Boğa Spermasının Çözdürme Sonrası Spermatolojik Kalitesi Üzerine Shilajit’in Etkileri

Öz

Sığır spermasının dondurularak saklanması oksidatif ve osmotik stres oluşturarak motilite, canlılık, morfoloji ve membran bütünlüğünde bozulmalara yol açmaktadır. Bu çalışmada, ticari bir sulandırıcıya Shilajit ilavesinin çözdürme sonrası sperma kalitesi üzerine etkisi araştırılmıştır. Dört sağlıklı boğadan elde edilen 16 ejakülat, kontrol ve %1, %2, %3 ile %4 Shilajit gruplarına ayrılmıştır. Çözdürme sonrası değerlendirmeler motilite ve kinematik parametreler (CASA), canlılık (Eosin–Nigrosin), morfoloji (Hancock), plazma membran bütünlüğü (HOST) ve oksidatif durum (TAS ve TOS) analizlerini içermiştir. Veriler tek yönlü ANOVA ve Tukey post hoc testi ile değerlendirilmiştir. Sonuçlar, Shilajit ilavesinin toplam motiliteyi %58,6’dan %79,2’ye, progresif motiliteyi ise %36,5’ten %51,6’ya çıkardığını göstermiştir (p < 0,001). Canlılık kontrol grubunda %80,5 iken %4 Shilajit grubunda %93,8’e yükselmiş, morfolojik anormallikler %10,5’ten %4,5’e düşmüştür (p < 0,001). Membran bütünlüğü tüm Shilajit gruplarında kontrol grubuna göre artmış (%61,9’a karşı %76,8), ancak dozlar arasında anlamlı fark bulunmamıştır. TAS ve TOS değerleri gruplar arasında istatistiksel olarak değişmemiştir; bu da Shilajit’in dondurma sürecinde oksidatif dengeyi koruduğunu göstermektedir. Sonuç olarak, Shilajit özellikle motilite, canlılık, morfoloji ve membran bütünlüğü gibi dondurma sonrası sperm kalite parametrelerini artırmış ve oksidatif dengeyi desteklemiştir. Bu bulgular, Shilajit’in boğa sperma sulandırıcılarında doğal bir katkı maddesi olarak umut verici potansiyele sahip olduğunu göstermektedir.

Anahtar Kelimeler

• Antioksidan
• Kriyoprezervasyon
• Shilajit
• Sığır
• Sperma Kalitesi

Effects of Shilajit on Post-Thaw Spermatological Quality of Bull Semen

Öz

Cryopreservation of bovine semen induces oxidative and osmotic stress, impairing motility, viability, morphology, and membrane integrity. This study investigated the effect of Shilajit supplementation in a commercial extender on post-thaw semen quality. Ejaculates from four clinically healthy bulls (n = 16) were divided into a control and four treatment groups supplemented with 1%, 2%, 3%, or 4% Shilajit. Post-thaw evaluations included motility and kinematic parameters (CASA), viability (Eosin–Nigrosin), morphology (Hancock), plasma membrane integrity (HOST), and oxidative status (TAS and TOS). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Results showed that Shilajit supplementation significantly improved total motility (58.6% in control vs. 79.2% in 4% group) and progressive motility (36.5% vs. 51.6%, p < 0.001). Viability increased from 80.5% in the control to 93.8% at 4%, while morphological abnormalities declined from 10.5% to 4.5% (p < 0.001). Membrane integrity also improved in all supplemented groups compared with the control (61.9% vs. up to 76.8%). TAS and TOS values remained statistically unchanged among groups, indicating that Shilajit maintained oxidative balance during cryopreservation. In conclusion, Shilajit enhanced multiple post-thaw sperm quality parameters, particularly motility, viability, morphology, and membrane integrity, while supporting oxidative stability. These findings highlight its potential as a natural additive in bovine semen extenders, with promising implications for artificial insemination outcomes.

