Veterinary Research Forum

Riociguat supplementation modulates post-thaw ram sperm quality in a dose-dependent manner through effects on motility, membrane integrity and oxidative balance

Document Type : Original Article

Authors

Recep Hakkı Koca 1
Serkan Ali Akarsu 2
Tutku Can Acisu 3
Zehra Coşkun Bahçe 1
Gamze Uçak 2
Cihan Gür 4
Ömercan Alat 4
Mehmet Akif Aydın 5

1 Department of Reproduction and Artificial Insemination, Veterinary Faculty, Bingol University, Bingol, Türkiye

2 Department of Reproduction and Artificial Insemination, Veterinary Faculty, Ataturk University, Erzurum, Türkiye

3 Department of Reproduction and Artificial Insemination, Veterinary Faculty, Fırat University, Elazıg, Türkiye

4 Department of Biochemistry, Veterinary Faculty, Ataturk University, Erzurum, Türkiye

5 Department of Reproduction and Artificial Insemination, Institute of Healt Sciences, Kafkas University, Kars, Türkiye

https://doi.org/10.30466/vrf.2026.2077381.4987
Abstract
The present study showed riociguat supplementation modulates in a Tris–egg yolk extender on post-thaw ram semen quality. Semen samples were collected from eight healthy, sexually mature Tuj rams (n = 48 ejaculates) during the breeding season and cryopreserved after supplementation with riociguat at concentrations of 0.00 (control), 0.50, 5.00, 50.00, and 250 µM. Post-thaw sperm quality was evaluated using computer-assisted sperm analysis for motility and kinematic parameters, alongside assessments of plasma and acrosomal membrane integrity, mitochondrial membrane potential, DNA integrity and oxidative stress markers including malondialdehyde and glutathione. Riociguat supplementation (5.00 - 250 µM) significantly modulated mitochondrial membrane potential and intracellular antioxidant capacity as evidenced by reduced malondialdehyde and increased glutathione levels. Moreover, all riociguat-treated groups exhibited significantly improved DNA integrity compared with the control. Moreover, acrosomal damage was declined at the highest concentrations (5.00, 50.00 and 250 µM). These findings suggest that riociguat exerted a protective effect on acrosomal membranes during cryopreservation. In conclusion, riociguat modulated post-thaw ram sperm function in a dose-dependent manner, exerting both beneficial and detrimental effects depending on concentration. Although fertility outcomes were not assessed in the present study, the observed biochemical and functional changes provided preliminary evidence that riociguat might influence key pathways associated with sperm resilience to cryoinjury, thereby, justifying further investigations incorporating in vivo fertility and reproductive performance assessments.

