Numerous antioxidants have been added to sperm extenders to reduce the damage caused by these stresses on spermatozoa and to maintain their quality during freezing, but the desired success has not yet been achieved^2-4.

The temperature changes that occur during the freezing-thawing processes cause physical and chemical stress in mammalian spermatozoa, leading to changes in the lipid composition of the plasma membrane^5,6. It is suggested that the damage occurring during freezing of ram semen is due to peroxidation of membrane lipids induced by increased oxidative stress levels due to ice crystal formation⁷. It has been reported that freezing and thawing causes many damage in ram sperm, including loss of motility, increased rates of dead and abnormal spermatozoa, spermatozoa with damaged plasma and acrosomal membranes, and increased LPO, an indicator of disruptions in the oxidant-antioxidant balance^8,9.

Ganoderma lucidum (GL) is a mushroom widely used in Chinese medicine that contains biologically active compounds, including polysaccharides, phenols, proteins, polysaccharide-protein complexes, lipid components, and terpenoids¹⁰. GL has been reported to have antioxidant^11, and anti-inflammatory effects¹².

GL has also been reported to have a protective effect against disorders associated with the male genital system¹³. Another study reported that GL protects testicular histology, improves sperm quality, enhances antioxidant status, and prevents apoptosis¹⁴. Tutku et al.¹⁵ found that in rats exposed to the endocrine-disrupting chemical bisphenol A, the GL treatment group provided protection against disrupted oxidant balance (p<0.001). Most of these studies indicate that Reishi extract can positively affect sperm quality and production due to its antioxidant capacity.

Scientific research has not yet directly examined the effects of Ganoderma lucidum (Reishi mushroom) on ram semen. There has been no study yet investigating how spermatological values change during freezing and thawing of ram semen and its effects on oxidative stress. However, the effects of the GL mushroom on male reproductive health have been extensively studied, particularly in experimental studies conducted on rats, and the positive results obtained in rats demonstrate its potential benefits for male reproductive health. This study aimed to determine the effects of adding different amounts of Ganoderma lucidum extract to a semen extender on antioxidant properties and cryopreservation quality in ram semen.

Animal Material: Six 2-year-old Akkaraman rams, clinically healthy, with no pathological findings during genital examination, and weighing an average of 60-65 kg, were used. Before and throughout the experimental studies, the rams were fed high-quality forage and concentrates. Drinking water was provided ad libitum. Semen samples were collected from 6 healthy Akkaraman rams by artificial vagina 3 times for two weeks (3 replications).

Experimental Design and Formation of Study Groups: For this purpose, a stock solution (SS) of GL liquid extract (Immunat, herbal, Turkey) was obtained. Stock solutions were diluted again with Tris+egg yolk extender (TYSS) [20 mL egg yolks + 80 mL Tris diluent (Tris hydroxymethylene aminomethane 3.63 g, fructose 0.50 g, citric acid 1.99 g] and in the final solution, GL was adjusted to 0.5%, 1%, 2%, 4%, and 8% before 5°C. Sperm were collected from rams using an artificial vagina. Motility of the sperm brought to the laboratory were determined using a computer-aided sperm analysis (CASA-ISASv1, Proiser, Spain). For the collected sperm to be used in the study, total motility was required to be above 70% and density above 3 billion. Sperm meeting these requirements were diluted 1:1 with previously prepared TYSS. The diluted sperm were pooled. The pooled samples were divided into 2 ml aliquots for each study group and combined with the study groups. In this way, experimental groups were created [Group 1: 0% GL (Control), Group 2: 0.5% GL, Group 3: 1% GL, Group 4: 2% GL, Group 5: 4% GL, and Group 6: 8% GL]. The temperature of the diluted semen was then lowered to 5°C in the cooling cabinet, and the experimental groups were gradually combined with 10% glycerol TYSS containing the same proportions of GL. Following this process, the semen was kept in the cooling cabinet for 4 hours and then drawn into 0.25 mL mini straws. They were frozen in an automatic freezing device (Microdigitcool, IMV, France). Frozen semen was stored in containers containing liquid nitrogen at -196°C. Twenty-four hours after freezing, the straws were thawed in 25 seconds in water at 38°C. Oxidative stress parameter levels were measured in thawed semen using spermatological analyses.

Spermatological Analyses
Motility and Kinematic Parameter Analysis: At this stage, motility and kinematic parameters were determined in sperm using the Computer-Assisted Sperm Analysis (CASA) system. Thawed semen was diluted 1:4 with Tris buffer, and 3 ?L was placed on a special slide (Spermtrack 20 ?m). Total, progressive motility rates (%), kinematic parameter values were recorded through the motility module.