Anahtar Kelimeler

• Antioxidant
• Bull
• Cryopreservation
• Semen Quality
• Shilajit

Kaynakça

1. Agarwal, A., Virk, G., Ong, C., & du Plessis, S. S. (2014). Effect of oxidative stress on male reproduction. World Journal of Men's Health, 32(1), 1–17. https://doi.org/10.5534/wjmh.2014.32.1.1
2. Aitken, R. J. (2020). Impact of oxidative stress on male and female germ cells: Implications for fertility. Reproduction, 159(5), R189–R201. https://doi.org/10.1530/REP-19-0452
3. Aitken, R. J., & Baker, M. A. (2006). Oxidative stress, sperm survival and fertility control. Molecular and Cellular Endocrinology, 250(1–2), 66–69. https://doi.org/10.1016/j.mce.2005.12.026
4. Aitken, R. J., Gordon, E., Harkiss, D., & Twigg, J. (1998). Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biology of Reproduction, 59(5), 1037–1046. https://doi.org/10.1095/biolreprod59.5.1037
5. Aitken, R. J., & Fisher, H. (1994). Reactive oxygen species generation and human spermatozoa: The balance of benefit and risk. BioEssays, 16(4), 259–267.
6. Alexander, J.H. (2008). Bull breeding soundness evaluation: a practitioner’s perspective. Theriogenology 70, 469–72.
7. Al-Mutary, M. G., Al-Ghadi, M. Q., Ammari, A. A., Al-Himadi, A. R., Al-Jolimeed, A. H., Arafah, M. W., Amran, R. A., Aleissa, M. S., & Swelum, A. A. A. (2020). Effect of different concentrations of resveratrol on the quality and in vitro fertilizing ability of ram semen stored at 5°C for up to 168 h. Theriogenology, 152, 139–146. https://doi.org/10.1016/j.theriogenology.2020.05.001
8. Alvarez, J. G., & Storey, B. T. (1995). Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Molecular Reproduction and Development, 42(3), 334–346.

