Optimizing Cryopreservation: The Effect of MitoTEMPO and Glycerol Ratios on Ram Semen Quality After Freezing and Thawing

Abstract

ABSTRACT

Background

MitoTEMPO is an important mitochondrial targeted and ROS scavenging antioxidant. It is often used as antiviral, anticancer and immunomodulator agents. In addition, there are studies proving its positive effect on sperm after thawing.

Objectives

The aim of our study was to determine the effects of mitoTEMPO addition, which has strong antioxidant and cryoprotective properties in different glycerol ratios, on motility, kinematics, sperm quality and oxidative stress in spermatozoa after freezing and thawing.

Materials and Methods

Semen was collected from seven rams twice a week for 3 weeks. The semen samples were pooled and extended using standard protocol. Experimental groups were formed (1 µM mitoTEMPO with 3% glycerol, 1 µM mitoTEMPO with 5% glycerol, 5 µM mitoTEMPO with 3% glycerol and 5 µM mitoTEMPO with 5% glycerol) and without (control with 3% glycerol and control with 5% glycerol) mitoTEMPO and they were frozen in mini straws. In this study, motility, kinematic parameters, plasma membrane integrity, mitochondrial membrane potential, acrosome integrity, viability, DNA fragmentation and oxidative stress parameters were determined.

Results

The increases in total motility, increases in plasma membrane integrity ratio, decreases in MDA level and decreases in DNA damage ratio in mitoTEMPO groups were statistically significant (p < 0.05).

Conclusion

Post‐thawed sperm, the addition of 1 µM mitoTEMPO to the 3% glycerol extender preserved the integrity of the spermatozoon membrane, increased total motility, prevented DNA damage and oxidative stress, and increased the rate of sperm with intact acrosome.

Introduction

Damages occurring during the cryopreservation of ram semen are attributed to the peroxidation of membrane lipids caused by increased oxidative stress due to ice crystal formation (Grötter et al. 2019; Kameni et al. 2021). The fact that the ram sperm membrane is rich in polyunsaturated fatty acids (PUFA) increases oxidative stress. Increased oxidative stress leads to the peroxidation of both lipids and proteins within the cell (Özer Kaya et al. 2021; Surai et al. 2001) and deoxyribonucleic acid (DNA) (Barzilai and Yamamoto 2004; Hamilton et al. 2018). Therefore, increased lipid peroxidation (LPO) has been associated with decreased motility, increased morphological disorders, increased acrosomal damage, impaired membrane integrity, increased proportion of dead sperm, decreased mitochondrial activity and increased DNA damage (Hassan et al. 2024; Khalil et al. 2024). There are studies that include many antioxidant substances added to ram semen extender to prevent damage to spermatozoon and maintain its long-term viability (Khalil et al. 2023; Shehab-El-Deen et al. 2022). Antioxidants have the ability to protect spermatozoons against reactive oxygen species (ROS) attack and LPO damage. Increased ROS levels during long-term storage can be potently eliminated by enzymatic and non-enzymatic antioxidant additives (Khalil et al. 2023; Silvestre et al. 2021).

Ram spermatozoa are equipped with a powerful antioxidant enzyme system in the form of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) to scavenge ROS. However, the endogenous antioxidant system is often unable to scavenge excess ROS produced from dead spermatozoon, leading to ROS production resulting in oxidative stress. To prevent ROS production, the addition of a suitable antioxidant to the semen extender may be beneficial in reducing oxidative stress. Thus, spermatozoa are protected during freezing and thawing (Türk 2015; Liu et al. 2020). In recent years, fatty acids, seminal plasma, sugars and various substances with antioxidant potential have been added to the base extender to reduce ROS production in semen and improve sperm quality during cryopreservation. In addition, supplementation of non-enzymatic antioxidants, also known as synthetic antioxidants, including ascorbic acid, carotenoids, GSH, amino acids, ubiquinones and vitamins, has been shown to benefit semen cryopreservation applications (Allai et al. 2018). Thus, even a small amount of antioxidants improves sperm functions during semen storage (Dikalova et al. 2010).

