Determination of the Effect of Epigallocatechin Gallate on Oxidative Stress, Apoptosis and Sperm Quality in Rabbit Semen

Abstract

Objectives;In this study, it was aimed to determine the effect of Epigallocatechin gallate (EGCG) on the cryopreservation of rabbit semen. Material and metod; For this purpose, semen was collected from six male New Zealand rabbits. Collected semen were divided into 5 groups as control, 25 µM, 50 µM, 100 µM and 200 µM. Semen samples were reconstituted and frozen after adding EGCG. Frozen straws were then thawed. Results; After freezing and thawing, total motility, progressive motility, and rate of fast and medium speed sperm were significantly higher in the 50 µM group than in the other groups. Static sperm ratio was found to be lower in the 50 µM group than in the other groups. When the flow cytometry results were examined, there was no statistical difference between the groups in terms of the ratio of dead and live sperm. However, the rate of dead sperm with acrosome damage was found to be the lowest in the 50 µM group. High mitochondrial membrane potential sperm ratio was found higher in 50 µM and 100 µM groups compared to other groups (p<0.05). The apoptosis rate was numerically the lowest in the 25 µM and 50 µM groups. While the rate of ROS was highest in the control group, it was significantly reduced in all EGCG support groups. Conclusion; In conclusion, the addition of EGCG rabbit semen to the cryodium improved semen quality.

Keywords

• Antioxidants
• cryopreservation
• epigallocatechin gallate
• rabbit
• semen

References

1. 1. Duranthon V, Beaujean N, Brunner M, et al. On the emerging role of rabbit as human disease model and the instrumental role of novel transgenic tools. Transgenic Res. 2012; 21(4):699-713.
2. 2. Nishijima K, Tanaka M, Sakai Y, et al. Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit. Cryobiology. 2014; 69(1):22-25.
3. 3. Mahadevan M, Trounson A. Effect of cooling, freezing and thawing rates and storage conditions on preservation of human spermatozoa. Andrologia. 1984; 16(1):52-60.
4. 4. 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):220-28.
5. 5. 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.
6. 6. Aksoy M, Cankat Lehimcioglu N, Akman O. Effect of seminal plasma on functional integrity of rabbit sperm membranes during storage at 4ºC or freezing. World Rabbit Sci. 2010; 16(1):1-6.
7. 7. Gandini L, Lombardo F, Paoli D, et al. Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod. 2000; 15(4):830-39.
8. 8. Mohammed AK, Khalil WA, Youssef HF, et al. Influence of adding zeolite loaded with different charges to semen extender on sperm quality in rabbits after cryopreservation. Cryobiology. 2021; 103:107-15.

