In the presented study, the effects of naringin added to diluent at doses of 1, 2 and 4 mM and diosmin at doses of 2 and 4 mM on motility, membrane integrity, acrosome integrity and mitochondrial activity rates were investigated using fluorescent staining techniques regarding to post-freeze-thawing ram spermatological parameters. In addition, sperm membrane lipid profile analysis was performed with the High Performance Layer Chromatography (HPLC) method and sperm membrane protein profiles analysis with SDS-PAGE method were performed. In terms of immunofluorescence, 8-OhDG antibody was used for DNA damage, and Cu - Zn SOD antibody was used for cell membrane integrity.
Motility, which is one of the success criteria of spermatozoa, is expressed as the ratio of all spermatozoon moving in any direction(Hafez, 1987; Januskauskas et al., 1996). Motility is one of the markers of viability and structural integrity of spermatozoa (Kathiravan et al., 2011). Motility is controlled from the flagella and main part. While the flagellar part provides motility, the main part is responsible for hyperactivation. [34–36]. In our study, a statistical difference was found between the D4 group and the control group in terms of motility findings (p < 0,05). No difference was observed between the other groups. Similarly, it has been stated that 200 nM to 5 µM fullerene added to ram semen increases the total and progressive motility value (Turk et al., 2022). In another study, gallic acid and carnosic acid added to ram semen were found to have the highest total motility value in gallic acid (Gungor et al., 2019). These differences are thought to be due to the antioxidants added and the doses used.
Ram spermatozoon is extremely sensitive to lipid peroxidation caused by reactive oxygen species, as its plasma membrane is rich in unsaturated fatty acids. Freezing of semen causes cold shock that develops during the freezing process, damage due to phase change in membrane structures and oxidative stress. Developing oxidative stress and cytotoxic aldehydes (malondialdehyde, etc.) that develop cause damage to spermatozoon functions . During the freezing of sperm cells, the formation of reactive oxygen species (ROS) is observed as a result of oxidative stress. These oxidative changes cause changes in sperm integrity and behavior. As a result, lipid, protein, DNA degradation, cell death, and ultimately a decrease in fertilization occur [61–65]. For this reason, the cold shock damage that develops in the environment can be minimized with some additives with cryoprotective and antioxidative properties, which are added to semen extender. In addition, antioxidants such as GPX, CAT and SOD that neutralize these species such as produced O2 and H2O2 are found in both the mitochondria and the secretions of the reproductive system [30, 31]. The fact that antioxidant compounds also have cryoprotectant properties provides better results from semen frozen with these substances [8, 32, 33]. Superoxide dismutase (SOD), which is in the family of metalloproteins, has two forms: Cu-Zn SOD, which is cytosolic, and Mn SOD, which is mitochondrial. SOD is common in mammalian tissues and has a significant protective effect against the harmful effects of superoxide anions [67, 68]. In the present study, Cu Zn SOD positivity was observed less frequently in the control group, while it was observed more intensely in the experimental groups, and it was thought that the applied antioxidants activate the protective mechanism in the sperm cells. In a study, 5 mM methionine, 5 mM cysteamine and 1 mM cysteine were added to ram semen and a lower MDA level was observed compared to the control group (Alçay et al., 2017). Similarly, in our study, the Cu-Zn SOD level, which is a sign of oxidative stress, was found to be decreased in the antioxidant added groups. This suggests that it is necessary to add antioxidants to semen extenders in rams. In their study with naringin, Akondi et al.(2011) applied naringin to rats with diabetes, and it was observed that sperm parameters improved and oxidative stress decreased compared to the r diabetes group. This situation is similar to the low level of Cu-Zn SOD seen in naringin groups, and it is thought to be due to the antioxidant property of naringin.
The plasma membrane envelops the spermatozoon and holds the organelles and intracellular contents, and with its semi-permeable feature, it allows the passage of some soluble substances and ions. Some plasma membrane proteins facilitate the transition of glucose and fructose from the extracellular environment into the spermatozoon [37–39]. In cases where the sperm plasma membrane is damaged, spermatozoon are considered dead and lose the ability to fertilize in vivo [40–42]. Therefore, it is essential to determine the plasma membrane integrity before the use of semen for assisted reproduction techniques such as in vitro fertilization (IVF) and artificial insemination (AI) . In our study, plasma membrane integrity was found to be lower in N1 and N2 groups compared to other groups (p < 0.05). However, plasma membrane integrity was found to be better in N4, D2 and D4 groups compared to other groups (p < 0.05). Güngör et al., (2019) in their study, observed that carnosic acid's plasma membrane and acrosome integrity were better in ram semen compared to other groups. In the same study, it was determined that gallic acid did not sufficiently protect the integrity of the plasma membrane and acrosome. In a study determining the effect of 7-dehydrocholesterol in ram semen, it was observed that it provided acrosome integrity compared to the control group (Inanç et al.,2018). This situation is similar to our study. It was observed that plasma membrane integrity from N1 and N2 groups was lower compared to N4 group. It can be interpreted that the dose increase preserves the integrity of the plasma membrane in the naringin groups. When the acrosome integrity was examined, it was observed that there was a statistical difference between the D4 group and the other experimental groups (p < 0.05). Diosmin groups were observed to preserve acrosome integrity compared to naringin groups. The dose used in the D4 group was thought to be more effective in providing acrosomal integrity in ram semen.