Oxidative Stress Analyses: Frozen ram sperm samples were thawed at room temperature. All samples were then centrifuged at 600 g x 10 minutes x 4°C to remove the semen extender, which was added to increase sperm volume, preserve sperm viability, protect sperm from sudden temperature changes, and prevent microorganisms from damaging the sperm. The cellular pellet was washed three times with distilled water and centrifuged each time to preserve the pellet (600 g x 10 minutes x 4°C). The cell pellet was then separated, weighed, and diluted with distilled water at a 1:10 (weight/volume) ratio. All samples were then homogenized with a Bullet Blender Tissue Homogenizer at speed 10 for 4 minutes. A homogenization bead (0.9-2.0 mm stainless steel bead (SSB14B)) was used in the same ratio as the sample volume. The homogenate was centrifuged at 3000 g for 15 minutes at 4°C to determine malondialdehyde (MDA), glutathione (GSH) levels, and catalase (CAT), glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities, and at 10000 g for 55 minutes to determine glutathione peroxidase (GSH-Px) activity (Hettich Mikro 200R, Tuttlingen, Germany). The supernatant was used for MDA, GSH, CAT, GST, SOD, and GSH-Px analyses^16-21.

Statistical Analyses: Data obtained from the study are presented as the mean ± standard error (SPSS 22 software). Statistical significance was assessed at p<0.05, which was considered significant. The Shapiro-Wilk normality test was performed to determine whether all measured parameters were normally distributed, and the results revealed that all parameters were normally distributed. The homogeneity of the variances was evaluated according to the Levene test, and according to the result of the Levene test, it was concluded that there was homogeneity among the variances. Based on these findings, one-way analysis of variance (ANOVA) was used to examine differences between groups, and post hoc Tukey test was used for pairwise comparisons.

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Table 1: Total and progresive motility (%)

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Table 2: Kinematic parameters

Oxidative Stress Values: Table 3 presents the findings for MDA and GSH levels, along with CAT, GSH-Px, GST, and SOD activities following the freeze?thaw process. In comparison with the control group, MDA concentrations were significantly elevated in the 8% GL group, whereas no meaningful differences were detected in the 0.5%, 1%, 2%, and 4% GL groups. GSH concentrations and CAT activity increased in the 1% and 2% GL groups, decreased in the 8% GL group, and remained unchanged at 0.5% and 4%. GSH-Px activity was enhanced in the 0.5%, 1%, and 2% groups, reduced at 8%, and showed no difference at 4% relative to the control. SOD activity was reduced in the 8% GL group but did not vary in the other treatment groups. GST activity showed no statistically significant variation across any of the groups.

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Table 3: MDA, GSH levels, CAT, GSH-Px, GST, and SOD activities determined after freezing and thawing in the control and experimental groups (Mean ± standard error)

Excessive production of oxidant molecules and a deficiency in the antioxidant defense system disrupt the oxidative balance²³. Oxidative stress causes various cellular and DNA damage, and also damages mitochondrial DNA, which lacks histone wrapping and repair mechanisms. Mitochondrial damage leads to increased oxidative stress and decreased ATP levels, which in turn leads to activation of the cell death pathway²⁴.

The antioxidant properties of GL extract are known, and our study supports this literature. The positive effects observed in spermatology are thought to be due to GL's basic antioxidant properties by reducing free radical production, scavenging superoxide and hydroxyl radicals, and suppressing the production of reactive oxygen species²⁵.

Acısu et al. ¹⁵ investigated the effects of GL on testicular dysfunction in rats exposed to Bisphenol A, an endocrine-disrupting chemical. For this purpose, they administered 25 mg/kg/bw BPA and 300 mg/kg/bw GL to rats via oral gavage for 8 weeks. They found that CAT, GPx activity, and GSH levels were reduced, while the GL treatment group provided protection against disrupted oxidant balance (p<0.001).

Yi et al. ²⁶ investigated the optimal concentration of GLP added to semen cryopreservation extenders at different concentrations after thawing, finding a GLP concentration of 0.8 mg/mL. They reported increased viability in goat sperm, significantly preserved plasma membrane integrity and mitochondrial activity after thawing compared to the control group, and significantly increased antioxidant capacity (p<0.05).

Ozmen et al. ²⁷ in their study investigating the effect of electromagnetic fields (EMF) generated by 10 kV (50 Hz) high-voltage power lines on epididymal sperm characteristics and the protective effects of GL and melatonin in male rats exposed to EMF, evaluated epididymal sperm concentration, motility, and morphology. Their study found that EMF exposure could have different effects on sperm quality depending on the duration of exposure; GL treatment increased sperm concentration and reversed the negative effect of EMF on sperm morphology in rats exposed to EMF for 26 or 52 days; and melatonin treatment had negative effects on sperm concentration and sperm morphology in rats exposed to EMF for 26 or 52 days.

Ghajari et al.²⁸ investigated the protective effect of Ganoderma lucidum on mice exposed to the antidepressant drug sertraline. For 35 days, rats receiving SRT were also given GL extract (at a dosage of 300 mg/kg). Furthermore, sperm parameter assessment using CASA results revealed that sperm volume, motility, and viability were significantly lower in the SRT group (p<0.001). Administration of GL extract to animals receiving SRT may have reduced histological changes. They found that GL significantly reduced lipid peroxidation in spermatozoa. They also determined that GL protected the testes of mice from damage caused by SRT, likely due to its ability to inhibit reactive oxygen species.

The addition of 0.5%, 1%, and 2% GL extract to semen extenders in rams protected spermatozoa against damage during freezing and thawing and improved freezing quality due to its antioxidant properties. Adding GL extract to ram semen extenders at the specified concentrations is recommended. This study determined that GL has a protective role against motility losses caused by freezing and thawing.

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