Permeating and non-permeating cryoprotective are utilized in cryobiology to shield gamete cells against osmotic, chemical and ice crystal formation stress. Preventing water molecules from turning into huge volume ice crystals during the freezing process is one of glycerol's most significant effects. In addition, because of the interaction between membrane proteins and glycoproteins (Gilmore et al. 1995), it can adhere to the membrane of cells and organelles, decreasing membrane mobility and causing a particulate clump to form into the membrane structure (Bucak et al. 2022). Furthermore, it has certain adverse effects by changing some proteins in spermatozoa, which impairs sperm function, and it is regrettably cytotoxic at concentrations beyond a certain threshold. The best course of action is to combine non-permeating cryoprotectants like trehalose with a low glycerol rate in an effort to resolve the issue (Bucak et al. 2022).

MitoTEMPO is a physicochemical compound that is one of the SOD mimics. It has the ability to selectively accumulate in mitochondria (Dikalova et al. 2010). Studies have shown mitoTEMPO to be a mitochondria-targeted antioxidant with superoxide and alkyl radical scavenging properties (Dikalova et al. 2010; Liang et al. 2010; Liu et al. 2010). MitoTEMPO can accumulate a thousandfold in the mitochondrial matrix due to its positive charge (Trnka et al. 2009). MitoTEMPO consists of a piperidine nitroxide TEMPOL (2-(2.2.6.6-tetramethylpiperidine-1-oxyl-4-ylamino)-2 oxoethyl) unit and a lipophilic triphenylphosphonium (triphenylphosphonium chloride) moiety (Jiang et al. 2015; Shetty et al. 2019). There are studies emphasizing that the addition of mitoTEMPO to sperm diluents has a positive effect on sperm quality in rabbits, pigs, goats, rams, bulls and even humans as an antioxidant (Kuželová et al. 2024; Cho et al. 2020; Barfourooshi et al. 2023; Elkhawagah et al. 2024; Lu et al. 2018).

In this study, the aim was to determine the effect of adding mitoTEMPO to the extender in vitro at different doses in rams on the quality of semen after thawing. This was done by assessing changes in motility, kinematics, plasma membrane integrity, oxidative stress, viability, mitochondrial membrane integrity, DNA damage and acrosomal membrane integrity parameters during long-term storage of semen. It was also aimed to see the effect of mitoTEMPO at a low glycerol ratio.

Materials and Methods

Animals and Semen Collection

As animal material in the study, six Akkaraman breed rams, 2 years old and clinically healthy with a live weight of 55–60 kg, were used. Sperm was collected with an artificial vagina (temperature: 42°C). Semen with at least 70% motility and sperm density over 2 billion/mL were pooled to be used in the study. The study was carried out during the breeding season (October–November). In the study, semen was collected twice a week for 3 weeks.

Dilution, Cooling and Freezing

Tris + egg yolk extender (tris (hydroxymethyl) aminomethane 0.3 M + glucose 0.027 M + citric acid 0.1 M + 15% egg yolk + distilled water 100 mL) was prepared and used as a diluent in semen storage.

After using this diluent for pre-dilution, diluted sperm with final glycerol concentrations of 3% and 5% were obtained by diluting with Tris + egg yolk diluent containing 6% and 10% glycerol at a 1:1 ratio at 5°C.

In the study, mitoTEMPO (Sigma-Aldrich, St. Louis, MO, USA; Cas no.: 1334850-99-5) semen extender groups were 0 µM mitoTEMPO + 3% glycerol (C–3G%), 0 µM mitoTEMPO + 5% glycerol (C–5G%), 1 µM mitoTEMPO + 3% glycerol (1 µM–3G%), 1 µM mitoTEMPO + 5% glycerol (1 µM–5G%), 5 µM mitoTEMPO + 3% glycerol (5 µM–3G%) and 5 µM mitoTEMPO + 5% glycerol (5 µM–5G%).

After the experimental groups were formed, they were gradually chilled from 38°C to 5°C. Following glycerol addition and equilibration, the straws were frozen in liquid nitrogen vapour using an automatic semen freezing tool (Microdigitcool, IMV, France) and kept at −196°C until analysis. Frozen sperm samples were thawed in a water bath at 38°C for 25 s after storage (Türk et al. 2022). Sperm motility, kinematic parameters, antioxidant parameters, flow cytometry viability rate, mitochondrial activity, acrosomal integrity and DNA damage of the semen in the straws were examined.

Spermatological Analyses

CASA Analysis

Motility determination was made with CASA (ISASv1, Proiser, Spain). For analysis, 3.5 µL of the samples extra diluted with Tris buffer was placed on a special slide (Spermtrack 20) designed for the device and examined with a phase-contrast microscope (Nikon Eclipse Ci, Tokyo, Japan) connected to the CASA system. At least five microscopic areas were examined using spermatozoon speed, immobile < 15 µm/s < slow < 50 µm/s < medium < 80 µm/s < fast (Özer Kaya et al. 2021).

