Animal Reproduction (AR)
Animal Reproduction (AR)

Effect of sulforaphane on long-term storage of rabbit semen

Serkan Ali Akarsu; İbrahim Halil Güngör; Aslıhan Çakır Cihangiroğlu; Tutku Can Acısu; Recep Hakkı Koca; Gaffari Türk; Mustafa Sönmez; Seyfettin Gür

Downloads: 1
Views: 264


In this study, it was aimed to determine the effect of sulforaphane (SFN) on rabbit semen cryopreservation. Semen collected from animals was divided into 5 equal volumes as Control, SFN 5 µM, SFN 10 µM, SFN 25 µM and SFN 50 µM groups. Afterwards, semen analyzes were performed. According to our results, there was no statistical difference between the groups at 4°C. However after freezing thawing, the highest total motility, progressive motility and rapid spermatozoa rate was seen in the 10 µM SFN group, while the lowest was observed in the 50 µM SFN group (P<0.05). Static sperm ratio was highest in the 50 µM group, while the lowest was observed in the 10 µM SFN group. When flow cytometry results examined the rate of acrosomal damaged and dead sperm was the lowest in the 10 µM SFN group, a statistical difference was observed between the control group (P<0.05). The highest rate of sperm with high mitochondrial membrane potential was seen in the 5 µM SFN and 10 µM SFN groups. Apoptosis and ROS rates were found to be lower in the experimental groups compared to the control groups (P<0.05). As a result, SFN supplementation at a dose of 10 µM increased the quality of sperm in the freezing and thawing processes of rabbit semen. In conclusion, 10 µM SFN improved the quality of cryopreservation of rabbit semen.


apoptosis, cryopreservation, oxidative stress, rabbit, semen


Abdelnour SA, Hassan MA, El‐Ratel IT, Essawi WM, El‐Raghi AA, Lu Y, Sheiha AM. Effect of addition of L‐carnitine to cryopreservation extender on rabbit post‐thaw semen parameters, antioxidant capacity, mitochondrial function, apoptosis, and ultrastructure changes. Reprod Domest Anim. 2022;57(8):902-11. PMid:35491499.

Abdelnour SA, Hassan MA, Mohammed AK, Alhimaidi AR, Al-Gabri N, Al-Khaldi KO, Swelum AA. The effect of adding different levels of curcumin and its nanoparticles to extender on post-thaw quality of cryopreserved rabbit sperm. Animals (Basel). 2020;10(9):1508. PMid:32858961.

Ahmad E, Naseer Z, Aksoy M. Glutathione supplementation in semen extender improves rabbit sperm attributes during refrigeration. World Rabbit Sci. 2021;29(2):81-6.

Bansal AK, Bilaspuri G. Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int. 2011;2011:1-7. PMid:20871827.

Castellini C. Semen production and management of rabbit bucks. In: 9th World Rabbit Congress; 2008 Jun 10-13; Verona, Italy. Valencia: World Rabbit Science; 2018. p. 265-78.

Chaveiro A, Santos P, Silva F. Assessment of sperm apoptosis in cryopreserved bull semen after swim‐up treatment: a flow cytometric study. Reprod Domest Anim. 2007;42(1):17-21. PMid:17214767.

Dogan S, Mason MC, Govindaraju A, Belser L, Kaya A, Stokes J, Rowe D, Memili E. Interrelationships between apoptosis and fertility in bull sperm. J Reprod Dev. 2013;59(1):18-26. PMid:22986927.

Domingo P, Olaciregui M, González N, De Blas I, Gil L. Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents. Cryobiology. 2018;81:174-7. PMid:29366763.

Georgikou C, Yin L, Gladkich J, Xiao X, Sticht C, de la Torre C, Gretz N, Gross W, Schäfer M, Karakhanova S, Herr I. Inhibition of miR30a-3p by sulforaphane enhances gap junction intercellular communication in pancreatic cancer. Cancer Lett. 2020;469:238-45. PMid:31678166.

Guerrero-Beltrán CE, Calderón-Oliver M, Pedraza-Chaverri J, Chirino YI. Protective effect of sulforaphane against oxidative stress: recent advances. Exp Toxicol Pathol. 2012;64(5):503-8. PMid:21129940.

Hagen D, Gilkey A, Foote R. Spermatozoal velocity and motility and its relationship to fertility in the rabbit inseminated with low sperm numbers. World Rabbit Sci. 2002;10(4):135-40.

Halo M Jr, Bułka K, Antos PA, Greń A, Slanina T, Ondruška Ľ, Tokárová K, Massányi M, Formicki G, Halo M, Massányi P. The effect of ZnO nanoparticles on rabbit spermatozoa motility and viability parameters in vitro. Saudi J Biol Sci. 2021;28(12):7450-4. PMid:34867049.

