Effects of resveratrol supplementation to equine sperm during semen cryopreservation

Natália de Castro Alves; Marina Morra Freitas; Jade Raquel Dias Farias; Cesar Lopes Horta; José de Oliveira Carvalho Neto; Olindo Assis Martins-Filho; Márcio Sobreira Silva Araújo; Ângela Quintão Lana; Guilherme Mattos Jardim Costa; Fernanda Radicchi Lobato de Almeida; Erica Azevedo Costa; Monique de Albuquerque Lagares

doi:10.1590/1984-3143-AR2025-0107

ORIGINAL ARTICLE

Effects of resveratrol supplementation to equine sperm during semen cryopreservation

Natália de Castro Alves; Marina Morra Freitas; Jade Raquel Dias Farias; Cesar Lopes Horta; José de Oliveira Carvalho Neto; Olindo Assis Martins-Filho; Márcio Sobreira Silva Araújo; Ângela Quintão Lana; Guilherme Mattos Jardim Costa; Fernanda Radicchi Lobato de Almeida; Erica Azevedo Costa; Monique de Albuquerque Lagares

http://dx.doi.org/10.1590/1984-3143-AR2025-0107 Anim Reprod, vol.23, n2, e20250107, 2026

PDF

Downloads: 0

Abstract

During equine semen cryopreservation, most of the seminal plasma is removed, making sperm more susceptible to oxidative stress. This study evaluated the effect of resveratrol supplementation on the quality of frozen-thawed equine sperm. Semen from ten stallions was frozen using a control INRA 96 extender and extenders supplemented with 5-, 10-, 100-, and 150- µM resveratrol. Post-thaw evaluations included motility, kinematic parameters, morphology, plasma and acrosomal membrane integrity, mitochondrial potential, lipid peroxidation, nitrite, hydrogen peroxide, malondialdehyde levels, total reactive oxygen species (ROS), chromatin protamine deficiency, chromatin condensation, sperm binding to bovine oviduct explants, and gene expression of apoptosis-related genes such as B cell lymphoma 2 (BCL2) and BCL2-associated X (BAX), mitochondrial ROS modulator 1 associated with mitochondrial ROS production (ROMO1), sperm acrosome-associated 3 related with sperm binding capacity to the zona pellucida (SPACA3), and DNA damage repair gene 8-oxoguanine DNA glycosylase 1 (OGG1). Data were analyzed by ANOVA and Tukey test (P < 0.05). The 10 µM resveratrol treatment significantly increased sperm motility, mitochondrial activity, SPACA3 expression, and the number of sperm bound to oviduct explants compared to the control. Additionally, 10 µM resveratrol reduced total ROS, ROMO1, and BAX gene expression, indicating reduced oxidative stress and apoptosis. In conclusion, 10 µM resveratrol supplementation improved sperm metabolic activity, enhanced oviduct binding capacity, and demonstrated antioxidant and anti-apoptotic effects. Thus, incorporating resveratrol into freezing extenders could be a promising strategy to improve the fertilizing capacity of equine sperm in artificial insemination programs.

Keywords

antioxidant, reproduction, cryopreservation, semen, stallion

References

Asadi N, Bahmani M, Kheradmand A, Rafieian-Kopaei M. The impact of oxidative stress on testicular function and the role of antioxidants in improving it: a review. J Clin Diagn Res. 2017;11(5):IE01-05. https://doi.org/10.7860/JCDR/2017/23927.9886. PMid:28658802.

Bang S, Qamar AY, Tanga BM, Fang X, Cho J. Resveratrol supplementation into extender protects against cryodamage in dog post-thaw sperm. J Vet Med Sci. 2021;83(6):973-80. https://doi.org/10.1292/jvms.21-0125. PMid:33896876.

Bucak MN, Ataman MB, Başpinar N, Uysal O, Taşpinar M, Bilgili A, Öztürk C, Güngör S, İnanç ME, Akal E. Lycopene and resveratrol improve post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA integrity. Andrologia. 2015;47(5):545-52. https://doi.org/10.1111/and.12301. PMid:24909239.

Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-10. https://doi.org/10.1016/S0076-6879(78)52032-6. PMid:672633.

Colégio Brasileiro de Reprodução Animal – CBRA. Manual para exame andrológico e avaliação de sêmen animal. Belo Horizonte: CBRA; 2013.

Córdova A, Strobel P, Vallejo A, Valenzuela P, Ulloa O, Burgos RA, Menarim B, Rodríguez-Gil JE, Ratto M, Ramírez-Reveco A. Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5′ AMP-activated protein kinase (AMPK). Cryobiology. 2014;69(3):473-81. https://doi.org/10.1016/j.cryobiol.2014.10.008. PMid:25445464.

Du M, Liu Y, Zhang L, Li X, Wang N, He Q, Cao J, Zhao B, Shi Y, Li B, Bou G, Dugarjaviin M. The effect of resveratrol on the cryopreservation of Mongolian horse semen. Arch Tierzucht. 2025;68(1):27-41. https://doi.org/10.5194/aab-68-27-2025.