Keywords

Membrane integrity
Oxidative stress
Ram sperm
Soluble guanylate cyclase stimulator
Subjects
1.     Ollero M, Perez-Pe R, Muiño-Blanco T, et al. Improvement of ram sperm cryopreservation protocols assessed by sperm quality parameters and heterogeneity analysis. Cryobiology 1998; 37(1): 1-12.
2.     Riesco MF, Alvarez M, Anel-Lopez L, et al. Multiparametric study of antioxidant effect on ram sperm cryopreservation - from field trials to research bench. Animals (Basel) 2021; 11(2): 283. doi: 10.3390/ ani11020283.
3.     Li Y, He M, Ran H, et al. ZnO NPs protect boar sperm in liquid storage through increasing the phosphorylation of PKAs. Anim Reprod 2025; 22(2): e20240025. doi: 10.1590/1984-3143-AR2024-0025.
4.     Saha A, Asaduzzaman M, Bari FY. Cryopreservation techniques for ram sperm. Vet Med Int 2022; 2022, 7378379. doi: 10.1155/2022/7378379.
5.     Türk G, Koca RH, Güngör İH, et al. Effect of hydrated C60 fullerene on lipid peroxidation, vitamins, and amino acid composition in frozen–thawed ram semen. Anim Reprod Sci 2022; 238: 106939. doi: 10.1016/j.anireprosci.2022.106939.
6.     Sheikholeslami SA, Soleimanzadeh A, Rakhshanpour A, et al. The evaluation of lycopene and cysteamine supplementation effects on sperm and oxidative stress parameters during chilled storage of canine semen. Reprod Domest Anim 2020; 55(9): 1229-1239.
7.     Banday MN, Lone FA, Rasool F, et al. Use of antioxidants reduces lipid peroxidation and improves quality of crossbred ram sperm during its cryopreservation. Cryobiology 2017; 74: 25-30.
8.     Akarsu SA, Güngör İH, Cihangiroğlu AÇ, et al. Effect of sulforaphane on long-term storage of rabbit semen. Anim Reprod 2023; 20(2): e20230001. doi: 10.1590/1984-3143-AR2023-0001.
9.     Ros-Santaella JL, Pintus E. Plant extracts as alternative additives for sperm preservation. Antioxidants (Basel) 2021; 10(5): 772. doi: 10.3390/antiox10050772.
10. Makowski CT, Rissmiller RW, Bullington WM. Riociguat: a novel drug for the treatment of pulmonary hypertension. Pharmacotherapy 2015; 35(5): 502-519.
11. Seker U, Kavak DE, Guzel BC, et al. Targeting soluble guanylate cyclase with Riociguat has potency to alleviate testicular ischaemia reperfusion injury via regulating various cellular pathways. Andrologia 2022; 54(11): e14616. doi: 10.1111/and.14616.
12. Aslanoglu B, Kaya S, Gunara SO, et al. Neuroprotective potential of a novel soluble guanylate cyclase stimulator the Riociguat alone or in a combination manner with resveratrol in experimental stroke model in rats. Int J Morphol 2024; 42(4): 984-990.
13. Soleimanzadeh A, Mohammadnejad L, Ahmadi A. Ameliorative effect of Allium sativum extract on busulfan-induced oxidative stress in mice sperm. Vet Res Forum 2018; 9(3): 265-271.
14. Baldi E, Luconi M, Bonaccorsi L, et al. Signal transduction pathways in human spermatozoa. J Reprod Immunol 2002; 53(1-2): 121-131.
15. Miraglia E, De Angelis F, Gazzano E, et al. Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway. Reproduction 2011; 141(1): 47-54.
16. Özmen MF, Cirit Ü, Arıcı R, et al. Evaluation of synergistic effects of iodixanol and trehalose on cryosurvival of electroejaculated ram semen. Andrologia 2020; 52(9): e13656. doi: 10.1111/ and.13656.
17. Özer Kaya Ş, Güngör İH, Dayan Cinkara S, et al. Effect of different doses of hydrated C60 fullerene nanoparticles on ram semen during cool storage. Turk J Vet Anim Sci 2021; 45(1): 139-147.
18. Izanloo H, Soleimanzadeh A, Bucak MN, et al. The effects of glutathione supplementation on post-thawed turkey semen quality and oxidative stress parameters and fertilization, and hatching potential. Theriogenology 2022; 179: 32-38.
19. Söderquist L, Madrid-Bury N, Rodriguez-Martinez H. Assessment of ram sperm membrane integrity following different thawing procedures. Theriogenology 1997; 48(7): 1115-1125.
20. Öztürk AE, Bodu M, Bucak MN, et al. Synergistic effects of trehalose and low concentrations of cryoprotectants on post-thaw ram sperm quality. Cryobiology 2020; 95: 157-163.
21. Koca RH, Güngör İH, Cihangiroğlu AÇ, et al. Optimizing cryopreservation: the effect of MitoTEMPO and glycerol ratios on ram semen quality after freezing and thawing. Vet Med Sci 2025; 11(4): e70406. doi: 10.1002/vms3.70406.
22. Akarsu SA, Gür C, Küçükler S, et al. Protective effects of syringic acid against oxidative damage, apoptosis, autophagy, inflammation, testicular histopathologic disorders, and impaired sperm quality in the testicular tissue of rats induced by mercuric chloride. Environ Toxicol 2024; 39(10): 4803-4814.
23. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990; 186: 407-421.
24. Akerboom TP, Sies H. Assay of glutathione, glutathione disulfide and glutathione mixed disulfides in biological samples. Methods Enzymol 1981; 77: 373-382.
25. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1): 265-275.
26. Stasch JP, Pacher P, Evgenov OV. Soluble guanylate cyclase as an emerging therapeutic target in cardiopulmonary disease. Circulation 2011; 123(20): 2263-2273.
27. Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal 2011; 14(3): 367-381.
28. Herrero M, Lamirande E, Gagnon C. Nitric oxide is a signaling molecule in spermatozoa. Curr Pharm Des 2003; 9(5): 419-425.
29. Evgenov OV, Pacher P, Schmidt PM, et al. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential. Nat Rev Drug Discov 2006; 5(9): 755-768.
30. Domínguez-Rebolledo AE, Fernández-Santos MR, Bisbal A, et al. Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments. Reprod Fertil Dev 2010; 22(5): 856-870.
31. Mostahsan Z, Azizi S, Soleimanzadeh A, et al. The protective roles of Mito-TEMPO on testicular ischemia‑reperfusion injury based on biochemical and histopathological evidences in mice. Iran J Vet Surg 2024; 19(2), 97-105.
32. Shakouri N, Soleimanzadeh A, Rakhshanpour A, et al. Antioxidant effects of supplementation of 3, 4‐dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen. Reprod Domest Anim 2021; 56(7): 1004-1014.
Veterinary Research Forum
Volume 17, Issue 9
September 2026
Pages 631-637

  • Receive Date 10 November 2025
  • Revise Date 27 December 2025
  • Accept Date 07 January 2026

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