Plasma Membrane Integrity

Post-thaw semen was rediluted ratio of 1:5 at 38°C with Tris buffer solution. Then, 50 µL of the semen samples diluted with Tris were taken and mixed with 500 µL of hypotonic solution and incubated at 38°C for 60 min. After incubation, 200 spermatozoa were counted at 400× magnification of the phase-contrast microscope (Nikon Eclipse Ci, Tokyo, Japan). The proportion of intact spermatozoa with swollen and curled tails was expressed as a percentage (Söderquist et al. 1997).

Flow Cytometry

Mitochondrial Membrane Potential

To determine the mitochondrial membrane potential in frozen-thawed semen, JC-1/PI (Invitrogen, T3168, Canada) was used. In the staining procedure performed in a dark environment at 38°C, 985 µL PBS was added to the Eppendorf tube. 10 µL of sperm (to have 1 × 10⁶ per mL spermatozoon in the final volume) was added and 2.5 µL of PI and 2.5 µL of JC-1 dye were added. The tube was vortexed and incubated in the dark at 38°C for at least 60 min. It was analysed with a flow cytometry device (Cytoflex, Beckman Coulter, Fullerton, USA). Then, the analysis results were given as percentages of high and low mitochondrial membrane potential (Inanc et al. 2018).

Viability

A commercial kit, sperm viability (SYBR-14/ PI, Molecular Probe: L7011 Invitrogen, Canada), was used to analyse frozen-thawed semen with a flow cytometry device. For dead spermatozoon analysis, all procedures were carried out in a dark environment in an oven at 38°C. A total of 980 µL of PBS and 10 µL of sperm were added to the Eppendorf tube (so that there would be 1 × 10⁶ per mL spermatozoon in the final volume). Then, 5 µL PI and 5 µL SYBR-14 dyes were added. The tube was vortexed and incubated in the dark at 38°C for at least 60 min. Analysis was performed with a flow cytometry device. Live sperm rate expressed as a percentage (Alvarez et al. 2012).

Acrosome Integrity

A commercial kit, lectin-PNA (peanut agglutinin) (Alexa Fluor 488; PNA-Alexa 488, L21409, Invitrogen, England), was used to analyse frozen-thawed semen with a flow cytometry device. For acrosomal integrity analysis, 38°C. All procedures were carried out in an incubator in the dark. 982 µL of PBS and 10 µL of sperm (1 × 10⁶ per mL spermatozoa in the final volume) were added to the Eppendorf tube. Then, 3 µL of PI and 5 µL of lectin-PNA dye were added. The tube was vortexed. Then, it was incubated for 60 min at 38°C. The analysis was performed with a flow cytometry device. Results expressed as a percentage (Standerholen et al. 2014).

DNA Fragmentation

Spermatozoon DNA damage TUNEL method was used, and analysis was performed with the commercial kit for this method, Apodirect (ABCAM ab66108, Cambridge, England). Briefly, the negative (N) and positive (P) controls included in the kit package were brought to room temperature. Two millilitres of negative and positive controls were divided into Eppendorf tubes, and to make sperm (S) tubes, 50 µL of semen was added to 950 µL of PBS and placed in Eppendorf tubes. N, P and S tubes were centrifuged at 300 × g for 5 min. The supernatant was discarded, and 0.5 mL of PBS was added to each tube. It was adjusted to 1% paraformaldehyde in 5 mL PBS per sample. 0.5 mL of PBS was added to the sperm tube and placed in a falcon tube. The final solution, paraformaldehyde semen, was incubated on ice in the dark for 15 min. After incubation, the supernatant was discarded by centrifugation at 300 × g for 5 min. Five millilitres of PBS was added again, and the washing process was repeated two times. Then, it was centrifuged at 300 × g for 5 min, the supernatant was discarded, and 0.5 mL PBS was added. Thereupon, 5 mL of 70% ethanol cooled on ice was added and kept on ice for 30 min. This mixture was divided into 1 mL tubes and centrifuged at 300 × g for 5 min. Afterwards, the ethanol supernatant was discarded. One millilitre wash buffer was added and centrifuged at 300 × g for 5 min. The supernatant was discarded. Once again, 1 mL wash buffer was added and centrifuged at 300 × g for 5 min. The supernatant was discarded again. Fifty-one millilitres of staining solution included in the kit package was prepared. This prepared solution was incubated for 60 min at 38°C in a dark environment to combine with the cells. Shaked every 15 min. One millilitre of Rinse Buffer included in the kit was added and centrifuged at 300 × g for 5 min and the supernatant was discarded. One millilitre of rinse buffer was added again, centrifuged at 300 × g for 5 min, and the supernatant was discarded. PI/RNase A included in the kit was added and incubated with P and N tubes at room temperature for 30 min. Analysed in flow cytometry within 3 h (Sharma et al. 2016).