Estrada E, Rodríguez-Gil JE, Rocha LG, Balasch S, Bonet S, Yeste M. Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen-thawed boar semen. Andrology. 2014;2(1):88-99. https://doi.org/10.1111/j.2047-2927.2013.00144.x. PMid:24123940.

Garcez ME, Santos Branco C, Lara LV, Pasqualotto FF, Salvador M. Effects of resveratrol supplementation on cryopreservation medium of human semen. Fertil Steril. 2010;94(6):2118-21. https://doi.org/10.1016/j.fertnstert.2010.01.058. PMid:20189559.

Green DPL. Three-dimensional structure of the zona pellucida. Rev Reprod. 1997;2(3):147-56. https://doi.org/10.1530/ror.0.0020147. PMid:9414478.

He WH, Zhai XH, Duan XJ, Di HS. Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing. J Zhejiang Univ Sci B. 2020;21(6):485-94. https://doi.org/10.1631/jzus.B1900520. PMid:32478494.

Hurtado De Llera A, Martin-Hidalgo D, Gil MC, Garcia-Marin LJ, Bragado MJ. AMP-activated kinase AMPK is expressed in boar sperm and regulates motility. PLoS One. 2012;7(6):e38840. https://doi.org/10.1371/journal.pone.0038840. PMid:22719961.

Jakop U, Engel KM, Hürland M, Müller P, Osmers JH, Jung M, Schulze M. Oxidative stress-induced lipid alterations increase detached acrosomes after cryopreservation of Holstein bull semen. Theriogenology. 2023;197:37-45. https://doi.org/10.1016/j.theriogenology.2022.11.036. PMid:36470108.

Kaeoket K, Chanapiwat P. The beneficial effect of resveratrol on the quality of frozen-thawed boar sperm. Animals. 2023;13(18):2829. https://doi.org/10.3390/ani13182829. PMid:37760229.

Kenney RM, Bergman RV, Cooper WL. Minimal contamination techniques for breeding mares: techniques and preliminary findings. In: 21st Annual Convection of the American Association of Equine Practitioners; 1975. Boston: AAEP; 1975. p. 327-49.

Kozyra A, Madalińska A. Resveratrol as a multifunctional bioactive compound. Aesthetic Cosmetology and Medicine. 2025;14(3):121-5. https://doi.org/10.52336/acm.2025.018.

Lagares MA, Petzoldt R, Sieme H, Klug E. Assessing equine sperm-membrane integrity. Andrologia. 2000;32(3):163-7. https://doi.org/10.1046/j.1439-0272.2000.00351.x. PMid:10863971.

Lecewicz M, Strzezek R, Kordan W, Majewska A. Effect of extender supplementation with low molecular weight antioxidants on selected quality parameters of cryopreserved canine spermatozoa. J Vet Res. 2018;62(2):221-7. https://doi.org/10.2478/jvetres-2018-0032. PMid:30364791.

Leemans B, Gadella BM, Sostaric E, Nelis H, Stout TA, Hoogewijs M, Van Soom A. Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion sperm. Biol Reprod. 2014;91(1):13. https://doi.org/10.1095/biolreprod.113.116418. PMid:24829033.

Leon PM, Campos VF, Kaefer C, Begnini KR, McBride AJ, Dellagostin OA, Seixas FK, Deschamps JC, Collares T. Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells. Zygote. 2013;21(3):279-85. https://doi.org/10.1017/S0967199411000554. PMid:21933470.

Leung MR, Zeng J, Wang X, Roelofs MC, Huang W, Zenezini Chiozzi R, Hevler JF, Heck AJR, Dutcher SK, Brown A, Zhang R, Zeev-Ben-Mordehai T. Structural specializations of the sperm tail. Cell. 2023;186(13):2880-2896.e17. https://doi.org/10.1016/j.cell.2023.05.026. PMid:37327785.

Li J, Zhao W, Zhu J, Ju H, Liang M, Wang S, Chen S, Ferreira-Dias G, Liu Z. Antioxidants and oxidants in boar spermatozoa and their surrounding environment are associated with AMPK activation during liquid storage. Vet Sci. 2023;10(3):214. https://doi.org/10.3390/vetsci10030214. PMid:36977253.

Lomeo AM, Giambersio AM. “Water test”: a simple method to assess sperm-membrane integrity. Int J Androl. 1991;14(4):278-82. https://doi.org/10.1111/j.1365-2605.1991.tb01093.x. PMid:1879962.

Mies A Fo. Tecnologia do semen I: exame e classificação. In: Mies A Fo, editor Reprodução dos animais e inseminação artificial. 3. ed. Porto Alegre: Sulina; 1975. p. 423-58. (vol. 2).

Nashtaei MS, Amidi F, Sedighi Gilani MA, Aleyasin A, Bakhshalizadeh S, Naji M, Nekoonam S. Protective features of resveratrol on human sperm cryopreservation may be mediated through 5′ AMP-activated protein kinase activation. Andrology. 2017;5(2):313-26. https://doi.org/10.1111/andr.12306.

