoa is due to the activity of antioxidants that oppose the lipid peroxidation, responsible for the integrity loss of the mitochondrial and the plasmatic membranes. Furthermore, it is known that adding ROS during thawing can also cause increased DNA fragmentation and reduced motility [68,69,70,71]. Our results are also supported by other studies, which show that the exposure to antioxidants, for example to Vitamin E, also present in the fruit of Opuntia sp., cause an increase in the motility and survival rate of spermatozoa after thawing [72,73,74]. Vitamin E and other antioxidants supplementation exert a stimulation on sperm motility and drop the concentration of Malondialdehyde (MDA) [75], a stable product of fatty acids oxidation and indirect index of intensity of this event into the cells [76].
These results can be attributed to gallic acid and rutin. The study conducted by Abarikwu et al. (2014) established that gallic acid leads to improvements in stereoidogenesis and spermatogenesis with a consequent increase in sperm quality [77]. However, some studies have pointed out that gallic acid can also have pro-oxidant effects, responsible for cell damage with a decrease of SOD [78]. The controversial role of gallic acid seems to be associated with its concentration, which if elevated, determines the oxidation of organic molecules [79]. Likewise, Rutin has an antioxidant power that allows not only to reduce the concentration of ROS but also to improve the physiological properties of spermatozoa. Xu et al. (2020), observed that exposure to rutin determines an increase in the integrity of the membrane and mitochondria that also improved the fertilization yield and blastocyst formation rate [80].    . DNA fragmentation test after exposure to extracts before cryopreservation: a) Halosperm test: 1) untreated sample (CTRL); 2) sample exposed to O. dillenii extracts; 3) sample treated with O. ficus indica cv red; 4) sample exposed to O. ficus indica cv yellow; b) percentage of fragmented DNA: CTRL; OFI cv yellow; OFI cv red; O. dillenii.

Conclusions
In conclusion, our experimental evidence demonstrates how the antioxidants compounds of Opuntia sp. improve the gametic quality and reduce the damage induced by ROS . Therefore, oxidative stress could be considered a fundamental parameter of the semen and marker of sperm quality [81]. Indeed, in recent years, numerous studies have found a correlation between ROS overproduction and idiopathic infertility [82,83]. Exposure to antioxidants has positive consequences on all parameters and improves the spermatozoa performance even after the freeze-thaw cycle. It is necessary the identification of substances for developing a medium that can optimize the sperm quality, namely of men with anomalies and reproductive difficulties.