Oxidative Stress Analysis

Semen samples were stored at −80°C until analysis. Before analyses, sperm samples were centrifuged at 600 × g and 4°C for 10 min and then the supernatant was removed. After weighing the cellular pellet, it was diluted 1:10 with distilled water and homogenized with the TissueLyser homogenization device. The resulting homogenates were centrifuged in a refrigerated centrifuge for 15 min at 1200 × g and 4°C for MDA, GSH, CAT, GSH-Px and glutathione S-transferase analyses, and the supernatants were used in the analyses.

MDA Analysis

MDA is known as the end product of LPO in cells. Its presence is indicative of increased ROS (Çelikezen and Ertekin 2008; Ersayit et al. 2009). MDA determination was made according to the method modified by Placer et al. (1966). It is based on the reaction of MDA, one of the aldehyde products of LPO and TBA. The resulting MDA forms a pink complex with TBA, and the level of LPO was determined by measuring the absorbance of this solution spectrophotometrically at 532 nm. 0.25 mL of the sample, 0.25 mL of 1,1,3,3-tetraethoxypropane and 0.25 mL of physiological saline are added to the colour reagent containing 2.25 mL of TBA, incubated at 37°C for 30 min and the turps are mixed. Then, its temperature is increased to 100°C and cooled under cold water. Then, it was centrifuged at 3000 rpm for 10 min and the upper phase absorbance was read at 532 nm (Placer et al. 1966).

GSH Analyses

GSH determination was made using the method reported by Ellman et al. (1961). This method was measured by an enzymatic cycling procedure in the presence of DTNB, NADPH and GR, based on the fact that sulfhydryl groups form a very stable yellow colour when DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)], which is a spectrophotometric method, is added. Take 1.5 mL of the precipitant solution (1.67 g glacial metaphosphoric acid, 0.20 g disodium EDTA, 30 g NaCl, weigh 100 mL distilled water), add 1 mL distilled water, add 1 mL standard solution to the second precipitant solution and 1 mL sample to the third precipitant solution is added and centrifuged. The filtrate is made, and 0.25 mL is taken. Then, 42.60 g Na₂HPO₄ was weighed on each of them, and 1 mL of the solution was diluted to 1 L, and 0.125 mL of the solution was diluted to 100 mL with 20 mg DTNB 1% Na-citrate solution and the filtrate was used in the experimental environment. After the test tubes were prepared, they were vortexed and absorbances were read against the blank at 412 nm (Ellman et al. 1961).

CAT Analysis

Briefly, for the determination of CAT activity, 2 mL of the prepared samples was taken and 1 mL of substrate (65 µmol/L H₂O₂ in 50 mmol/L PBS, pH 7.0) was added and incubated at 37°C for 60 s. On the other hand, for the blank tube, 1 mL of phosphate buffer was added to 2 mL of the sample, and after setting zero with the blank at 240 nm, CAT activity was calculated by measuring the absorbance difference of the sample at 0 and 30 s (Aebi 1984).

GSH-Px Analysis

For the determination of GSH-Px activity, 10 µL of the sample, 670 µL of distilled water, 100 µL of Tris buffer, 20 of GSH and 100 µL of GR were taken for the blank tube. For the sample tube, 10 µL of the sample, 660 µL of distilled water, 100 µL of Tris buffer, 20 µL of GSH and 100 µL of GR were taken. After incubating at 37°C for 10 min, 10 µL of t-BOOH was taken onto the sample tube and the decrease in the adsorbance of the mixture at 0 and 2.5 min was determined at 340 nm (Lawrence and Burk 1976).