Nguyen TM, Seigneurin F, Froment P, Combarnous Y, Blesbois E. The 5′ AMP-activated protein kinase (AMPK) is involved in the augmentation of antioxidant defenses in cryopreserved chicken sperm. PLoS One. 2015;10(7):e0134420. https://doi.org/10.1371/journal.pone.0134420. PMid:26222070.

Nouri H, Shojaeian K, Samadian F, Lee S, Kohram H, Lee JI. Using resveratrol and epigallocatechin-3-gallate to improve cryopreservation of stallion sperm with low quality. J Equine Vet Sci. 2018;70:18-25. https://doi.org/10.1016/j.jevs.2018.07.003.

Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolff SP. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenilphosphine. Anal Biochem. 1994;220(2):403-9. https://doi.org/10.1006/abio.1994.1357. PMid:7978285.

Partyka A, Lukaszewicz E, Nizański W, Twardoń J. Detection of lipid peroxidation in frozen-thawed avian sperm using C₍₁₁₎-BODIPY_(581/591). Theriogenology. 2011;75(9):1623-9. https://doi.org/10.1016/j.theriogenology.2011.01.002. PMid:21396693.

Pérez-Rico A, Crespo F, Sanmartín ML, De Santiago A, Vega-Pla JL. Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen. Anim Reprod Sci. 2014;149(3–4):204. https://doi.org/10.1016/j.anireprosci.2014.08.007. PMid:25192831.

Pillet E, Batellier F, Duchamp G, Furstoss V, LE Vern Y, Kerboeuf D, Vidament M, Magistrini M. Freezing stallion semen in INRA96®-based extender improves fertility rates in comparison with INRA82. Dairy Sci Technol. 2008;88(2):257-65. https://doi.org/10.1051/dst:2008002.

Pollard CL, Gibb Z, Swegen A, Grupen CG. NAD+, Sirtuins and PARPs: enhancing oocyte developmental competence. J Reprod Dev. 2022;68(6):345-54. https://doi.org/10.1262/jrd.2022-052. PMid:36171094.

Roca J, Martinez-Alborcia MJ, Gil MA, Parrilla I, Martinez EA. Dead sperm in raw semen samples impair in vitro fertilization outcomes of frozen-thawed sperm. Fertil Steril. 2013;100(3):875-81. https://doi.org/10.1016/j.fertnstert.2013.05.020. PMid:23768987.

Shin JA, Chung JS, Cho SH, Kim HJ, Yoo YD. ROMO1 expression contributes to oxidative stress-induced death of lung epithelial cells. Biochem Biophys Res Commun. 2013;439(2):315-20. https://doi.org/10.1016/j.bbrc.2013.07.012. PMid:23867822.

Simões R, Feitosa W, Mendes C, Marques M, Nicacio A, Barros F, Visintin JA, Assumpção M. Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency. Biotech Histochem. 2009;84(3):79-83. https://doi.org/10.1080/10520290902843595. PMid:19306222.

Takeo S, Sato D, Kimura K, Monji Y, Kuwayama T, Kawahara-Miki R, Iwata H. Resveratrol improves the mitochondrial function and fertilization outcome of bovine oocytes. J Reprod Dev. 2014;60(2):92-9. https://doi.org/10.1262/jrd.2013-102. PMid:24390595.

Xu Y, Zhang X, Liu H, Li J, Wang Z. Antioxidant strategies to mitigate oxidative stress during sperm cryopreservation. Frontiers in Endocrinology. 2025;16:1520835. https://doi.org/10.3389/fendo.2025.1520835.

Yánez-Ortiz I, Catalán J, Rodríguez-Gil JE, Miró J, Yeste M. Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep. Anim Reprod Sci. 2022;246:106904. https://doi.org/10.1016/j.anireprosci.2021.106904. PMid:34887155.

Zhu Z, Li R, Ma G, Bai W, Fan X, Lv Y, Luo J, Zeng W. 5′ AMP-activated protein kinase regulates goat sperm functions via energy metabolism in vitro. Cell Physiol Biochem. 2018;47(6):2420-31. https://doi.org/10.1159/000491616. PMid:29991051.

Zhu Z, Li R, Fan X, Lv Y, Zheng Y, Hoque SAM, Wu D, Zeng W. Resveratrol improves boar sperm quality via 5′ AMP-activated protein kinase activation during cryopreservation. Oxid Med Cell Longev. 2019;2019:5921503. https://doi.org/10.1155/2019/5921503. PMid:31565152.

Zhu Z, Zhao H, Cui H, Adetunji AO, Min L. Resveratrol improves the frozen-thawed ram sperm quality through antioxidant and AMPK-related pathways. Animals. 2023;13(24):3887. https://doi.org/10.3390/ani13243887. PMid:38136923.

Submitted date:
07/20/2025

Accepted date:
02/20/2026

Effects of resveratrol supplementation to equine sperm during semen cryopreservation

Abstract

Keywords

References

Links

Share

Anim Reprod