9. Armstrong, J. S., Rajasekaran, M., Hellstrom, W. J., & Sikka, S. C. (1998). Antioxidant potential of human serum albumin: Role in the recovery of high quality human spermatozoa for assisted reproductive technology. Journal of Andrology, 19(4), 412–419.
10. Assunção, C. M., Mendes, V. R. A., Brandão, F. Z., Batista, R. I. T. P., Souza, E. D., de Carvalho, B. C., Quintão, C. C. R., Raposo, N. R. B., & Camargo, L. S. A. (2021). Effects of resveratrol in bull semen extender on post-thaw sperm quality and capacity for fertilization and embryo development. Animal Reproduction Science, 226, 106697. https://doi.org/10.1016/j.anireprosci.2021.106697
11. Bansal, A. K., & Bilaspuri, G. S. (2011). Impacts of oxidative stress and antioxidants on semen functions. Veterinary Medicine International, 2011, 686137. https:// doi. org/ 10. 4061/ 2011/ 686137
12. Bhattacharya, S. K., Sen, A. P., & Ghosal, S. (1995). Effects of shilajit on biogenic free radicals. Phytotherapy Research, 9(1), 56–59.
13. Bucak, M. N., Ataman, M. B., Başpınar, N., Uysal, O., Taşpınar, M., Bilgili, A., ... & Akal, E. (2015). Lycopene and resveratrol improve post‐thaw bull sperm parameters: Sperm motility, mitochondrial activity and DNA integrity. Andrologia, 47(5), 545–552.
14. Bucak, M. N., Tuncer, P. B., Sarıözkan, S., Başpınar, N., Taşpınar, M., Çoyan, K., ... & Öztuna, D. (2010). Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: Antioxidants protect DNA integrity against cryodamage. Cryobiology, 61(3), 248–253.
15. Büyükleblebici, O., Büyükleblebici, S., Taşdemir, U., & Tuncer, P. B. (2016). The effects of different antioxidants on post-thaw microscopic and oxidative stress parameters in the cryopreservation of Brown-Swiss bull semen. Turkish Journal of Veterinary and Animal Sciences, 40(4), 404–412.
16. Chatterjee, S., & Gagnon, C. (2001). Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Molecular Reproduction and Development, 59(4), 451–458.
17. Ghosal, S. (1990). Chemistry of shilajit, an immunomodulatory Ayurvedic rasayan. Pure and Applied Chemistry, 62(7), 1285–1288.
18. Güngör, Ş., Ata, A., İnanç, M. E., & Kastelic, J. P. (2019). Effect of various antioxidants and their combinations on bull semen cryopreservation. Turkish Journal of Veterinary and Animal Sciences, 43(5), 590–595.
19. Halliwell, B. (1990). How to characterize a biological antioxidant. Free Radical Research Communications, 9(1), 1–32.
20. Halliwell, B., & Gutteridge, J. M. C. (2015). Free radicals in biology and medicine (5th ed.). Oxford University Press. https://doi.org/10.24496/intnaturalprod.1988.0_524
21. Kong, Y., But, P., Ng, K., Cheng, K., Cambie, R., & Malla, S. (1987). Chemical studies on a Nepalese panacea shilajit (I). International Journal of Crude Drug Research, 25(3), 179–182.
22. Lewis, S. E. M., & Aitken, R. J. (2005). DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell and Tissue Research, 322, 33–41. https://doi.org/10.1007/s00441-005-1097-5
23. Lewis, S. E. M., Sterling, E. S. L., Young, I. S., & Thompson, W. (1997). Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men. Fertility and Sterility, 67(1), 142–147. https://doi.org/10.1016/S0015-0282(97)81871-7
24. Li, Y., Kalo, D., Zeron, Y., & Roth, Z. (2016). Progressive motility–a potential predictive parameter for semen fertilization capacity in bovines. Zygote, 24(1), 70-82.
25. Maneesh, M., & Jayalekshmi, H. (2006). Role of reactive oxygen species and antioxidants on pathophysiology of male reproduction. Indian Journal of Clinical Biochemistry, 21(2), 80–89.
26. Moazamian, R., Polhemus, A., Connaughton, H., & Aitken, R. J. (2015). Oxidative stress and human spermatozoa: Diagnostic and functional significance of aldehydes generated as a result of lipid peroxidation. Molecular Human Reproduction, 21(6), 502–515. https://doi.org/10.1093/molehr/gav014
27. O’Flaherty, C., & Scarlata, E. (2022). Oxidative stress and reproductive function: The protection of mammalian spermatozoa against oxidative stress. Reproduction, 164(6), F67–F78. https://doi.org/10.1530/REP-22-0200
28. Olfati Karaji, R., Daghigh Kia, H., & Ashrafi, I. (2014). Effects of combined antioxidant supplementation on microscopic and oxidative parameters of freeze–thaw bull sperm. Cell and Tissue Banking, 15, 461–470.
29. Selcuk, M., Akal, E., Esin, B., Nizam, M. Y., & Genc, M. D. (2020). Comparative evaluation of the effects of different thawing methods on bull spermcharacteristics with computer-assisted semen analysis. Turkish Journal of Veterinary & Animal Sciences, 44(6), 1316-1321.
30. Sharma, R., Agarwal, A., Mohanty, G., et al. (2013). Proteomic analysis of seminal fluid from men exhibiting oxidative stress. Reproductive Biology and Endocrinology, 11, 85. https://doi.org/10.1186/1477-7827-11-85
31. Sikka, S. C. (1996). Oxidative stress and role of antioxidants in normal and abnormal sperm function. Frontiers in Bioscience, 1, e78–e86.
32. Simões, R., Feitosa, W. B., Siqueira, A. F. P., et al. (2013). Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome. Reproduction, 146(5), 433–441. https://doi.org/10.1530/REP-13-0123
33. Srivastava, R. S., Kumar, Y., Singh, S. K., & Ghosal, S. (1988). Shilajit: Its source and active principles. In International Symposium on the Chemistry of Natural Products (p. 524).
34. Sultan, J., Awan, M. A., Rakha, B. A., Waqar, S. A., Ansari, M. S., Naz, S., Iqbal, S., & Akhter, S. (2021). Asphaltum improves the post-thaw quality and antioxidant status of Nili Ravi buffalo bull sperm. Biopreservation and Biobanking, 19(3), 194–203.
35. Tasdemir, U., Tuncer, P., Buyukleblebici, S., Ozgurtas, T., Durmaz, E., & Buyukleglebici, O. (2014). Effects of various antioxidants on cryopreserved bull sperm quality. Kafkas Universitesi Veteriner Fakultesi Dergisi, 20(2).
36. Tuncer, P. B., Sariözkan, S., Bucak, M. N., & Büyükleblebici, S. (2021). Antioxidant supplementation ameliorates bull sperm parameters and fertilizing abilityfollowing the freeze-thaw process. Turkish Journal of Veterinary & Animal Sciences, 45(3), 457-462.
37. Tvrdá, E., Lukáč, N., Lukáčová, J., Hashim, F., & Massányi, P. (2015). In vitro supplementation of resveratrol to bovine spermatozoa: Effects on motility, viability and superoxide production. Journal of Microbiology, Biotechnology and Food Sciences, 4(4), 336–341. https://doi.org/10.15414/JMBFS.2015.4.4.336-341
38. Ugur, M. R., Saber Abdelrahman, A., Evans, H. C., Gilmore, A. A., Hitit, M., Arifiantini, R. I., Purwantara, B., Kaya, A., & Memili, E. (2019). Advances in cryopreservation of bull sperm. Frontiers in Veterinary Science, 6, 268. https://doi.org/10.3389/fvets.2019.00268
39. Ünal, İ., & Uysal, H. (2024). Seminal oxidative stress index can be used as a marker in the prediction of bull semen cryotolerance. Animal Reproduction Science, 270, 107618.
40. Wang, A. W., Zhang, H., Ikemoto, I., Anderson, D. J., & Loughlin, K. R. (1997). Reactive oxygen species generation by seminal cells during cryopreservation. Urology, 49(6), 921–925.
41. Xiao, Y., Wu, Z., & Wang, M. (2018). Effects of fulvic acids on goat sperm. Zygote, 26(3), 220–223.