Hernández AF, Gil F, Lacasaña M, Rodríguez-Barranco M, Tsatsakis AM, Requena M, Parrón T, Alarcón R. Pesticide exposure and genetic variation in xenobiotic-metabolizing enzymes interact to induce biochemical liver damage. Food Chem Toxicol. 2013;61:144-51. PMid:23688862.

Houghton CA. Sulforaphane: its “coming of age” as a clinically relevant nutraceutical in the prevention and treatment of chronic disease. Oxid Med Cell Longev. 2019;2019:2716870. PMid:31737167.

Iaffaldano N, Di Iorio M, Rosato MP, Manchisi A. Cryopreservation of rabbit semen using non-permeable cryoprotectants: effectiveness of different concentrations of low-density lipoproteins (LDL) from egg yolk versus egg yolk or sucrose. Anim Reprod Sci. 2014;151(3-4):220-8. PMid:25465902.

Iaffaldano N, Di Iorio M, Rosato MP. The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: dimethylacetamide versus dimethylsulfoxide. Theriogenology. 2012;78(6):1381-9. PMid:22898020.

Ishiura Y, Ishimaru H, Watanabe T, Fujimuro M. Sulforaphane exhibits cytotoxic effects against primary effusion lymphoma cells by suppressing p38MAPK and AKT phosphorylation. Biol Pharm Bull. 2019;42(12):2109-12. PMid:31787726.

Jiménez-Rabadán P, García-Álvarez O, Vidal A, Maroto-Morales A, Iniesta-Cuerda M, Ramón M, del Olmo E, Fernández-Santos R, Garde JJ, Soler AJ. Effects of vitrification on ram spermatozoa using free-egg yolk extenders. Cryobiology. 2015;71(1):85-90. PMid:26004240.

Johinke D, De Graaf S, Bathgate R. Quercetin reduces the in vitro production of H2O2 during chilled storage of rabbit spermatozoa. Anim Reprod Sci. 2014;151(3-4):208-19. PMid:25465901.

Johinke D, de Graaf S, Bathgate R. The effect of sperm concentration and storage vessel on quercetin‐supplemented rabbit semen during chilled storage. Reprod Domest Anim. 2015;50(4):567-73. PMid:25939767.

Keum YS. Regulation of the Keap1/Nrf2 system by chemopreventive sulforaphane: implications of posttranslational modifications. Ann N Y Acad Sci. 2011;1229(1):184-9. PMid:21793854.

Kim S, Lee Y, Kim Y. Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 C. Anim Reprod Sci. 2011;124(1-2):118-24. PMid:21349666.

Küçük N, Raza S, Matsumura K, Uçan U, Serin İ, Ceylan A, Aksoy M. Effect of different carboxylated poly l-lysine and dimethyl sulfoxide combinations on post thaw rabbit sperm functionality and fertility. Cryobiology. 2021;102:127-32. PMid:34242651.

Lavara R, Mocé E, Lavara F, Castro MPV, Vicente JS. Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits? Theriogenology. 2005;64(5):1130-41. PMid:16125557.

Lopes S, Jurisicova A, Sun J, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod. 1998;13(4):896-900. PMid:9619544.

Maya-Soriano MJ, Taberner E, Sabés-Alsina M, Piles M, Lopez-Bejar M. Absence of beneficial effects on rabbit sperm cell cryopreservation by several antioxidant agents. Zygote. 2015;23(1):1-10. PMid:23965603.

Mocé E, Vicente J, Lavara R. Effect of freezing–thawing protocols on the performance of semen from three rabbit lines after artificial insemination. Theriogenology. 2003;60(1):115-23. PMid:12620585.

Mocé E, Vicente JS. Rabbit sperm cryopreservation: a review. Anim Reprod Sci. 2009;110(1-2):1-24. PMid:18805659.

Mohammed AK, Khalil WA, Youssef HF, Saadeldin IM, Gabr SA, Hammad ME, Mehrez AZ. Influence of adding zeolite loaded with different charges to semen extender on sperm quality in rabbits after cryopreservation. Cryobiology. 2021;103:107-15. PMid:34480940.

Parrish JJ, Foote R. Fertility of cooled and frozen rabbit sperm measured by competitive fertilization. Biol Reprod. 1986;35(2):253-7. PMid:3768452.

Peña FJ, Johannisson A, Wallgren M, Rodríguez-Martínez H. Assessment of fresh and frozen–thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity. Theriogenology. 2003;60(4):677-89. PMid:12832017.

