The inability of low endogenous antioxidant levels in sperm to provide adequate protection against excessive ROS production during the cryopreservation process causes irreversible damage to the sperm (Mazzilli et al. 1995). Therefore, endogenous natural antioxidant compounds are needed to prevent or minimize the oxidative damage of sperm during the cryopreservation process (Yeste 2016). Thanks to its polyphenolic compounds, P content can be effective in protecting reproductive health by preventing or minimizing stressful conditions that may arise from sudden temperature changes in the cell (Hashem et al. 2013). However, it is thought that P may cause toxic effects in high concentrations, as in all honey bee products (Fikri et al. 2019). Evaluating the results of the current study, it can be easily said that antioxidant P doses do not cause any changes in sperm motility properties. On the contrary, it significantly limited the motility capability of semen in the highest dose (P200). Tail abnormalities were significantly reduced in P25 and P50 treatments, while total abnormality values were not decreased in all P doses at the end of the freeze-thawing. Our results showed a gradual decrease in semen abnormalities upon the addition of high levels of P concentration.
There are some studies that examine motility changes by adding bee products and other antioxidant substances in various concentrations to the sperm diluent, and the results of these studies support our findings. Moraes et al. (2014) reported that 1.25 g powdered P added to the rabbit diet did not affect progressive spermatozoa motility and reduced spermatozoa tail abnormalities. Honey added to sperm at different concentrations (2%, 3%, 4%, and 5%) did not show significant differences in sperm motility (El-Sheshtawy et al. 2016). Research results of Amini et al. (2019) revealed that royal jelly added to Tris-egg yolk extender did not affect ram sperm motility after dilution. Royal jelly at various amounts (0, 0.05, 0.1, 0.2, 0.3, and 0.4%) added to bull semen did not show any change in sperm motility properties (Shahzad et al. 2016). Parallel to the conclusions reached here, the research findings of Inanç et al (2019) revealed that green tea extract supplemented to the tris diluent did not change the motility and kinetic parameters in bull semen. Considering the studies that support our results, it is thought that even if there are differences in mammalian species or antioxidant agents, the cause of similar effects on sperm motility may be antioxidant dose choices. In line with the current study, Malik's (2018) study showed that adding the honey solution to bull semen cryoprotective medium at concentrations of 0.1%, 0.2%, 0.3, and 0.4%, had no advantage on sperm motility. In another study, powdered P extract added to ram semen extender decreased semen motility parameters during the 6 h incubation period compared to the 0. h (Mohamed and Zanouny 2017). The fact that the positive effects of the bee products used on motility were not different from the C in their studies. The first is the cryoprotective effect of the substances in the stock solution, the second is the inability of the bee products used to integrate well into the cell membrane phospholipid structure, and the last is the variety of stress factors in in-vitro conditions.
Contrary to our conclusions, in studies conducted in different breeds, some researchers have announced that P causes positive changes in sperm motility (Capucho et al. 2012; Hashem et al. 2013; Gabr 2013; Handayani and Gofur 2019). El-Battawy and Brannas (2015) reported that 10% concentration provides the best motility from P added to mice semen. El-Sherbiny (2015) reported that royal jelly, honey, and P significantly increased the percentage of sperm progressive motility in New Zealand White male rabbits, while the percentages of dead and abnormal sperm increased. Research results reported that royal jelly applied as a sperm protectant caused a significant increase in sperm motility of male mice (Karacal and Aral 2008), hamster sperm cell concentration (Kohguchi et al. 2004), and in sperm count and viable sperm ratios of adult male rats (Hassan 2009). Olayemi et al. (2011) showed that honey added to the egg yolk extender increases the motility of goat semen after cold storage. Fakhrildin and Alsaadi (2014) reported that the addition of 10% honey as a cryoprotectant to the extender showed positive effects on spermatological parameters after thawing in human sperm. We believe that the main reasons for obtaining different motility results in semen cryopreservation studies in different mammalian species may be due to differences in extraction methods, dose differences, active ingredient differences, and freezing times, even if the antioxidant components are the same. In addition, possible harmful effects of the cryopreservation process, including cooling, freezing, and thawing, or the inability of the exogenous antioxidant agent to perform adequate treatment can be argued.