GST Analysis

According to the method of Habig et al. (1974), which is based on measuring the absorbance of the product [1-(S-glutathionyl)-2,4-dinitrobenzene)] resulting from the conjugation of GST activity, GSH and 1,2-dichloro-4-nitrobenzene (CDNB) at a wavelength of 340 nm, in a spectrophotometer was carried out. 0.1 mL of CDNB, GSH and sample was taken, and 2.2 mL of Tris buffer was added and measured on a spectrophotometer (Habig et al. 1974).

Protein Analysis

For protein analyses, 1 mL of alkaline copper reagent was placed in three tubes, the sample was added to one, the standard solution to one, and 1 mL of distilled water was added to the other; all tubes were mixed and incubated for 10 min at room temperature. Then, 4 mL of phenol reagent was added, and the tubes were immediately mixed thoroughly in the vortex and kept at 55°C for 5 min. After incubation, it was immediately cooled under tap water. Then, the absorbance of the sample tubes was read against the blank tube at 650 nm (Lowry et al. 1951).

Statistical Analysis

All statistical analyses were performed using SPSS (Version 22.0, Inc., Chicago, USA). Values are presented as mean ± standard error (SEM). In the study, non-parametric Kruskal–Wallis analysis of variance was used to determine the differences between the control group and mitoTEMPO-supplemented semen samples, and the non-parametric Mann–Whitney U test was used for pairwise comparisons. A value of p < 0.05 was considered statistically significant.

Results

CASA Analysis

The average motility and other spermatozoon kinematic values in the control group and mitoTEMPO-containing groups after freezing and thawing the sperm are shown in Table 1. When total motilities were examined, the increase in the trial groups containing 1 µM–3G% glycerol, 1 µM–5G% and 5 µM–3G% was found to be statistically significant compared to the control groups containing 3G% and 5G% (p < 0.05). When the kinematic parameters VCL, VSL and VAP values were examined, it was determined that the difference between the control groups containing 3G% and 5G% glycerol and the group containing 1 µM–3G% glycerol was statistically significant (p < 0.05). Also, 5 µM–5G% was lower than the other mitoTEMPO groups (p < 0.05). VCL value at 1 µM–3G% was higher than in other mitoTEMPO groups.

TABLE 1 Motilities and kinematic parameters in control and mitoTEMPO containing semen after freezing and thawing.

	C–3G%	C–5G%	1 µM–3G%	1 µM–5G%	5 µM–3G%	5 µM–5G%
TM	34.89 ± 3.76a	34.31 ± 5.41a	45.33 ± 4.61b	44.71 ± 5.55b	40.21 ± 4.51b	38.45 ± 5.67a
PM	18.30 ± 3.48	16.75 ± 2.89	22.41 ± 2.01	22.08 ± 2.90	18.72 ± 3.05	17.65 ± 3.80
VCL	104.31 ± 6.41a	109.55 ± 10.48a	131.61 ± 9.84b	116.28 ± 12.3a	107.21 ± 12.13a	109.95 ± 12.51a
VSL	66.16 ± 10.04a	70.85 ± 9.74a	90.08 ± 4.10b	82.31 ± 7.83a	72.48 ± 10.57a	69.61 ± 9.94a
VAP	77.05 ± 7.76a	83.65 ± 11.36a	109.13 ± 9.61b	95.51 ± 10.85a	85.25 ± 12.59a	86.83 ± 12.82a
LIN	62.65 ± 7.43	63.43 ± 4.37	69.4 ± 3.35	71.46 ± 3.25	65.83 ± 4.81	62.51 ± 5.68
STR	84.26 ± 6.33	84.76 ± 2.56	84.33 ± 4.90	87.23 ± 3.35	85.2 ± 3.84	81.7 ± 5.81
WOB	73.40 ± 4.47	74.71 ± 4.22	82.56 ± 1.31	81.91 ± 1.68	77.33 ± 4.63	76.68 ± 4.34
ALH	3.21 ± 0.27	3.20 ± 0.10	3.18 ± 0.07	2.91 ± 0.18	2.81 ± 0.23	2.85 ± 0.21
BCF	9.55 ± 0.21	9.23 ± 0.48	9.11 ± 0.31	9.08 ± 0.29	8.65 ± 0.84	8.50 ± 0.74

Plasma Membrane Integrity

HOS test values of the semen after freezing and thawing in the control and groups containing different doses of mitoTEMPO are given in Figure 1. Compared to the control groups, the increase in the trial groups containing 1 µM–3G%, 1 µM–5G% and 5 µM–3G% is statistically significant (p < 0.05).