Petruska P, Capcarova M, Sutovsky P. Antioxidant supplementation and purification of semen for improved artificial insemination in livestock species. Turk J Vet Anim Sci. 2014;38(6):643-52.

Qin Z, Tang J, Han P, Jiang X, Yang C, Li R, Tang M, Shen B, Wang W, Qin C, Zhang W. Protective effects of sulforaphane on di-n-butylphthalate-induced testicular oxidative stress injury in male mice offsprings via activating Nrf2/ARE pathway. Oncotarget. 2017;8(47):82956-67. PMid:29137315.

Rosato MP, Iaffaldano N. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology. 2013;79(3):508-16. PMid:23218394.

Salisbury GW, VanDemark NL, Lodge JR. Physiology of reproduction and artificial insemination of cattle. San Francisco: WH Freeman and Company; 1978.

Sanocka D, Kurpisz M. Reactive oxygen species and sperm cells. Reprod Biol Endocrinol. 2004;2(1):12. PMid:15038829.

Sarıözkan S, Bucak MN, Tuncer PB, Büyükleblebici S, Cantürk F. Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters, DNA integrity and fertilizing ability. Cryobiology. 2014;68(1):129-33. PMid:24468272.

Sarıözkan S, Türk G, Cantürk F, Yay A, Eken A, Akçay A. The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen. Cryobiology. 2013;67(1):1-6. PMid:23597426.

Shibahara H, Obara H, Ayustawati, Hirano Y, Suzuki T, Ohno A, Takamizawa S, Suzuki M. Prediction of pregnancy by intrauterine insemination using CASA estimates and strict criteria in patients with male factor infertility. Int J Androl. 2004;27(2):63-8. PMid:15149462.

Valipour J, Nashtaei MS, Khosravizadeh Z, Mahdavinezhad F, Nekoonam S, Esfandyari S, Amidi F. Effect of sulforaphane on apoptosis, reactive oxygen species and lipids peroxidation of human sperm during cryopreservation. Cryobiology. 2020;99:122-30. PMid:33248050.

Vaux DL, Korsmeyer SJ. Cell death in development. Cell. 1999;96(2):245-54. PMid:9988219.

Viudes-De-Castro MP, Mocé E, Lavara R, Marco-Jiménez F, Vicente JS. Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate. Theriogenology. 2014;81(9):1223-8. PMid:24629591.

Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril. 2003;80(3):531-5. PMid:12969693.

Wang Y, Zhang Z, Guo W, Sun W, Miao X, Wu H, Cong X, Wintergerst KA, Kong X, Cai L. Sulforaphane reduction of testicular apoptotic cell death in diabetic mice is associated with the upregulation of Nrf2 expression and function. Am J Physiol Endocrinol Metab. 2014;307(1):E14-23. PMid:24801392.

Warwick E, Cassidy A, Hanley B, Jouni ZE, Bao Y. Effect of phytochemicals on phase II enzyme expression in infant human primary skin fibroblast cells. Br J Nutr. 2012;108(12):2158-65. PMid:22424477.

Wu DM, Zheng ZH, Fan SH, Zhang ZF, Chen GQ, Lu J. Sulforaphane administration alleviates diffuse axonal injury (DAI) via regulation signaling pathway of NRF2 and HO‐1. J Cell Biochem. 2020;121(1):430-42. PMid:31232487.

Xu Y, Han X, Li Y, Min H, Zhao X, Zhang Y, Qi Y, Shi J, Qi S, Bao Y, Nie G. Sulforaphane mediates glutathione depletion via polymeric nanoparticles to restore cisplatin chemosensitivity. ACS Nano. 2019;13(11):13445-55. PMid:31670945.

Yang S, Yu L-H, Li L, Guo Y, Zhang Y, Long M, Li P, He JB. Protective mechanism of sulforaphane on cadmium-induced sertoli cell injury in mice testis via Nrf2/ARE signaling pathway. Molecules. 2018;23(7):1774. PMid:30029485.

Zhang Y, Talalay P, Cho C, Posner GH. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci USA. 1992;89(6):2399-403. PMid:1549603.

Zhu Z, Fan X, Lv Y, Zhang N, Fan C, Zhang P, Zeng W. Vitamin E analogue improves rabbit sperm quality during the process of cryopreservation through its antioxidative action. PLoS One. 2015;10(12):e0145383. PMid:26700473.

Zhu Z, Fan X, Pan Y, Lu Y, Zeng W. Trehalose improves rabbit sperm quality during cryopreservation. Cryobiology. 2017;75:45-51. PMid:28223021.

Submitted date:

Accepted date:

64747caaa953953763448e24 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections