Yeni Zelanda Tavşanı Spermasının Çözdürme Sonrası Spermatolojik ve Biyokimyasal Yönden İncelenmesi

Öz

Sunulan çalışmada antioksidan madde trehaloz pozitif kontrol olarak kullanılarak kafeik asitin tavşan sperması üzerine etkileri araştırıldı. Deneyde, 4 erkek ve 1 dişi tavşan kullanıldı. Ejakülatlar 4 hafta boyunca haftada 2 tekrar şeklinde suni vajen yöntemi ile alındı. Alınan spermalar pooling yapıldıktan sonra dört eşit gruba ayrılarak sulandırıldı: temel sulandırıcı +%6 DMSO+kafeik Asit (25µM ve 50µM), temel sulandırıcı+%6 DMSO+trehaloz (pozitif kontrol) (50mM) ve temel sulandırıcı+%6 DMSO (Kontrol) içeren sulandırıcılar ile seyreltildi. Sulandırma işlemin sonrasında sperma örnekleri payetlere çekilerek, +4°C de 1 saat ekilibrasyon uygulandı. Numuneler, sıvı azot buharında donduruldu ve sıvı azotta saklandı. Çözüm sonrası sperma motilite değerlerinde en yüksek değere trehaloz 50 mM grubunda (%46.25±1.25) ulaşıldı ve kontrol grubu ile (%35.63±1.99) istatistiksel fark belirlendi (p<0.05). Sperma membran bütünlüğü (SYBR 14/PI) sonuçları kafeik Asit 25 µM kafeik Asit 50 µM ve trehaloz 50 mM gruplarında sırasıyla %47.11±0.96; %49.05±0.85; %49.79±1.04 olarak belirlenmiş ve katkı grupları ile kontrol grubu (%39.01±1.21) arasında istatistiksel fark bulunmuştur (p<0.05). Akrozom bütünlüğü (FITC-PNA) sonuçlarında ise Trehaloz 50 mM %48.70±1.03 gurubunda en yüksel değere ulaşılmış ve kontrol grubu %41.48±0.80 ile istatistiksel fark görülmüştür. Total antioksidan kapasite ve total oksidan seviye değerlerinde, Trehalose 50 mM grubunda kontrol grubuna kıyasla anlamlı fark gözlendi (p<0.05). Sonuç olarak, tavşan sperması dondurulmasında sulandırıcıya eklenen kafeik asit in yüksek dozda daha iyi koruyucu etki gösterdiği belirlendi.

Anahtar Kelimeler

• kafeik asit
• kriyoprezervasyon
• floresan boyama
• oksidatif stres
• tavşan sperması

Proje Numarası

The Scientific and Technological Research Council of Turkey (TÜBİTAK) with number 1919B011802699

Spermatological and Biochemical Examination of New Zealand Rabbit Semen After Thawing

Öz

In the present study, the effects of caffeic acid on rabbit semen were investigated using the antioxidant substance trehalose as a positive control. In the experiment, 4 male rabbits and 1 female rabbit were used. Ejaculates were collected via the artificial vaginal method 2 times a week for 4 weeks. Pooled semen samples were divided into four groups and diluted as follows: basic diluent+6%DMSO+caffeic acid (25µM and 50µM), basic diluent+6%DMSO+trehalose (positive control) (50mM), and basic diluent+6%DMSO (Control). After dilution, the semen samples were aspirated into straws and allowed to equilibrate at +4°C for 1 h. The samples were frozen in liquid nitrogen vapor and stored in liquid nitrogen. The highest value of sperm motility was detected in the 50 mM trehalose group (46.25±1.25%), which was significantly different from that in the control group (35.63±1.99%) (p<0.05). The sperm membrane integrity (SYBR 14/PI) results were determined to be 47.11±0.96%, 49.05±0.85%, and 49.79±1.04% in the 25 µM caffeic acid, 50µM caffeic acid, and 50 mM trehalose groups, respectively, and a significant difference was found between the supplement groups and the control group (39.01±1.21%) (p<0.05). In terms of acrosome integrity (FITC-PNA), the highest value was reached in the trehalose 50 mM group (48.70±1.03%), and a significant difference was observed compared with that in the control group (41.48±0.80%). A significant difference was observed in the total antioxidant capacity and total oxidant level values between the 50 mM trehalose group and the control group (p<0.05). As a result, caffeic acid added to the extender when rabbit semen was frozen had a better protective effect at higher doses.