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Acrosomal Integrity

Acrosomal integrity rates in the control and mitoTEMPO groups after freezing and thawing are given in Table 2. The spermatozoa with damaged acrosomes and dead (PNA+ PI+) were higher in the control group containing 5% glycerol than in the other groups, and the difference between the groups was found to be statistically significant (p < 0.05). The decrease in acrosome-damaged and live (PNA+ PI−) spermatozoa in the mitoTEMPO groups compared to the control groups was found to be statistically significant (p < 0.05).

TABLE 2 Acrosomal integrity parameters in control and mitoTEMPO containing semen after freezing and thawing (%).

	C–3G%	C–5G%	1 µM–3G%	1 µM–5G%	5 µM–3G%	5 µM–5G%
PNA+ PI+	50.03 ± 2.75a	64.89 ± 5.41b	47.47 ± 3.34a	50.55 ± 6.07a	49.75 ± 6.07a	54.56 ± 1.12a
PNA− PI+	21.18 ± 3.61a	9.41 ± 1.99b	19.79 ± 2.32c	22.91 ± 7.32ac	25.67 ± 7.76ac	14.29 ± 2.49abc
PNA− PI−	28.68 ± 2.03	25.51 ± 3.99	32.52 ± 4.48	26.45 ± 4.82	24.48 ± 2.26	31.05 ± 2.63
PNA+ PI−	0.12 ± 0.04a	0.17 ± 0.08a	0.05 ± 0.00b	0.06 ± 0.02b	0.06 ± 0.01b	0.07 ± 0.02b

DNA Fragmentation

Figure 2 shows the DNA damage rate in control groups and groups containing different doses of mitoTEMPO after freezing and thawing. The difference between the control groups containing 3% and 5% glycerol in DNA damage values and the decrease in the DNA damage rate in the experimental groups compared to the control groups were found to be statistically significant (p < 0.05). In addition, the difference between the other trial groups, except the 1 µM–3G% trial group and the 5 µM–5G% trial group, is statistically significant (p < 0.05).

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Mitochondrial Membrane Potential

Mitochondrial membrane potential findings of sperm after freezing and thawing are given in Figure 3. The difference between the groups in terms of both high mitochondrial membrane potential and low mitochondrial membrane potential was found to be statistically insignificant (p > 0.05).

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Viability

Viability rate of sperm after freezing and thawing are given in Figure 4. There were no statistical differences between all groups (p > 0.05).

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Oxidative Stress Parameters

Oxidative stress parameters in control and mitoTEMPO groups after freezing and thawing are given in Table 3. While there was no statistical difference between the control groups containing different amounts of glycerol in MDA values, a statistical difference was detected between the control groups and the experimental groups containing different doses of mitoTEMPO and different amounts of glycerol, and the decrease in the mitoTEMPO groups was found to be significant (p < 0.05). In terms of GSH values, the increase in the control group containing 5G% and the 1 µM–5G% and 5 µM–5G% trial groups were found to be statistically significant (p < 0.05). The increase in GSH-Px values in the group containing 5 µM–3G% compared to the control group containing 3G% is statistically significant (p < 0.05).

TABLE 3 Oxidative stress parameters in control and mitoTEMPO containing semen after freezing and thawing.

	MDA (nmol/g)	GSH (µmol/mL)	CAT (k/g prot.)	GSH-Px (U/g prot.)	GST (U/mg prot.)
C–3G%	0.47 ± 0.01a	5.94 ± 0.07ab	14.40 ± 0.81	89.50 ± 4.53a	20.13 ± 0.63ab
C–5G%	0.48 ± 0.01a	5.82 ± 0.08b	12.74 ± 0.66	95.08 ± 4.14ab	20.71 ± 0.82ab
1 µM–3G%	0.38 ± 0.01b	6.12 ± 0.08ac	15.16 ± 1.52	100.51 ± 7.10a	18.54 ± 0.66a
1 µM–5G%	0.38 ± 0.01b	6.48 ± 0.12d	14.69 ± 0.93	112.71 ± 8.15b	19.32 ± 0.82a
5 µM–3G%	0.41 ± 0.01b	6.33 ± 0.16acd	13.60 ± 0.98	116.12 ± 7.79b	21.08 ± 1.20ab
5 µM–5G%	0.41 ± 0.01b	6.24 ± 0.06cd	13.89 ± 1.69	102.97 ± 3.86b	22.28 ± 0.99b

Discussion

In our previous study, only these two doses were selected in the diluting and freezing phase of semen because the doses of mitoTEMPO between 0.1 and 200 µM doses of 1 and 5 µM doses increased the antioxidant effect and had statistically the highest values in total motility, progressive motility and plasma membrane integrity ratio up to 96 h after short-term storage (Koca et al. 2024).