9. 9. Stewart AJ, Mullen W, Crozier A. On‐line high‐performance liquid chromatography analysis of the antioxidant activity of phenolic compounds in green and black tea. Mol Nutr Food Res. 2005; 49(1):52-60.
10. 10. Ding J, Wang H, Wu Z-B, Zhao J, Zhang S, Li W. Protection of murine spermatogenesis against ionizing radiation-induced testicular injury by a green tea polyphenol. Biol Reprod. 2015; 92(1):6.
11. 11. Wang L-F, Kim D-M, Lee CY. Effects of heat processing and storage on flavanols and sensory qualities of green tea beverage. J Agric Food Chem. 2000; 48(9):4227-32.
12. 12. Skrzydlewska E, Ostrowska J, Farbiszewski R, Michalak K. Protective effect of green tea against lipid peroxidation in the rat liver, blood serum and the brain. Phytomedicine. 2002; 9(3):232-38.
13. 13. Pena F, Plaza Davila M, Ball B, et al. The impact of reproductive technologies on stallion mitochondrial function. Reprod Domest Anim. 2015; 50(4):529-37.
14. 14. Schroeder EK, Kelsey NA, Doyle J, et al. Green tea epigallocatechin 3-gallate accumulates in mitochondria and displays a selective antiapoptotic effect against inducers of mitochondrial oxidative stress in neurons. Antioxid Redox Signal. 2009; 11(3):469-80.
15. 15. Ahmed H, Jahan S, Riaz M, Khan BT, Ijaz MU. Epigallocatechin-3-gallate (EGCG) addition as an antioxidant in a cryo-diluent media improves microscopic parameters, and fertility potential, and alleviates oxidative stress parameters of buffalo spermatozoa. Cryobiology. 2020; 97:101-109.
16. 16. De Amicis F, Santoro M, Guido C, Russo A, Aquila S. Epigallocatechin gallate affects survival and metabolism of human sperm. Mol nutr food res. 2012; 56(11):1655-64.
17. 17. 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.
18. 18. Plaza Davila M, Bucci D, Galeati G, et al. Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding. Reprod Domest Anim. 2015; 50(6):1011-16.
19. 19. Gadani B, Bucci D, Spinaci M, Tamanini C, Galeati G. Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization. Theriogenology. 2017; 90:88-93.
20. 20. Guvvala PR, Ravindra JP, Rajani CV, Sivaram M, Selvaraju S. Protective role of epigallocatechin-3-gallate on arsenic induced testicular toxicity in Swiss albino mice. Biomed Pharmacother. 2017; 96:685-94.
21. 21. Küçük N, Raza S, Matsumura K, et al. Effect of different carboxylated poly l-lysine and dimethyl sulfoxide combinations on post thaw rabbit sperm functionality and fertility. Cryobiology. 2021; 102:127-32.
22. 22. Halo Jr M, Bułka K, Antos PA, et al. The effect of ZnO nanoparticles on rabbit spermatozoa motility and viability parameters in vitro. Saud J Biol Sci. 2021; 28(12):7450-54.
23. 23. Salisbury GW, VanDemark NL, Lodge JR. Physiology of reproduction and artificial insemination of cattle. WH Freeman and Company. 1978.
24. 24. 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):1223-28.
25. 25. Jiménez-Rabadán P, García-Álvarez O, Vidal A, et al. Effects of vitrification on ram spermatozoa using free-egg yolk extenders. Cryobiology. 2015; 71(1):85-90.
26. 26. Peña FJ, Johannisson A, Wallgren M, Rodrı́guez-Martı́nez 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.
27. 27. Abdelnour SA, Hassan MA, El‐Ratel IT, et al. 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.
28. 28. Kim S, Lee Y-J, Kim Y-J. 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.
29. 29. Li Z, Lin Q, Liu R, Xiao W, Liu W. Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation. J androl. 2010;31(5):437-444.
30. 30. Nishijima K, Kitajima S, Matsuhisa F, Niimi M, Wang C-c, Fan J. Strategies for Highly Efficient Rabbit Sperm Cryopreservation. Animals. 2021;11(5):1220.
31. 31. Ömür AD, Akarsu SA. Tavşanlarda Spermanın Alınması, İşlenmesi ve Saklanması. Lab Hayv Bil Uyg Derg. 2022; 2(2):105-10.
32. 32. Greifová H, Tvrdá E, Jambor T, Lukáč N. Dose-and time-dependent effects of epicatechin on bovine spermatoza in vitro. J Microbiol Biotechnol Food Sci. 2021; 2021:235-39.
33. 33. Sarıözkan S, Özdamar S, Türk G, Cantürk F, Yay A. In vitro effects of L-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage. Cryobiology. 2014; 68(3):349-53.
34. 34. Dias TR, Alves MG, Casal S, Silva BM, Oliveira PF. The single and synergistic effects of the major tea components caffeine, epigallocatechin-3-gallate and l-theanine on rat sperm viability. Food Funct. 2016; 7(3):1301-05.
35. 35. Nouri H, Shojaeian K, Samadian F, Lee S, Kohram H, Lee JI. Using resveratrol and epigallocatechin-3-gallate to improve cryopreservation of stallion spermatozoa with low quality. J Equine Vet Sci. 2018; 70:18-25.
36. 36. Kumar A, Prasad J, Srivastava N, Ghosh S. Strategies to minimize various stress-related freeze–thaw damages during conventional cryopreservation of mammalian spermatozoa. Biopreserv Biobank. 2019; 17(6):603-12.
37. 37. Isachenko E, Isachenko V, Katkov II, Dessole S, Nawroth F. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success. Reprod Biomed Online. 2003; 6(2):191-200.
38. 38. Kubovicova E, Makarevich AV, Balazi A, Vasicek J, Chrenek P. Factors affecting rabbit sperm cryopreservation: a mini-review. Zygote. 2022; 30(1):1-8. 39. Amidi F, Pazhohan A, Shabani Nashtaei M, Khodarahmian M, Nekoonam S. The role of antioxidants in sperm freezing: a review. Cell Tissue Bank. 2016; 17(4):745-56.
39. 40. Raza S, Uçan U, Aksoy M, Erdoğan G, Ceylan A, Serin I. Silk protein sericin pretreatment enhances osmotic tolerance and post-thaw sperm quality but reduces the ability of sperm cells to undergo in vitro induced acrosome reaction in rabbit. Cryobiology. 2019; 90:1-7.
40. 41. Zhang Y, Lin H, Liu C, Huang J, Liu Z. A review for physiological activities of EGCG and the role in improving fertility in humans/mammals. Biomed Pharmacother. 2020; 127:110186.
41. 42. Lombardo F, Sansone A, Romanelli F, Paoli D, Gandini L, Lenzi A. The role of antioxidant therapy in the treatment of male infertility: an overview. Asian J Androl. 2011; 13(5):690.
42. 43. Long L, Li Y, Wang YD, et al. The preventive effect of oral EGCG in a fetal alcohol spectrum disorder mouse model. Alcohol Clinic Exp Res. 2010; 34(11):1929-36.
43. 44. Chen M, Liu W, Li Z, Xiao W. Effect of epigallocatechin-3-gallate (EGCG) on embryos inseminated with oxidative stress-induced DNA damage sperm. Syst Biol Reprod Med. 2020; 66(4):244-54.
44. 45. Qu W, Kasprzak KS, Kadiiska M, et al. Mechanisms of arsenic-induced cross-tolerance to nickel cytotoxicity, genotoxicity, and apoptosis in rat liver epithelial cells. Toxicol Sci. 2001; 63(2):189-95.
45. 46. Flores E, Ramió-Lluch L, Bucci D, Fernández-Novell J, Peña A, Rodríguez-Gil J. Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm. Theriogenology. 2011; 76(8):1450-64.
46. 47. Wang J, Gao X, Wang L, et al. Establishment of the first cell line from the small yellow croaker (Larimichthys polyactis) and its application in unraveling the mechanism of ROS-induced apoptosis under hypoxia. Aquaculture. 2023; 563:738900.
47. 48. Asadi A, Ghahremani R, Abdolmaleki A, Rajaei F. Role of sperm apoptosis and oxidative stress in male infertility: A narrative review. Int J Reprod BioMed. 2021; 19(6):493.