Anahtar Kelimeler

• caffeic acid
• cryopreservation
• fluorescence staining
• oxidative stress
• rabbit sperm

Destekleyen Kurum

This research was funded by The Scientific and Technological Research Council of Turkey (TÜBİTAK) with number 1919B011802699

Proje Numarası

The Scientific and Technological Research Council of Turkey (TÜBİTAK) with number 1919B011802699

Etik Beyan

This study was conducted at the Experimental Animal Application and Research Center of Aksaray University. The research protocol was approved by the Aksaray University Local Ethics Committee for Animal Experiments (Approval date: 11/2021; Decision No: 2021-7/14).

Teşekkür

This research was funded by The Scientific and Technological Research Council of Turkey (TÜBİTAK) with number 1919B011802699

Kaynakça

1. Agarwal, A., Virk, G., Ong, C., & Du Plessis, S. S. (2014). Effect of oxidative stress on male reproduction. The world journal of men's health, 32(1), 1.
2. Akyol, S., Akbas, A., Butun, I., Toktas, M., Ozyurt, H., Sahin, S., & Akyol, O. (2015). Caffeic acid phenethyl ester as a remedial agent for reproductive functions and oxidative stress-based pathologies of gonads. Journal of intercultural ethnopharmacology, 4(2), 187.
3. Ball, B. A. (2008). Oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse. Animal reproduction science, 107(3-4), 257-267.
4. Benaroudj, N., & Goldberg, A. L. (2001). Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. Journal of Biological Chemistry, 276(26), 24261-24267.
5. Bilodeau, J. F., Chatterjee, S., Sirard, M. A., & Gagnon, C. (2000). Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Molecular Reproduction and Development: Incorporating Gamete Research, 55(3), 282-288.
6. Boiti, C., Castellini, C., Besenfelder, U., Theau-Clément, M., Liguori, L., Renieri, T., & Pizzi, F. (2005). Guidelines for handling of rabbit bucks and semen.World Rabbit Science, 13(2), 71-91.
7. Budgude, P., Kale, V., & Vaidya, A. (2021). Cryopreservation of mesenchymal stromal cell-derived extracellular vesicles using trehalose maintains their ability to expand hematopoietic stem cells in vitro. Cryobiology, 98, 152-163.
8. Dovgan, B., Barlič, A., Knežević, M., & Miklavčič, D. (2017). Cryopreservation of human adipose-derived stem cells in combination with trehalose and reversible electroporation. The Journal of membrane biology, 250(1), 1-9.