Mitochondria in the spermatozoon are localized in the middle part above the main part of the flagellum. Mitochondria produce ATP through oxidative phosphorylation . Mitochondria are located in the neck of the spermatozoon, and the mitochondria in the sperm are morphologically and biochemically different from somatic cell organelles. These differences are related to the finding of specific enzymes in organelles. Spermatozoa use different energy sources and thus their metabolic and physicochemical states change . It is important to evaluate spermatozoon before fertilization process with assisted reproduction techniques in terms of mitochondrial functional integrity. If a sperm cell has a functional mitochondria, after staining with a fluorescent dye, the entire middle part is stained; if it does not have a functional mitochondria, none of the middle part is stained . It has been reported that antioxidant substances used in ram semen increase the mitochondrial membrane potential (Güngör et al., 2019). In our study, N4, D2 and D4 groups were found to have higher mitochondrial activity (p < 0,05). However, no difference was observed in the N1 and N2 groups compared to the other groups. In this case, it can be thought that diosmin is an exogenous energy source for spermatozoa. However, low-dose naringin appears to be ineffective. In a similar study with different antioxidants (methionine, curcumin, ellagic acid; 1, 2, 4 mM; each), the antioxidant-containing groups showed higher motility value and acrosome integrity ratio than the control group in ram semen. The doses of methionine (1 mM), curcumin (1, 2 mM) and ellagic acid (1, 2 mM) gave a higher rate of sperm plasma membrane integrity than the control group. In addition, in terms of mitochondrial activity, it was determined that 1 mM doses of antioxidant groups provided higher protection compared to other groups .
Lipids are important components of the cell membrane. Membrane lipid composition has been associated with the different functions of spermatozoa. Many researchers associate membrane lipids with the survival success of sperm cells after cryopreservation or cold shock. Because while sperm cell maturation or acrosome reaction is a natural phenomenon, cryopreservation or cold shock is not. Sperm cells are unnatural for such an alteration and are not programmed in such a situation and are directly under stress. Therefore, the change in membrane lipid composition due to cold shock or cryopreservation can be seen as the response of sperm cells to a certain stressful situation . The greatest damage during cryopreservation is the peroxidation of lipids, especially phospholipid-bound polyunsaturated fatty acids (PUFAs) [52, 53]. Increased free cholesterol, free fatty acids, triacylglycerol and cholesterol ester concentrations cause negative effects on sperm functions . The biggest problem associated with the cryopreservation of sperm cells is the loss of viability as a result of freezing- thawing process [55–57]. Loss of viability is associated with membrane damage induced by peroxidation of sperm phospholipids [58, 59]. In the study, a statistical difference was observed in CholE, TAG, FFA, CHOL, MAG, PL levels compared to the control group. This can be interpreted as naringin and diosmin change the lipid composition of semen and make it resistant to oxidative stress. However, there was no difference between the groups in terms of DAG level.
Cryopreservation changes sperm protein composition and sperm quality. In a study, they observed that the protein content of sperm membrane and seminal fluid changed in the freezing process of semen taken from Murrah buffaloes . In our study, the highest protein amounts were obtained from the N4 and D4 groups. However, sperm total protein amounts of all groups with antioxidant added were higher than the control group (Table 3). When the ratios of 30 individual proteins in the total protein content were examined to control why this situation occurred, it was observed that each antioxidant prevented oxidative protein degradation during cryopreservation by exerting different levels of protective effect on different proteins (Table 3).
It has been reported that 8-OhDG is a marker showing DNA degradation in sperm . In the present study, the detection of 8-OhDG positivity in the control group and the other experimental groups in immunofluorescence staining at a similar and very low level was interpreted as no statistically significant DNA damage occurred in the sperm cells during the dilution and freezing of the semen samples.
In conclusion in order to increase the fertility ability of frozen semen in rams, some antioxidant substances are added to the extenders. Although they have positive effects in general, the effectiveness of antioxidants varies according to animal species, breed, season, diluent components and freezing protocols. In addition, the findings obtained in spermatological parameters after freezing-thawing may also vary depending on the techniques used in reconstitution and freezing of semen, changes in solution time and temperature, or the person performing the analysis. In our presented project study, it was observed that the protective effect of diosmin on plasma membrane integrity (2. 4 mM), acrosome integrity (4 mM), lipid profile (4 mM) and protein profile (4 mM) came to the fore. It was observed that naringin, applied at a dose of 1 mM, increased mitochondrial activity compared to the control group. It is thought that it would be meaningful to support in vitro examination parameters with in vitro/vivo fertility parameters in further studies to be carried out.