Lu et al. (2018) stated that they determined motility with CASA after freezing and thawing human semen and that 5 and 50 µM mitoTEMPO doses improved total motility and progressive motility after freezing and thawing. Zhang et al. (2019) in their study investigating whether mitoTEMPO was effective in preventing the damage that occurred during the freezing of spermatozoa taken from people with asthenospermia, they stated that 10 µM dose was more effective in total and progressive motility. Cho et al. (2020) stated that sperm motility was higher at the 5 µM dose of mitoTEMPO in pig semen and the increase was found to be statistically significant (p < 0.05). Asadzadeh et al. (2021) stated in their study on rams that the doses of 5 and 50 µM mitoTEMPO added to the semen extender containing soybean lecithin + Tris were higher than the control group and other groups in both total motility and progressive motility, and the difference between the doses was statistically significant. Masoudi et al. (2021) stated in their study on frozen rooster semen that 5 and 50 µM doses increased sperm motility compared to the control group. Esmaeilkhanian et al. (2021) reported in their study on frozen goat semen that after thawing, 1 and 10 µM mitoTEMPO doses significantly improved spermatozoon motility compared to the control group.

The increase in total motility of mitoTEMPO groups may be due to the fact that mitoTEMPO, a powerful mitochondria-targeted antioxidant, causes the inhibition or elimination of LPO and mitochondrial oxygen radical production and the regulation of antioxidant-related enzyme activities in the cells by adding mitoTEMPO to semen extenders (Du et al. 2017). In addition, researchers have reported that the use of mitoTEMPO provides an increase in motility by regulating the level of GPI, which is an important enzyme in the glycolytic pathway and closely related to spermatozoon quality and provides the necessary energy to the spermatozoon (Lu et al. 2018).

In their study in humans, Lu et al. (2018) stated that 5 and 50 µM doses of mitoTEMPO protected membrane integrity after freezing and thawing compared to other doses. Zhang et al. (2019), in their study investigating whether mitoTEMPO, a mitochondrial-targeted antioxidant, was effective in preventing damage in freezing semen taken from people with asthenospermia, stated that 10 and 100 µM mitoTEMPO doses were better in protecting mitochondrial membrane integrity. Masoudi et al. (2021) stated in their study on frozen rooster semen that 5 and 50 µM mitoTEMPO doses preserved membrane integrity after freezing and thawing. The membrane structure of ram spermatozoon is sensitive and sensitive to LPO, and the cooling and freezing-thawing process causes an increase in the MDA level, thus damaging the membrane lipid structure (Liu et al. 2010). It is thought that mitoTEMPO protects the plasma and acrosomal membrane by reducing ROS resulting from freezing and thawing semen.

Cho et al. (2020) added different doses of mitoTEMPO (0.5, 5, 50 and 500 µM) to pig semen diluted with lactose egg yolk sperm extender and investigated the effect of different doses of mitoTEMPO on kinematic parameters after freezing and thawing. They determined that there was no difference between the kinematic parameters. The differences between studies may be due to differences in mitoTEMPO doses, different animal species in the studies, different diluent contents, different glycerol ratios and different study plans. In addition, the addition of mitoTEMPO significantly preserves the mitochondrial activity of sperm and reduces the rate of damaged mitochondria during storage. On the other hand, the use of mitoTEMPO is thought to improve kinematic parameters because it regulates the level of GPI, which is an important enzyme in the glycolytic pathway and is closely related to spermatozoon quality.

Some researchers, Zhang et al. (2019), in their study investigating whether mitoTEMPO, a mitochondrial-targeted antioxidant, is effective in preventing damage in freezing semen taken from people with asthenospermia, added mitoTEMPO at doses of 1, 10 and 100 µM along with the control and thawed the semen and then analysed DNA damage. They stated that 10 and 100 µM doses were better (p < 0.05). In their study on rams, Asadzadeh et al. (2021) examined DNA damage by adding 0.5, 5, 50 and 500 µM doses of mitoTEMPO to the extender along with the control group and freezing and thawing and found that the DNA damage was 12.2 ± 0.8%, 10.3 ± 0.8%, 10.0 ± 0.8%, respectively. They found the values to be 9.5 ± 0.8 and 11.9 ± 0.8 and reported that the DNA damage of the control group was higher and more significant than the other groups (p < 0.05).