9. Ehtiati, S., Alizadeh, M., Farhadi, F., Khalatbari, K., Ajiboye, B. O., Rahimi, V. B., & Askari, V. R. (2023). Promising influences of caffeic acid and caffeic acid phenethyl ester against natural and chemical toxins: A comprehensive and mechanistic review. Journal of Functional Foods, 107, 105637.
10. El-Mazoudy, R. H., Abd ElGawad, H. S., & El-Shenawy, N. S. (2011). Evaluation of protective effect of propolis against testicular oxidative damage, lipid peroxidation and infertility induced by chlorpyrifos in albino rats, Egypt. J. Exp. Biol. (Zool.), 7(2), 129-138.
11. Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical biochemistry, 38(12), 1103-1111.
12. Erel, O., & Neselioglu, S. (2014). A novel and automated assay for thiol/disulphide homeostasis. Clinical biochemistry, 47(18), 326-332.
13. Foote, R. H., & Carney, E. W. (2000). The rabbit as a model for reproductive and developmental toxicity studies. Reproductive Toxicology, 14(6), 477-493.
14. Garner, D. L., & Johnson, L. A. (1995). Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biology of Reproduction, 53(2), 276–284.
15. Iwatani, M., Ikegami, K., Kremenska, Y., Hattori, N., Tanaka, S., Yagi, S., & Shiota, K. (2006). Dimethyl sulfoxide has an impact on epigenetic profile in mouse embryoid body. Stem cells, 24(11), 2549-2556.
16. Jain, N. K., & Roy, I. (2009). Effect of trehalose on protein structure. Protein Science, 18(1), 24-36.
17. Khaled, F. A., Yousef, M. I., & Kamel, K. I. (2016). The protective role of propolis against the reproductive toxicity of mono-sodium glutamine in male rabbits. IJCS, 4(2), 4-9.
18. Khan, I. M., Cao, Z., Liu, H., Khan, A., Rahman, S. U., Khan, M. Z., Sathanawongs A., & Zhang, Y. (2021). Impact of cryopreservation on spermatozoa freeze-thawed traits and relevance OMICS to assess sperm cryo-tolerance in farm animals. Frontiers in Veterinary Science, 8, 609180.
19. Kus, I., Colakoglu, N., Pekmez, H., Seckin, D., Ogeturk, M., & Sarsilmaz, M. (2004). Protective effects of caffeic acid phenethyl ester (CAPE) on carbon tetrachloride-induced hepatotoxicity in rats. Acta histochemica, 106(4), 289-297.
20. Lan, Q., Xue, L. E., Cao, J., Xie, Y., Xiao, T., & Fang, S. (2022). Caffeic acid phenethyl ester (CAPE) improves boar sperm quality and antioxidant capacity in liquid preservation (17 C) linked to AMPK activity maintenance. Frontiers in Veterinary Science, 9, 904886.
21. López-Gatius, F., Sances, G., Sancho, M., Yániz, J., Santolaria, P., Gutiérrez, R., Núñez M., Núñez J., & Soler, C. (2005). Effect of solid storage at 15 C on the subsequent motility and fertility of rabbit semen. Theriogenology, 64(2), 252-260.
22. Mandumpal, J. B., Kreck, C. A., & Mancera, R. L. (2011). A molecular mechanism of solvent cryoprotection in aqueous DMSO solutions. Physical Chemistry Chemical Physics, 13(9), 3839-3842.
23. Martínez-Páramo, S., Pérez-Cerezales, S., Gómez-Romano, F., Blanco, G., Sánchez, J. A., & Herráez, M. P. (2009). Cryobanking as tool for conservation of biodiversity: effect of brown trout sperm cryopreservation on the male genetic potential. Theriogenology, 71(4), 594-604.
24. Mehdipour, M., Daghigh-Kia, H., Najafi, A., Mehdipour, Z., & Mohammadi, H. (2022). Protective effect of rosiglitazone on microscopic and oxidative stress parameters of ram sperm after freeze-thawing. Scientific reports, 12(1), 13981.
25. Nagy, S., Jansen, J., Topper, E. K., & Gadella, B. M. (2003). A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in a field situation. Theriogenology, 59(9), 1801–1815.