Protection of sperm DNA damage is one of the most important factors affecting fertility capacity (Rathke et al. 2007) and also negatively affects the implantation ability and development of the embryo (Zini and Libman 2010). The lipid peroxidation reactions caused by an excessive increase of semen ROS cause sperm DNA damage, and eventually its loss causes sperm dysfunctions (De Lamirande et al. 1997). Therefore, it is necessary to control ROS for normal acrosomal reactions, sperm capacity, and reproductive physiology. In the current study, antioxidant dose groups P25 and P50 showed positive effects on DNA integrity, while toxic effects were detected at P100 and P200 doses. Following our data, different amounts of cysteine (0.0, 0.5, 1.0 mM) added to the frozen bull semen extender have been reported to have a positive effect on the preservation of DNA integrity (Ansari et al. 2016). Khalifa and Mohamed (2016) have proven that propolis is effective in protecting DNA integrity thanks to its antioxidant and antimicrobial properties by including it in the ram semen medium. Alcay et al. (2017) reported that 0.5 and 0.75% royal jelly administered groups in 6 hours incubation significantly prevented cryodamage by preserving the DNA integrity of buck semen. Caffeic acid, one of the most effective components of P, was found to reduce DNA damage in bull semen at 50 and 100 μM doses after freezing-thawing (Soleimanzadeh et al. 2020). In our previous studies, we found that various plant extracts protect DNA integrity by reducing oxidative damage that may occur after freezing-thawing in bull semen (Avdatek et al. 2018; Taşdemir et al. 2020). Our results, and other studies supporting us, have revealed the positive effect of dose-dependent antioxidant supplements on DNA damage as a result of the synergistic effect with diluents and other substances in the intracellular endogenous antioxidant system. In contrast to the results obtained, Taşdemir et al. (2014) stated that antioxidant substances (fetuin and cysteine) added to the bull semen did not affect DNA integrity. The reason for the conflicting findings with our study is that DNA damage may be related not only to oxidative damage but also to osmotic damage.
Due to the high concentration of unsaturated fatty acids in the spermatozoa membrane, lipid peroxidation chain reactions can be induced. This situation plays a crucial role in the etiology of male infertility (Sharma and Agarwal 1996), causing decreased fluidity of the sperm plasma membrane and functional losses (Aitken 2002). MDA concentration levels are defined as one of the most basic and essential markers of oxidative stress (Ottolenghi et al. 2019). High MDA concentration levels are indicative of the oxidative stress state of semen after thawing.Small molecules such as GSH assist endogenous antioxidants or could eliminate ROS products as co-factors (Ugur et al. 2019). In our study, it is found that MDA levels increased in all P dose applications, with the highest levels in P100 and P200 groups (p<0.001) while GSH levels did not change in any of the P treatments (p>0.05). The reason for the high MDA levels in the treatment groups depending on the dose increase may be the oxidative reactions caused by the unstable molecules in the P content. Our study data revealed a significant increase in sperm TAC levels with P supplementation. The TAC level reached its highest level when P200 was applied (p<0.001). These results showed clearly that P has a stimulating effect by triggering intracellular enzymatic reactions. Possible ROS damages in sperm cells after thawing may probably help to provide oxidative balance by increasing antioxidant defense capacity with P treatment. Studies with contrary findings to us have shown that antioxidants therapeutically applied to the bull semen diluent increase TAC and contribute to fertilization ability (Avdatek et al. 2018; Soleimanzadeh et al. 2020). El-Seadawy et al. (2017) reported that 0.8-2.0 mg of P/5 mL added to Tris extender reduces lipid peroxidation in rabbit semen after chilling for up to 72 hours. Soleimanzadeh et al. (2020) reported that caffeic acid reduced MDA levels at 50 and 100 μM after freezing-thawing in bull semen, increased TAC and GSH activities. In the study of Büyükleblebici et al. (2014), glycerol and ethylene glycol were added at 3, 5, and 7% concentrations. It was reported that 3% ethylene glycole+5 mM cysteine was the combination that reduced MDA levels in bull semen after freezing-thawing. Taşdemir et al. (2020) stated that Pinus brutia Ten extract, which they added as a cryoprotectant to Simmental bull semen, decreased MDA levels and increased GSH activity. Inanç et al. (2019) found that the addition of green tea extract bull semen did not affect MDA levels and showed the highest total antioxidant activity in the 50 μg/mL green tea extract. Avdatek et al. (2018) reported that quercetin therapeutically reduces MDA levels and has positive effects on GSH and TAC levels in bull semen, especially at 25 μg/mL doses. The results of researchers have shown that bee products and other natural antioxidant agents reduce or prevent oxidative damage by having positive effects on some oxidative stress parameters depending on the concentration.
Due to its many positive effects, natural substances have been preferred instead of synthetic cryoprotectants in semen cryopreservation. It has also been shown that the phenolic, flavonoid, and terpenoid components of honey bee products are widely used among natural products. However, high doses of natural products can have adverse effects. Therefore, careful determination of the dose of the antioxidant agent to be used therapeutically is highly critical. As a result, based on all the research conducted in this study, it can be clearly said that P25 and P50 doses are effective in maintaining some biochemical and physiological properties of bull sperm, while P200 doses cause toxic effects.