The researchers' results were found to be compatible with our study, and it was stated that this positive effect of mitoTEMPO on DNA was due to mitoTEMPO reducing oxidative stress and preventing DNA damage in different cell types (Trnka et al. 2009; Hu and Li 2016). In addition, it has been found that glycerol, which is used as an intracellular cryoprotectant when freezing spermatozoa, reduces DNA damage in spermatozoa even at low rates when used with mitoTEMPO. It has been observed that it prevents intracellular toxications caused by glycerol in ram spermatozoon and mitoTEMPO can be used as a cryoprotectant.

MitoTEMPO may be a helpful tactic to preserve mitochondrial membrane potential and viability throughout the freezing process since it is believed to limit mitochondrial Bax translocation. MitoTEMPO's structure, which is identical to that of hydroxylamine, allows it to prevent ROS overflow and excess production during the cryopreservation process (Trnka et al. 2009). In addition, mitoTEMPO generates nitroxide radicals, which function similarly to SOD to maintain the stability of the electron transport chain and the integrity of the phospholipid membrane bilayer (Yang et al. 2018). However, in our study, we did not find statistical significance between mitochondrial membrane potential and viability. This may be due to the necessity of ensuring the optimum level when adding antioxidants because if, for any reason, isotonic balance is not achieved, either the dose is ineffective, or a toxic effect may be observed (Bucak et al. 2009).

Asadzadeh et al. (2021) reported in their study on rams, in which they added mitoTEMPO and froze and thawed, that MDA values of 5 and 50 µM doses decreased compared to the control and that this decrease was statistically significant. In addition, Zhang et al. (2019) stated that the addition of mitoTEMPO to semen caused a decrease in the formation of oxidation products (ROS and MDA) and a significant increase in antioxidant enzyme activities. In our study, it was determined that MDA levels decreased after freezing-thawing in the experimental groups to which mitoTEMPO was added. This decrease may be important evidence that mitoTEMPO, a mitochondrial antioxidant, protects spermatozoa against cryopreservation-induced oxidative stress with its ROS scavenging feature.

Conclusions

It was determined that the addition of 1 µM mitoTEMPO to the 3% glycerol extender, when preserved by freezing, preserved membrane integrity, increased total motility, improved kinematic parameters (VCL, VSL, VAP), increased the rate of spermatozoon with undamaged acrosomes and prevented DNA damage and oxidative stress. Therefore, semen extenders, which generally contain 5% glycerol for rams, can be adjusted to 3% with the addition of mitoTEMPO. It has been observed that these reported doses of mitoTEMPO can be added to semen extenders and can be used successfully as a cryoprotectant during the storage of ram semen.

Author Contributions

Conceptualization: Recep Hakkı Koca. Methodology: Recep Hakkı Koca, İbrahim Halil Güngör, Aslıhan Çakır Cihangiroğlu, Tutku Can Acısu, Nida Badıllı, Serkan Ali Akarsu, Şeyma Özer Kaya, Emre Kaya, Gaffari Türk, Mustafa Sönmez and Seyfettin Gür. Data curation: Recep Hakkı Koca. Investigation: Recep Hakkı Koca, İbrahim Halil Güngör, Aslıhan Çakır Cihangiroğlu, Tutku Can Acısu, Nida Badıllı, Serkan Ali Akarsu, Şeyma Özer Kaya, Emre Kaya, Gaffari Türk, Mustafa Sönmez and Seyfettin Gür. Formal analysis: Recep Hakkı Koca, İbrahim Halil Güngör, Aslıhan Çakır Cihangiroğlu, Tutku Can Acısu, Nida Badıllı, Serkan Ali Akarsu, Şeyma Özer Kaya, Emre Kaya, Gaffari Türk, Mustafa Sönmez and Seyfettin Gür. Supervision: Seyfettin Gür. Funding acquisition: Seyfettin Gür. Visualization: Recep Hakkı Koca. Project administration: Seyfettin Gür. Writing–original draft: Recep Hakkı Koca. Writing–review and editing: Recep Hakkı Koca.

Ethics Statement

The approval for the study was taken from Fırat University Animal Experiments Local Ethics Committee (Protocol No: 2019/24).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data available on request from authors.

Peer Review

The peer review history for this article is available at .

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