26. Namula, Z., Hirata, M., Wittayarat, M., Tanihara, F., Thi Nguyen, N., Hirano, T., Nii M., & Otoi, T. (2018). Effects of chlorogenic acid and caffeic acid on the quality of frozen‐thawed boar sperm. Reproduction in Domestic Animals, 53(6), 1600-1604.
27. Notman, R., Noro, M., O'Malley, B., & Anwar, J. (2006). Molecular basis for dimethylsulfoxide (DMSO) action on lipid membranes. Journal of the American Chemical Society, 128(43), 13982-13983.
28. Ntai, A., La Spada, A., De Blasio, P., & Biunno, I. (2018). Trehalose to cryopreserve human pluripotent stem cells. Stem cell research, 31, 102-112.
29. Ogeturk, M., Kus, I., Colakoglu, N., Zararsiz, I., Ilhan, N., & Sarsilmaz, M. (2005). Caffeic acid phenethyl ester protects kidneys against carbon tetrachloride toxicity in rats. Journal of ethnopharmacology, 97(2), 273-280.
30. Ozturk, C., & Ataman, M. B. (2018). Effect of paclitaxel and resveratrol on New Zealand rabbit semen. Asian Pacific Journal of Reproduction, 7(6), 266-269.
31. Petričáková, K., Janošíková, M., Ptáček, M., Savvulidi, F. G., & Zita, L. (2024). In Vitro and In Vivo Evaluation of the Fertilization Capacity of Frozen/Thawed Rooster Spermatozoa Supplemented with Different Concentrations of Trehalose. Animals, 14(24), 3586.
32. Richards, A. B., Krakowka, S., Dexter, L. B., Schmid, H., Wolterbeek, A. P. M., Waalkens-Berendsen, D. H., Shigoyuki A., & Kurimoto, M. (2002). Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food and chemical toxicology, 40(7), 871-898.
33. Rosato, M. P., & Iaffaldano, N. (2013). 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, 79(3), 508-516.
34. Rusco, G., Słowińska, M., Di Iorio, M., Cerolini, S., Maffione, A. B., Ciereszko, A., & Iaffaldano, N. (2022). Proteomic analysis of rabbit fresh and cryopreserved semen provides an important insight into molecular mechanisms of cryoinjuries to spermatozoa. Theriogenology, 191, 77-95.
35. Russo, A., Troncoso, N., Sanchez, F., Garbarino, J. A., & Vanella, A. (2006). Propolis protects human spermatozoa from DNA damage caused by benzo [a] pyrene and exogenous reactive oxygen species. Life sciences, 78(13), 1401-1406.
36. Sariozkan, S., Canturk, F., Yay, A., & Akcay, A. (2012). The effect of different storage temperature on sperm parameters and DNA damage in liquid stored new zealand rabbit spermatozoa. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 18(3).
37. Soleimanzadeh, A., Talavi, N., Yourdshahi, V. S., & Bucak, M. N. (2020). Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process. Cryobiology, 95, 29-35.
38. Soylu, M. K., Ak, K., Akçay, E., Baran, A., Evecen, M., & Tırpan, M. B. (Ed.). (2023). Hayvanlarda reprodüksiyon, androloji ve yardımcı üreme teknikleri (1. baskı). Ankara: Nobel Tıp Kitabevleri.
39. Takalani, N. B., Monageng, E. M., Mohlala, K., Monsees, T. K., Henkel, R., & Opuwari, C. S. (2023). Role of oxidative stress in male infertility. Reproduction and Fertility, 4(3).
40. Valdivia, M., Bravo, Z., Reyes, J., & Gonzales, G. F. (2021). Rescue and conservation of male adult alpacas (Vicugna pacos) based on spermatogonial stem cell biotechnology using atomized Black Maca as a supplement of cryopreservation medium. Frontiers in Veterinary Science, 8, 597964.
41. Yousef, M. I., & Salama, A. F. (2009). Propolis protection from reproductive toxicity caused by aluminium chloride in male rats. Food and chemical toxicology, 47(6), 1168-1175.
42. Yousef, M. I., Kamel, K. I., Hassan, M. S., & El-Morsy, A. M. (2010). Protective role of propolis against reproductive toxicity of triphenyltin in male rabbits. Food and Chemical Toxicology, 48(7), 1846-1852.
43. Zhu, Z., Fan, X., Pan, Y., Lu, Y., & Zeng, W. (2017). Trehalose improves rabbit sperm quality during cryopreservation. Cryobiology, 75, 45-51.